Arch Engg Orientatn
Arch Engg Orientatn ARE 100
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BIOL 687 Spring 2005 Introduction Microbial Ecology Spring 2005 Walter Dodds Lecture Notes Errors will be present test will cover information given during lecture and these are just to help you study and cannot be considered a complete substitute for attending class Please do not duplicate Without permission BIOL 687 Spring 2005 Introduction Microbial Ecology Topic 1 History and Introduction I A B C D E m A 13 III 3 Course Introduction Will cover the ecology of microorganisms in the natural environment Will use basic ecology basic microbiology and things that are more speci c to microbial ecologists As Louis Pasteur said quotThe role ofthe in nitely small is in nitely largequot Recent estimates of biomass in cellular carbon suggest that plants and bacteria have about the same amount There are about 5 X 1030 bacterial cells on earth Since bacterial cells are nutrient rich N and P and they represent the single largest pool of these nutrients on earth Whitman et al 1998 PNAS 9565786583 Will attempt to super cially cover all aspects of microbial ecology but Dr Rice s course covers the microbial ecology of soil so I will emphasize the aquatic aspects more heavily He also offers the laboratory which will help with methods for those with interest Basic mechanics Syllabus text of ce hours grade breakdown class schedule etc What is a microorganism De ned subjectively as anything smaller than the human eye can see We cannot easily see objects smaller than 01 mm diameter History of Microbial Ecology Early Microbiology 1 Robert Hooke 1664 described fruiting structure of fungi and some protozoa 2 Antoni van Leeuwenhoek used less ornate microscopes and was able to increase his resolution signi cantly Saw individual sperm after performing quotgross experiments upon himself quot He was also able to see single bacteria 1 pm resolution In a letter to the Royal Society of London in 1674 he wrote quotTho my teeth are kept usually very clean nevertheless when I view them in a Magnifying Glass I nd growing between them a little whiter matter as thick as wetted ower and then to my great surprise perceived that the aforesaid matter contained very many small Animals which moved themselves very extravagantly The biggest sort darted themselves thro the water of spittle as a Jack or Pike goes thro the water I took in my mouth some very strong wineVinegar and closing my Teeth I gargled and rinsed them very well with fair water but there were an innumerable quantity of Animals yet remaining in the scurf upon the Teeth The number of these Animals in the scurf of a man s Teeth are so many that I believe they exceed the number of Men in a kingdom quot Little else occurred in the eld of microbiology until the 1800 s microbiology for the most part remained descriptive and a parlor game The next major change in the eld of 39 generation i Spallanzani was able to show that meat which was heated and sealed did not putrefy rot but there was some inconsistency in his experiments because of heat resistant spores Work led to understanding canning food ii The controversy continued until 1860 s when Louis Pasteur the father of microbiology Showed that air contains microorganisms Filtered large amount of air through cotton Extracted cotton with alcohol and found animicules Made swan necked asks which he was able to boil and leave open to air The bacteria and fungi could not enter and the contents remained sterile His work seriously wounded the ideas of spontaneous generation 39 39 39 was the over 1 L V A evvv BIOL 687 Spring 2005 Introduction in John Tyndall Showed that boiling for as long as 5 12 hours did not kill all bacteria in a hay solution A 3 b Found resistant spores of bacteria in hay c Showed that if hay was boiled and cooled bacteria would start growing d Could kill bacteria by discontinued heating e Finally laid to rest theory of spontaneous generation 4 Fermentation and putrefaction i During l 39 l quot noticed that ill smelling products and gasses formed ii In 1837 CagnairdLatour Schwann and Kiitzing all proposed that yeast forms alcohol from sugars in Pasteur and others showed that microorganism could make a variety of chemical transformations iv Found fermentation is a result of breakdown of carbohydrates v Putrefaction result of breakdown of proteins such as rotting meat 5 Anaerobic life i Pasteur discovered that some microbes only lived in the absence of oxygen 21 He de ned fermentation as life without air b Showed that alcohol formed from sugar by yeast with no oxygen present Pasteur was working for brewers distillers vinegar producers and winemakers and with his pure cultures characterized fermentations for the rst time Around this time late 1800 s working in Carlsberg brewery isolated pure yeasts allowed control over beer fermentations 0 Only carbon dioxide formed with air present 6 Microorganisms as the cause of disease i Joseph Lister 1864 a Anesthesia allowed more complicated procedures b Lister impressed with Pasteur showing bacteria in air 0 Sterilized instruments dressing and air around surgical procedures d Dramatically reduced infection rates e Gave indirect evidence of microorganisms as cause of human disease ii Robert Koch 18431910 a Showed anthrax common in domestic animals was a disease caused by microbes b Isolated rod shaped bacteria from infected animals 0 Transferred isolated bugs in sterile medium 8 times d reinfected animals with the bacteria e Finally reisolated the disease f Became known as Koch s postulates 1 Microorganism always present when disease is 2 Must be isolated from diseased organism 3 Must cause disease when reinoculated in healthy organism 4 Must be able to recover the microbe from the reinfected organism g It was discovered that some diseases could be transmitted by uid passed through lters able to remove bacteria These particles were later shown to be viruses B Brief Environmental microbiologists BIOL 687 Spring 2005 Introduction 1 Microbial activity in the natural environment in the 1800 s i Soil would oxidize hydrogen gas but this ability would disappear with heating or acidi cation ii Ammonium changes to nitrate in sewage when passing through a sand column Processes stopped with chloroform vapors but could be restored by inoculation with soil 2 Sergei Winogradsky 18561953 i Isolated nitrifying bacteria take ammonium to nitrate ii Described the oxidation of hydrogen sul de and sulfur and oxidation of ferrous iron in De ned the idea of chemoautotrophy using chemicals for energy iv Described anaerobic nitrogen xing bacteria 3 Martinius Beijernck 18511931 i Isolated agents of symbiotic and aerobic nitrogen xation ii Believed that all microorganisms were everywhere that to culture them only the proper isolation technique was necessary in According to text was a man with no life other than microbiology 4 Numerous phycological mycological and protozoan taxonomists C History of ecology People have been interested in how the environment works from day 1 To survive knowledge of the environment was essential 2 Some of the earliest writers discuss environmental issues such as plagues 3 Greeks Herodotus and Plato had the idea of harmony in nature That a natural balance occurred and species outbreaks were punishments by spirits 4 Leeuwenhoek studied the reproductive rates of grain beetles carrion ies and human lice 5 Malthus published an early book on population growth Essay on Population 1798 in which he described geometric growth of populations 6 Malthus Darwin and others challenged the idea of harmony in nature in the day which ultimately led to the idea of natural selection 7 Much of early ecology came from the applied elds i In 1762 the Mynah bird was introduced from India to the island of Mauritius to control the red locust ii Similar biological introductions occurred from this time forward using ecology to protect food crops from pest invasions Medical work on malaria in late 1800 s centered on vectors and mathematics of epidemiology 9 S A Forbes and Karl Mobius among others started describing organisms in their community context in the late 1800 s 10 In 1899 H C Cowles described secession of plants on sand dunes and the idea of succession was taken by Clements 1916 and developed into a eld of study was controversial for years 11 The 1950 s saw the emergence of ecology as a de ned eld Major occurrences then include but are not limited to r V so V 1 Arguments between Nicholson and Baily about the relative importance of biotic or abiotic interactions controlling populations ii R MacArthur had ideas about the structuring of communities by competition in Odum pioneered the study of ecosystems theory looking at mass ux of materials and energy to describe system dynamics D Environmental issues and technology as the spur to contemporary development of microbial ecology 1 N V BIOL 687 Spring 2005 Introduction Awareness that we were messing up the environment came about during the 1960 s Ecologists work became perceived as important and problems such as water pollution and toxic chemicals could only be 39 b 39 39 39 39 working with ecologists This increased interest vastly expanded the number of people in the eld Within the last 40 years general application of new technologies has vastly increased the abilities to study microbes i Microscopes and electron microscopes vastly improved allowing distinction between microorganisms Counting techniques have allowed for direct enumeration of bacteria Analytical capabilities have improved allowing determination of metabolic pathways and ux rates of natural material in ecosystems Plastics have allowed for increasing sampling capabilities better incubation chambers greater ease of laboratory analysis Computers allow for much more rapid analysis of data Molecular techniques have increased the ability of investigators to identify organisms in their natural environment and determine taxonomic affinities between organisms vi Global sensing and sampling allows us to consider global biogeochemical cycles We are now to the point where we can start to gure out how gene activity relates to the biogeochemistry of the earth microbes drive it we can just start to gure out how Newman and Ban eld 2002 Science 29610711077 ii iii iv V Microbial Ecology Topic 2 Systematics 1 Names of organisms A Binomial system two names used mainly for eukarya 1 First name genus name 2 Second name species name 3 Usually Latin or Greek but often descriptive 1 Dictionary of roots can help 4 Will require students to underline binomial species names in this course 11 Small particles that can barely be considered organisms because they can not reproduce or metabolize by themselves A Prions infective protein molecules scrapie Kuru etc controversial if they are even infectious agents B C BIOL 687 Spring 2005 Systematics Virooids viruses containing only RNA Naked viruses Viruses RNA or DNA single or double stranded with a protein coat important in controlling microbial communities The viruses are de ned based on the size of their genome the capsid proteinaceous coat and what they infect D Much more complex viruses found recently Raolt et al Science 30613441350 1 Infects protozoa 2 Has 12 million bases 3 Evolved early as a eukaryotic virus 111 Prokaryotes now should be considered Archaea and Bacteria and Eukaryotes A Early classi cation of organisms split all organism into either plants or animals B Microbiology proved that there was no simple dichotomy 1 Some microscopic plants are motile and swim like animals 2 Some quotplantsquot such as fungi were able to live non photosynthetically 3 Fungi which are like plants have motile stages C Microorganisms were all classed as protists until electron microscopy increased the resolving power to much less than 1 pm D Electron microscopy and biochemical techniques con rmed light microscopy and solidi ed idea that that there are two major types of organisms Characteristic Bacteria Eukaryote Typical cell size 0150 um 1200 um nuclear body None nucleus nuclear membrane mycoplasmas DNA single molecule chromosomes histone bound Ribosomes small 70S large 80S except chloroplasts and mitochondria 70S Organelles None many cell wall peptidoglycan or none thick or absent chemically variable BIOL 687 Spring 2005 Systematics ll Movement small agella complex large agella m V Prokaryotes Archaea and Bacteria Prokaryotes make up what we call Bacteria 1 type of algae blue green algae cyanobacteria and Archaea Bergey s Manual of Determinative Bacteriology is the classi cation scheme that many professional microbiologists use Taxonomy is generally determined on the basis of morphology and a series of metabolic tests In this system it is important to get an organism into culture Instead of the binomial system there is a genus and a strain number or a genus and a species and a strain number How to de ne species 1 Classical de nition of species Problem with microorganisms 2 Are there distinct microbial species or just a continuum The problem of bacterial taxonomy Most cells very small and simple with little to differentiate one from another Bacterial species do not exhibit strict sexual reproduction but do exchange genetic information i This is used to a de ning characteristic for species in higher organisms such as plants and animals The idea that organisms are only distinct species if they can mate and produce viable offspring leaves out a large number of species ii Structural and chemical properties used on bacteria more recently molecular techniques stressed Cellular characteristics used in identi cation Size can depend upon culture medium ca 15 pm some are much less mycoplasmas have no cell wall and can squeeze through a 02 pm pore size lter Capsule i Appears as a halo around cell when stained with India ink ii Slime layer made of carbohydrates Cell wa i Gram staining reaction often used as the rst major grouping of Bacteria 21 Gram positive cells do not decolorize when exposed to alcohol after being stained with Gram s stain b Gram negative cells decolorize c Gram positive cells have peptidoglycan layers with tiechoic acid near the surface d Gram negative cells have a single layer of peptidoglycan associated with phospholipid lipoproteins and lipopolysaccharides complex lipids which make up a outer membrane 1 Cell wall structure gram gram 2 Some bacteria have no cell walls Mycoplasma Flagella some bacteria have speci c arrangements of agella some lack them completely Cell membrane lipid analysis can lead to classi cation scheme Pili mbriae protein extensions Environment some species only isolated from specialized environments Note this may be tricky For example the genes to switch an organism from obligate aerobe to a facultative optional aerobe are few an may be easily transferred Holzman 1998 ASM NH VV V N V L V IQMb VVVV so V 6 BIOL 687 Spring 2005 Systematics News 64552 Nutrition some species have very speci c nutritional requirements or speci c byproducts See table for more complete list of groups from Bergey s manual This has been taken advantage of recently with the Biolog system In this system numerous potential C sources are presented in a multiwell plate A redox indicator is used to determine if respiration is occurring The pattern of spots can be related to the taxonomy of the organism F Molecular methods 1 Recent methods make use of biochemical techniques to show degree of relatedness This could cause problems because some divergence is related directly to bacterial recombination Guttman amp Dykhuizen 1994 Science 26613801383 0 V 2 DNA hybridization i When strands are separated will try to bind with matching pairs ii if two cultures are of the same species most of DNA will bind most genes are the same in Degree of similarity can be shown by the percentage of DNA which binds iv In very different species ie bacteria and humans only a small percentage of the DNA will bind v an older version of this is CG content of DNA Considered not related if varies by more than 10 However this can cause problems because two fairly different species can converge on the same GampC composition Steel et al 1993 Nature 364440442 3 RNA methods i Ribosomal RNA is used to build proteins ii Each species has its own type of RNA rRNA iii rRNA is very stable evolutionarily changes very slowly unlike DNA or proteins this is because small changes in the machinery which makes all proteins can be devastating iv Degree of similarity shows degree of relatedness v The longer the two organisms separated the less similar the rRNA is Ahem 1994 AMS News 11600603 look at handout vi Some specieis dif cult to separate with rRNA may use proteins for these Palys et al 2000 Int J Systemat Evol Microbiol 5010211028 viiPotential problem with rRNA is that some genes may be identical in some widely divergent microbes Jaspers and Overmann 2004 AEM 7048314839 presentation 4 Lipid analysis i High pressure liquid chromatography has made possible determination of a characteristic ngerprint for each species many variable lipids in each cell ii There is currently a company which will attempt an identi cation on the basis of a library they have on lipid pro les Woose and his colleagues found a new groups of organism using this technique Archaea Includes therrnoacidophylic methanogenic organisms Originally thought to mainly inhabit extreme environments but more recently a number of reports suggest that they can be common in more benign habitats including soil and lakes Aravalli et al TREE 13190194 3 Very common in deep ocean Recent data suggest that 13 or more of bacteriasized particles in open oceans are archaebacteria LopezGarcia et al 2001 Nature 409603607 der Staay et al 2001 Nature 409507510 Kamer et al 2001 Nature 409507510 These deep ocean bacteria probably arose from thermophilic ancestors that invaded low temperature enivronements DeLong 2003 ASM news 69503511 C1 V NH VV 3 V gt lt V lt3 5 55 BIOL 687 Spring 2005 Systematics 5 Previously thought to be prokaryotes 6 According to rRNA analysis are as different from prokaryotes as prokaryotes are from eukaryotes 7 Chemical analysis tends to support this however Hori et al 1991 Evolution of Life pp 325 336 suggest that 5s RNA analysis reveals that bacteria and Archaea are in the same group That 16s RNA analysis is more misleading because of higher substitution rates in 16 s rrna several hundred bases have been added in Eukarya whereas 5 s rRNA remains invariant This is probably not true Archaea amp Eukarya both have similar transcription factors for RNA polymerase I II amp III Bacteria don t have it Rowlands et al 1994 Science 2641326 1329 8 Now have complete genome sequenced of an archaebacterium Bult et al 1996 Science 27310581073 Genes related to energy production metabolism and cell division most similar to those in bacteria Those for transcription translation and replication more similar to those found in Eukarya 9 Amino acid sequence data from 57 different enzymes using protein sequence changes as molecular clock suggest that Bacteria and Eubacteria diverged about 2 billion years ago Archaebacterial sequences were more similar to Eukarya than Bacteria Doolittle et al 1996 Science 271 470477 10 See book for evolutionary classi cation based on rRNA One major dif culty with using molecular methods to establish a tree of life is that genes have moved across broadly divergent types of bacteria Cohan 2001 Syst Biol 50513524 1 The more complete genomes we look at the more of this we see 2 Furthermore Eukaryotes may have different rates of gene change so lineage may be older than previously thought Pennisi E 1999 Science 28413051307 3 It appears that the eukaryotic genome formed from fusion of two prokaryotic genomes Rivera and Lake 2004 Nature 431 152155 presentation Photosynthetic organisms Please note that this taxonomy of bacteria does not necessarily represent phylogenetic relationships as that of higher organisms but organisms are grouped for convenience Cyanobacteria Oxygenic photosynthesis water occasionally H2 S as the electron donor Green bacteria Anoxygenic photosynthesis H2 HZS or S as the electron donor 1 39 39 39 39 r39 39 39 only use to create energy and to assist in carbon acquisition Prochlorobacteria Oxygenic photosynthesis water as the electron donor contain chl a and b like higher plants Purple photosynthetic bacteria anoxygenic photosynthesis H2 HZS or S as the electron donor can use CO2 or organic carbon as the carbon source Aerobic anoxygenic photosynthetic bacteria closely related to purple photosynthetic bacteria Can be heterotrophic or photosynthesize Sensing bacteriochlorophyll suggests that can make up to 11 of bacterial community in open ocean Kolber 2001 Science 29224922495 Taxonomy of photosynthetic bacteria can be dif cult because of lateral transfer of genes Raymond et al 2002 Science 29816161619 presentation Gliding bacteria Mybacteriales and Cytophagales are able to glide on solid substrates play an important ecological role in breaking down organic compounds Sheathed bacteria can attach themselves to solid substrates with sheaths Budding bacteria 1 Have appendages V 2 3 4 Lquot V V O V U V T U V W X Y BIOL 687 Spring 2005 Systematics 2 Can be useful in increasing surface area and nutrient uptake at low concentrations 3 Caulobacter uses its appendage to attach to solid substrate a common indicator of oligotrophic conditions Spirochetes move by exing their body and locomote well in high viscosity solutions T reponema pallidum causes syphilis in people other species are common in aquatic environments Important gut inhabitants of termites Spiral and curved bacteria Bdellovibrio included in this group they can penetrate and infect other prokaryotic cells do not usually think of bacteria as active predators but this one is Gram negative aerobic rods and cocci Pseudomonas common in soil and water Azotobacter xes nitrogen aerobically Rhizobium xes nitrogen as a symbiote with plants Methylotrophic bacteria utilize 1 carbon compounds methane methanol and CO important in global carbon cycling Probably more bacteria of this type than any other in the world Species of Proteobacteria and the Cytophaga Flavobacterium cluster are often the most abundant in marine surface waters Kirchman 2002 FEMS Micrbiol Ecol 3991100 Gram negative facultative anaerobic rods 1 Enterobacteriacea includes E coli and several intestinal pathogens 2 Vibrionaceae curved rods aquatic Vibrio cholerae causes cholera 3 Luminescent vibrios inhabit the light organs of sh Gram negative anaerobes include organisms important in the intestines of many animals other species important in the sulfur cycle Gram negative chemolithotrophs includes ammonium and nitrite oxidizing forms and sulfur compound oxidizers Gram positive cocci included Streptococcus Endospore forming rods and cocci form heat resistant spores Gram positive Asporogenous rods include Lactobacillaceae produce lactic acid as main byproduct yogurt Actinomycetes irregular morphology of cellssome resemble fungi lamentous Rickettsias include bacterium which causes rocky Mountain spotted fever and chlamydia Mycoplasmas lack a cell wall cause some animal diseases importance in natural environment not well known because they are hard to detect Evolution of eukaryotic cells the theory of serial endosymbiosis formation of cells by V bWN VVV Lquot V combination of preexisting cells the original proponent of this idea is Lynn Margulis from the 1960 A s The initial cells could have been of any type ie Archael Bacterial or Eukaryotic All cells have a similarity of ribosomal rRNA of at least 50 suggesting that all arose from a common ancestor There are numerous similarities between Archaea and Bacteria see table handout Archaea share a number of common characteristics with Eucarya not shared with Bacteria making it possible that Eucarya came from Archaea sometime after the split between Archaea and Bacteria Formation of the cytoskeleton and nucleus pure unadulterated speculation with some from other peoples studies 1 Cytoskeleton crosses the entire cell actin gel needs to be able to sever and rebuild the gel to allow cells to grow and respond to changes in osmotic pressure 2 Microtubules form columns which resist compression caused by the actin micro laments V 2 3 O V 9 E V BIOL 687 Spring 2005 Systematics With a cytoskeleton in place phagocytosis in both of the regions where the bacterial chromosome attaches which subsequently becomes permanently detached from the plasma membrane remains fused to the rough endoplasmic reticulum with compartmentalization of the DNA by the rough endoplasmic reticulum protecting the DNA from damage Initially this cover would have quotnuclear poresquot allowing for free molecular transport but eventually the pores would be speci c channels further protecting the DNA Another possibility is that the nucleus is simply a cell that was ingested That the nuclear membrane is a double membrane suggests that this is not as likely the case 5 There is no clear agreement upon the origin of the nucleus in Eucarya Formation of mitochondria 1 rRNA of mitochondria is more similar in both size and sequences to that of Bacteria than that of Eucarya 2 The SS rRNA sequences of mitochondria are most similar to those of the purple non sulfur photosynthetic bacteria as are the cytochrome and ferredoxin sequences i Could have initially been nonoxygenic photosynthetic endosymbionts similar to chloroplasts in today s plants but as the oxygen levels in the earth s early atmosphere rose they lost their ability to photosynthesize and become respiratory machines ii Extant purple nonsulfur photosynthetic bacteria loose the ability to photosynthesize and solely respire in the presence of oxygen However some recently discovered freeliving in the ocean capable of photosynthesis in oxic waters 3 Mitochondria are not able to live separately and have lost much of their ability to code for essential products but they still contain DNA and code for some of their own gene products 4 The idea that mitochondria arose from endosymbionts is accepted for the most part Origins of plant chloroplasts 1 Have rRNA more similar to that of cyanobacteria than that of Eucarya 2 Chloroplasts have lost the ability to survive on own but still contain own DNA Some of the products speci c for photosynthetic function are coded for in nuclear DNA suggesting migration of genes 3 Eukaryotic plants for the most part contain chl a and b and but even in those Eucarya that don t have chl b proteins associated with light harvesting complex suggest a common origin Wolfe et al 1994 Nature 367566568 chlorophyll b is not seen in the cyanobacteria Prochloron a bacterial oxygenic photosynthetic organism was found recently which contains chlorophyll a and b and was proposed to be the progenitor to chloroplasts rRNA analysis has since suggested that Prochloron is no more related to chloroplasts than to other cyanobacteria so it is probably just a descendant of some type of organism which became chloroplasts or something related to the type that became chloroplasts 4 Dino agellates have the habit of going around and ingesting different species of small algae and retaining them as chloroplasts They contain most of the known photosynthetic pigments of oxygenic photosynthetic organisms as endosymbiotic organelles An extant organism shows how it can happen They also have very bizarre chloroplast genes numerous small circular DNA gene circles one gene for each mini circular gene circle Zhang et al 1999 Nature 400155159 5 The idea that chloroplasts arose from endosymbionts is accepted for the most part 6 Major groups of phytoplankton diatoms coccolithopores and dino agellates Falkowksi et al 2004 Science 305354 arose from ingesting a red alga Cell walls of plants and many algae seem to be derived from genes transferred from cyanobacterial endosymbiont Niklas 2004 Bioscience 54831841 L V 3 V F Much recent molecular work veri es endosymbiosis but many issues still remain to be G H VI BIOL 687 Spring 2005 Systematics understood Dyall et al 2004 Science 304253257 presentation Flagella 1 It has been observed that Mixotrichia which is a motile protozoan from termite guts has numerous spirochetes attached to the surface with bracket like basal bodies They move in unison Some have proposed a similar origin for complex eukaryotic agella This theory does not receive widespread support 2 It has also been suggested that there were a series of steps of reorganization in a eukaryote that led to the formation of agella Other organelles found in individual organisms particularly protozoa Funes et al 2002 Science 2982155 presentation Eukaryotic organisms Note kingdom protozoa includes much more diversity than animals fungi or plants Probably will be divided into 6 or more kinddoms Baldauf et al 2000 Science 290972976 A Fungi 1 Molds i Filamentous fungi a Single lament hypha b Lots of laments called mycelium 0 Have sexual and asexual spores ii Among the largest organisms in the world Armillaria bulbosa Genetic means to describe single individual in a Michigan forest that covers 15 ha weighs over 10000 kg and has been stable genetically for 1500 years Smith et al 1992 Nature 356428431 iii Penicillin comes from a lamentous fungi iv Some important diseases are due to fungi are some of the most important decomposers in terrestrial nutrient cycles Important association with roots of plants Rare in marine and freshwaters 2 Yeasts i Single celled fungi ii Few diseases caused by yeasts beer and other types of alcohol produced by yeasts 3 Mushrooms Basidiomycetes 4 Large fruiting bodies are one stage i Commercially important edible Algae Oxygenic photosynthetic microscopic nonvascular Contain chlorophyll usually No sterile covering over gametangia Most need light to survive Appear green under the microscope Traditionally include cyanobacteria but we will consider them seperately Green Algae Chlorophytes i Eukaryotic algaechl a and b 1 V Immnwm VVVVVV 8 9 BIOL 687 Spring 2005 Systematics ii Chloroplast a bag of thylakoids pigments mostly chlorophyll iii Some use agella to pull along like breast stroke iv Many stages of life cycle are agellated v Can reproduce by dividing or sexual conjugation vi Forms 21 Unicellular b Filamentous viiFound in plankton suspended and benthic aquatic sediments habitats and soils Diatoms i Do not have agella ii Have a brown color due to carotenoids iii About 23 of the oceans production is formed by these algae so about 14 of the worlds primary production is done by diatoms iv Wall is composed of silica oxide glass Two types of silica walls called frustules a Centric like a petri dish and pennate v Of the basic shapes there is a wide variation some form spines which slow their sinking and protect them from grazing all have holes to let in nutrients the holes vary tremendously vi Tremendous variety of morphology The shapes are made by many repetitions of nm sized patterns Sumper 2002 Science 29524302433 viiVictorian upperclass women collected diatoms and arranged them into pictures on microscope slides creating a microscopic art form and earning the moniker diatomaniacs viii Found in plankton or benthos pennate forms commonly found in benthos able to glide on solid surfaces not motile in plankton also common in moist soils ix First diatom sequenced T halassiosira pseudonana unigue genes for silicon synthesis Arrnbrust et al 2004 Science 3067986 presentation x Pathologists use to determine drowning Victims Dino agellates i 2 agella ii Silica armor like plates iii Though many of these are photosynthetic some have lost their chloroplasts and go around ingesting bacteria predators Others have lost the usual chloroplast but seem to have a bluegreen algal cell or more living inside them which provide energy 12 BIOL 687 Spring 2005 Systematics iv Some species form a toxin called saXitoxin which causes red tide Similar bloom occurred several years ago in Melvem Reservoir Gymnodim39um a The organism that causes red tide is a dino agellate calledMesodim39um rubrum It eats another kind of algae a cryptophyte and uses its chloroplasts as its own rather than reproducing its own photosynthetic organelle Gustafson et al 2000 Nature 40510491052 1 During periods of heavy rain lots of nutrients wash into the ocean 2 These nutrients cause a bloom of these algae to form they cause poisoning of other organisms around them 3 The other organisms release more nutrients when they die fueling the red tide 4 Lots of dead sh was up on the beach during these episodes b This same toxin causes shell sh poisoning on the Oregon coast Mollusks concentrate the algae and become poisonous mainly during MayAugust 0 Most notorious recent bloom former is P steria has potent neurotoxin discovered when lab researchers had neurological symptoms Blooms off east coast kills sh hurts sherman closes swimming Controversial Reason hog farms are moving to Kansas v Mostly planktonic species but can have a varied and strange life cycle see handout 10 Chrysophytes golden brown algae i The pigments B carotene and xanthophyll carotenoids give these algae their special yellow coloration ii Generally planktonic species in Dinobryon is the most common planktonic species in local lakes iv Interesting because like the dino agellates they can ingest particles bacteria sized The differentiation between plant and animal gets quite fuzzy in this case 11 Crypotomonads i Interesting because they have 4 genomes nucleus mitochondria chloroplast and nucleomorph was a red alga The nucleomorph helps synthesize proteins for the chloroplast unusual because have chlorophyll C It is the most genedense genome ever documented Douglas et al 2001 Nature 41010911096 12 Euglenoids i Unicellular ii Capable of motility by agella and by a creeping motion in Many colorless iv Commonly found in highly enriched waters sewage sediments etc Lichens 1 Fungi and algae form lichens mutualists 2 Fungi protects algae 3 Algae provides food for fungi through photosynthesis Protozoa 1 Unicellular O V 9 BIOL 687 Spring 2005 Systematics 2 Motile most ingest particles broken into three phyla i Mastigophora those with few agella a Include photosynthetic agellates such as dino agellates Phytomastigophora b Include colorless agellates Zoomastigophora The heterotrophic nano agellates are important bacterial predators and are a part of this group This also includes several important human parasites such as trypanosomes ii Sarcodina amoebae important microbial predators A form of meningitis caused by one of these in Ciliophora those with many agella 21 Includes Paramecium also Vorticella E Rotifers Phylum Rotifera 1 Range from size of ciliated protozoa to much larger 2 Some 2000 species most fresh water 3 About 1000 cells show cilia mouth mastaX intestine salivary glands digestive glands ovary toe i Photoreceptors to sense light ii Population is all female through most of the year reproduces by self fertilization parthenogenesis During the fall when conditions start to deteriorate a few males are produced and so are unfertilized eggs Eggs that are fertilized by males are more resistant to harsh conditions allowing the population to survive for another year in Bdelloid rotifers never reproduce sexually have survived without seX for millions of years seX not absolutely necessary for animals 4 Cnideria polyps include Hydra 5 Gastrothricha gastrotrichs 50800 um long Distinct head with sensory appendages F Phylum Arthropoda includes insects some small enough to be considered microbes 1 Class Crustacea i Includes bamacles lobsters cray sh clams etc ii Two extremely important groups in fresh water are the microcrustaceans orders Cladocera and Copepoda iii Daphnia Cladocera water eas 023mm long BIOL 687 Spring 2005 Systematics Main swimming organs 2nd antenna Main sense organs 1st antennae Mandibles crunch up cells Median eye is in middle of head Rudimentary heart for circulation Thoracic legs push food toward mouth Brood chamber for eggs No lung oxygen diffuses in Ocellus is sensitive to different wavelengths than is the eye Eggs are in various stages in the brood chamber female for most of the time and self fertilize young actually develop inside the mother During fall some males are produced the male grasps the back of the female and copulates the sperm is stored al the brood chamber develops into a hard dark object called the epihippium This object contains the eggs and after it is fertilized it falls off the back It is resistant to drought etc and can over winter iv Life span lseveral weeks These are some of the most ef cient grazers of phytoplankton and suspended bacteria 2 Order Copepoda copepods i About same size as cladocerans draw 0Q AAAAA A vv ampampe3 a 1st antennae stability organ BIOL 687 Spring 2005 Systematics b Median eyespot 5 pairs of paddle shaped legs External egg sacs Hard exoskeleton where segments join there is exibility Eggs in general are fertilized each time g Complex series of development 6 nauplius stages 5 copepodite stages leading up to adult 3 Ostracoda ostracods or seed shrimp i Widespread benthic species ii Feed with lters on bacteria algae and detritus iii Encase in two shells with their feet in between G Nematodes 1 Small round worms generally microscopic 2 Vary from herbivores to detritivores to predators parasites 3 Most commonly found in sediments and soils A9 ovampv BIOL 687 Spring 2005 Origins of Life Microbial Ecology Topic 3 Origins of Life I The rst problem is how to de ne life A B C Does life have to be carbon based Example of a computer program that reproduces itself is that life Is a mule alive What were the early conditions on earth B C The earth formed 45 billion years ago Early conditions were probably reducing there was not free molecular oxygen but there was probably some oxidizing condition Early temperature was high the earth had to cool enough for liquid water to form After this point life could probably exist because life now exists where there is liquid water see table After earth cooled the young sun was dim however earth s temperature could have been maintained because methanogenic bacteria increased methane a greenhouse gas in the atmosphere Kastin and Siefert 2002 Science 29610661067 Because there was no oxygen levels of UV radiation were high There was no ozone to remove damaging UV rays Photosynthesis produced oxygen and perhaps hydrogen gas that oxidized the early earth s atmosphere Hoehler et al 2001 Nature 412324237 First fossils appear 335 billion years ago similar to stromatolites Stromatolites were actually hypothesized to be cyanobacterial before extant examples were described There are extant stromatolites are found in areas where grazers are rare 2 First contemporary stromatolites were described from shark bay in Australia This is a hypersaline bay in western Australia Infrequent contact with the ocean only during very high tides so salinity builds through evaporation No grazers 3 Stromatolites have layers hence stroma made from seasonal cycles They are generally built up of calcium carbonate deposited as a result of cyanobacterial photosynthesis Extant communities also contain eukaryotic microalgae 4 When stromatolites were rst described few people believed that they were biogenic structures There were massive fossil beds in the oldest of rocks Subsequent analysis showed that there were microfossils in them similar to 1 quot 39 organization 5 Other stromatolites have been described from lakes oceans and microbial mats Generally places where there is little grazing pressure 6 Fine grain carbonate production by cyanobacteria now similar to that found in stromatolites Kazmierczak and Altermann 2002 Science 2982351 presentation Molecular evidence for early life Molecular evidence conservation of protein sequences suggest that low GC bacteria diverged 3 billion years ago Tailliez 2001 Lait 81111 2 Molecular phyologeny suggests Eukarya and Archaea diverged early in earth s history Sterols now found in Eukarya and depletion in 13C indicative of methanogeneisis by Archaea apparently found in 27 million year old rock Fossils 15 million years ago are probably Eukarya Javaux et al 2001 Nature 4126669 Lipid micelles protein microspheres 1 Lipids are able to form small cells When they are repeatedly dried with DNA all of the DNA ends up inside of the cells 17 III BIOL 687 Spring 2005 Origins of Life 2 Proteins are also able to make up small spheres some have hypothesized that proteins were the rst cells Some interesting recent results on formation of organic chemicals When glycine is repeatedly cycled through hot and cold regions oligopeptides formed B When copper ions were present hexglycine formed C Under moderately reducing conditions and 100 C synthesis of all 20 amino acids is energetically favorable whereas it costs signi cant energy with 02 present at more moderate temperatures Amend and Shock 1998 Science 281 16541662 D Volcanic gas can catalyze polymerization of amino acids to peptides Leman et al 2004 Science 306283286 presentation E Isotope analysis of pyritesulfur from 34 billion years ago suggests that bacteria were present that were reducing sulfate to sul de Ohmoto et al 1993 Science 262555557 It is possible that iron reduction was an early form of metabolism because molecular taxonomy suggests early origins Vargas et al 1990 Nature 3956567 F A review by Schopf Prokaryotic Development 2000 pp105129 suggests methanogens at least 28 billion years ago and gramnegative sulfate reducers 27 billion years ago Methanogenesis may have been important in early atmosphere because the sun was weak and a greenhouse effect would be required to keep the earth s atmosphere warm Catling et al 2001 Science 293839843 G Some researchers claim that fossils of magnetosomes of bacteria can be seen in martian meteorites very controversial TomasKrepta et al 2002 AEM 6836633672 H Were earliest ancestors hyperthermophilic Early earth temperature was high see handouts but molecular evidence is not supportive of this idea Galtier et al 1999 Science 283220221 However organisms that populate deep sea hotsprings are separated by the deepest phylogenetic branches molecular Reysenbach and Shock 2002 Science 2961077 The jury is still out I We are all familiar with the spark experiments that created amino acids in a ask with repeated sparking of cyanide and ammonia gas Other people have done other experiments that have created the molecules other ways freezing and thawing drying and rewetting It was improbably that there were signi cant levels of cyanide in the early environment Organic compounds are contained in meteorites and could have seeded the earth with complex organic material early in earth history Remember that many organic compounds may be very stable in absence of metabolism so even if organic compounds built up slowly they could have accumulated to fairly high concentrations Thermodynamics dissipative structures order disorder and information Some have said that life violates the second law of thermodynamics that requires a closed system tends toward more entropy Why is this not necessarily true B When energy is put through systems the systems tend to become ordered in the processes of dissipating For example when water is boiled cells form where water is going down others where heated water is going up These hexagonal cells are stable as long as the heat keeps coming in Some have hypothesize that this feature describes the organization that life C The concept of life as highly ordered is not exactly correct Crystals are very highly ordered but are in no way life On the other hand a certain degree of order is necessary so as to not allow for death D Information is a vital aspect of life Passing information from generation to generations is a key characteristic of life How do we assess information Completely random code 18 VI VII E Genetic BIOL 687 Spring 2005 Origins of Life has no information completely regular pattern has no information However some degree of order is required to pass information If information were passed perfectly from generation to generation what would happen to life takeover CairnsSmith is responsible for the hypothesis that clays were the progenitors of life but that genetic information took over and they clays were dropped out of the picture Clays have a remarkable variety of crystal structures patterns that repeat errors that can be induced by crystal inclusions but these errors are propagated through the succeeding crystals These crystals are layered so they can fracture and produce progeny Clays are more stable than organic molecules so may have been more suitable for carrying information in the early earth were things did not happen so fast and UV inputs and temperature were high Clays are good at catalyzing organic reactions Hypothesized that clays were catalyzing the reactions and were using RNA like molecules to make proteins and the messengers took over and dropped the clays out of the picture There is no solid data for or against this hypothesis just as there is not info for or against most of the other hypotheses Metabolism or reproduction rst RNA w A orld Now that catalytic RNA s have been described there has been much excitement over RNA as a rst molecule That it could replicate and have enzymatic activity is very attractive Many people believe this Also many very small RNA s have biological activities initiating protein synthesis etc see Science breakthrough of the year 2002 Probably the most important is the eXistence of RNAcatalyzed RNA polymerization allows for selfreplication Johnston et al 2001 Science 29213191325 Some problems It is hard to imagine making RNA s just from a soup of organic molecules Do not remain stable for very long Early on the rates of reproduction may not have been very high BIOL 687 Spr 2005 Functional roles ecosystems Microbial Ecology Topic 4 Functional Roles Environments and Ecosystems I A Environments O About 25 of the world is terrestrial a large portion of this is covered by soil Many different types and classi cations of soils can vary in mineral and organic content in size of soils etc Agronomy classes and soil microbiology cover this much better thanI could Essentially all microorganisms in soil require water to be present in a small layer on the particle surface to function thus all microbes are essentially aquatic Sediments 1 Essentially saturated soils or maybe soils are subsaturated sediments Tend to become anaerobic with increased depth 2 Vary widely as soils in particle size content and organic matter content 3 Important how much water is contained porosity makes a difference in the way that solutes can be transferred 4 Often sediments are anaerobic anoxic with depth and aerobic at the surface and this interface occurs over pm to a few cm in depth As drastic effects on what organisms can survive and what biogeochemical processes can occur 5 Rate of transfer of materials is usually related to rates of diffusion 6 Note bacterial diversity in sediments and soils probably at least 10 times greater than in water column Torsvik et al 2002 Science 29610641065 Oceans 1 Salinities are similar worldwide but vary some 2 35 of planet s surface is covered by oceans 3 Central part of the ocean very nutrient poor oligotrophic light at surface drives photosynthesis nutrient inputs from atmosphere are minimal i Cold water tends to sink so a general tendency for water to move out of polar regions in deep and into polar regions from shallow ii Deep ocean has a number of different strata with temperature decreasing with depth Density differences in the water keeps these strata stable and for the most part water transfer between them is rare iii Nutrients tend to sink out in the form of organisms What happens to them iv Extreme storms changes in major circulation patterns EL Nino can alter these strata and allow for deep nutrient rich water to come up and stimulate photosynthesis in places where it does not normally It has been demonstrated that hurricanes can alter the rates of C02 ux across the surface of the ocean 4 Margins of oceans are more nutrient rich mesotrophic to eutrophic de ne there is input from the land and upwelling of nutrient rich waters occurs primarily near these areas 5 As upwelling occurs cold core rings can spin off into the deeper ocean and remain separate for a number of months 6 Estuaries interface between fresh and saline waters high currents sediments very important can completely dry with tides extremely productive 7 Intertidal habitats i Rocky intertidal a High wave energy b High biomass and productivity ii Sand 85 bl VV 20 BIOL 687 Spr 2005 Functional roles ecosystems a Fairly unproductive b Diatoms and invertebrates associated with sand episammic D Lakes and Streams 1 Lakes and streams show extreme variation from oligotrophic to eutrophic Chemistry varies from very low salinities to very high salinities 2 Physical features of lakes i Water has maximum density at 4 0C this works in oceans as well ii Consequently thermal strati cation occurs during the summer in Productivity of the lake algal production determines how fast oxygen disappears from the hypolimnion In eutrophic lakes ion the summer there is no oxygen in hypolimnion thus we see anaerobic microbial processes dominate we will talk more about this later 3 Streams i Characterized by ow ii Many of the microbes are attached to the bottom benthic Periphyton mixed assemblage of producers and consumers on bottom called bio lm by engineering and microbial types not associated with streams or lakes H Ways for organisms to make a living A Autotroph primary producers 1 Photoautotrophy photosynthesis i C02 H20 light gt CHZO Oz must know this ii Give the equation in words in Plants photosynthesize iv Light supplies energy chlorophyll harvests it v In some special cases His is substituted for water in this case oxygen is not an end product This is thought to be the primitive form of photosynthesis 2 Early history of measuring primary productivity i 384322 BC Aristotle said soil provides predigested material for plants to take up through their roots ii 1450 AD Nicolai de Cusa quot water thickens within the soil sucks off soil substances and becomes condensed to herb by action of the sunquot in ca 1600 van Helmont aside from trying to nd a method of obtaining mice from junk and sawdust he grew a 5 lb willow branch in a pot with 300 lbs of soil He watered it 21 BIOL 687 Spr 2005 Functional roles ecosystems with rainwater for 5 years Got a 164 lb tree with only 2 oz of soil lost Concluded that water condenses to form plants 3 Chemoautotrophy chemosynthesis i Special types of bacteria use chemical energy to x carbon dioxide ii HZS 12 Oz ADP P gt S H20 ATP in This is how communities or organisms form under the ocean near deep hydrothermal vents and in some deep groundwaters 21 There is no light as an energy source b The hot water from the vents contains hydrogen sul de which provides energy to the bacteria c The rest of the animals feed on this energy base will talk more about this under extreme environments B Heterotrophs most bacteria and other organisms that are not plants including us 1 Heterotrophs rely upon sugars and other carbon containing compounds for energy 2 Aerobic respiration CHZO Oz gt C02 H20 energy i This equation is the reverse of photosynthesis 3 Fermentation i Breakdown of organic carbon to yield energy a net oxidation in the absence of oxygen III Transfer of Energy A Study of ecosystems is in part the study of how efficiently energy is transferred B Biomass pyramids P producers H consumers or primary consumers C consumers rst level predators TC top consumers D decomposers Dry biomass per square meter Silver Springs Florida TC15 C11 D5 H37 P809macrophytes Coral Reef Emiwetok Atoll H13 2 P703 Old Field Georgia C001 H06 P470 Weber Lake Wisconsin C4 H11 P96 English Channel H21 22 BIOL 687 Spr 2005 Functional roles ecosystems p4 What are the differences in shape How does this relate to the global data given above Energy pyramids Give the amount of production or energy ow through each compartment Numbers in parentheses give the amount actually retained as biomass the rest is lost as part of the cost of doing business For Silver Springs Florida see gures TC 21 67174 H3368D5060 460 909 1478438 P 20810 8833 424 Note that all of the energy available is not used Note that about 90 of the energy disappears per level Note that the actually incorporated of total energy throughput decreases with the carnivores Note that the biomass does not necessarily re ect the energy throughput PB pyramid total energybiomass silver springs TC14 C348 H91D1012 P257 1 Which has the highest rates of metabolism 2 Why do plants move so little energy per unit biomass TV Global partitioning of biomass and productivity carbon ow handout table A What is the difference between productivity and biomass Why is this an important concept in understanding the structure and function of ecosystems Why might this information be important in global environmental issues B Points to consider on the handout Ecosystem area column 2 Marine 2 times terrestrial open ocean is the real biggie 2 Terrestrial systems column 3 range from almost no productivity per unit area to high levels in wetlands croplands tropical forests Productivity in the open ocean column 3 is very low areas associated with coastal features upwelling of nutrient rich water from the deep ocean estuaries coral reefs can have productivity on par with the more productive terrestrial environments Total production of the marine systems column 5 is about 50 of the total terrestrial production This means that the oceans are not an inexhaustible source of food for humans we only see the richest parts coastal areas These numbers have recently been redone with satellite radiometer data suggest 4550 GTC year391 for oceanic phytoplankton 4568 GTC year1 for land plants Longhurst et al 1995 J Plankt Res 1712451271 The total marine biomass amount of biological material column 8 is 02 of that in terrestrial systems Why is this V L V 3 V Lquot V 23 BIOL 687 Spr 2005 Functional roles ecosystems 6 How can the total productivity of the ocean per unit area be Within the ranges given on land 7 and the biomass in the ocean so much lower Where does the biomass go in the ocean There is more efficient transfer of energy to higher levels of the food chain There are differences in the productivity to biomass ratios it is much higher in the open ocean 24 BIOL 687 Spring 2005 Properties of water Microbial Ecology Topic 5 Physical and Chemical Properties ofWater Life in the Small 1 The molecule water A Water is an excellent solvent It can dissolve more ions with greater temperature eg try to put sugar into cold ice tea It dissolves less gas with increased temperature B Water is unusual in that it is one of the few compounds that expands when it freezes has high surface tension high heat capacity is liquid etc C The reason for this is hydrogen bonding causes water to assume a crystal structure D Curve for density of water with temperature did in previous section II Life in the water Reynolds numbers viscosity and scale A Microorganisms experience aquatic media different than we do This is a matter of physics 1 The number is a ratio between viscosity and inertia i Inertia F psz the higher the velocity of a uid of a given density the more inertia for example a bowling ball has more inertia when traveling at the same speed than does a basketball More inertia with more speed is intuitively obvious ii Viscosity FV uSvl the higher the coefficient of viscosity or the velocity the higher the viscosity the smaller the distance the more important become attractive forces within the liquid and the higher the viscosity Example wading up stream paddling with a rod or a paddle See handout for relationship of viscosity to temperature iii p density u the coefficient of viscosity 1 characteristic length v velocity S area of the ow being considered Reynolds numbers indicate the physical properties of the medium Re FiFV plvu Reynolds number is a ratio between viscosity and inertia if inertia is high and viscosity is low then the Reynolds number is high iv This means that for microorganisms the relative viscosity is high and the inertia is low and vice versa for larger organisms 2 The effect is that microorganisms need to contend with locomotion in an extremely viscous solution The analogy is a human swimming in warm tar or molasses 1 In spite of this handicap microorganisms are able to swim about 100 body lengths second a fast swimming sh such as a tuna can only sustain 10 body lengths per second ii There is no inertia at this scale If a bacterium stops swimming immediately it only glides for the distance of a hydrogen atom one angstrom 3 Below a certain size streamlining has no effect so we do not see streamlined microorganisms it is only the large algae such as seaweeds that tend to streamline against ow 4 This also means that paddle shaped cilia have no advantage Therefore circular agella are used tubes are quite strong as well Rather agella and cilia are pulled forward laterally through the medium and crosswise on the pull stroke Addition of hairs additional cylinders can increase the effective cross sectional area of the cilia signi cantly This is because the high viscosity 25 BIOL 687 Spring 2005 Properties of water allows little uid to ow between the hairs 5 All organisms should have more trouble swimming in colder water It is more dif cult for protozoan grazers to get particles in cold water because it is more viscous Another effect of life in the small is that extremely distant objects relatively effect locomotion and hydromechanical cues are passed rapidly 1 It has been calculated that a wall can slow a bacterium appreciably up to 50 cell diameters away This is because of the laminar nature of ow and the incompressibility of uids 03 V 2 This is interesting because virtually all of our observations of bacterial locomotion are made on slides where there are walls in the vicinity 3 Furthermore prey and predators may be able to sense each other from a long distance if they are moving or the prey are moving Copepods were shown to be able to sense presence of inert particles as small as 10 um across 10 u away Visser 2001 Marine Ecol Prog Ser 222124 4 In another experiment a ciliate and two agellates were exposed to predatorgenerated feeding currents a copepod that lter feeds and a stationary ciliate Jakobsen 2002 Aquat Microb Ecol 26 27128 1 i At this scale acceleration is too weak to sense but differences in velocity across the organisms generates a signal ii The ciliate would jump away when exposed to the ow eld iii Could respond over about 30 pm 111 Size ow and diffusion boundaries Flow boundary due to viscous forces near a wall or solid object 1 You can think of this as a no slip region due to viscosity that happens near solid surfaces 2 Smaller with higher ow 3 Small in areas which protrude into ow B Effects on morphology 1 As mentioned microorganisms do not need to be streamlined but larger organisms which protrude into ow do need streamlining if they are not to be washed away 2 Cladophora tends to fold up in ow and air out in still just like leaves on a tree C Diffusion effects 1 Flow controls diffusion 3 1 Eddy diffusion or convection move molecules at least 1000 times faster than molecular diffusion ii Eddy diffusion important on larger scale molecular diffusion in boundary layers iii Eddy diffusion extreme in streams less so in lakes but still important Leads to a diffusion boundary It is inside of the ow boundary There is a no slip condition at the surface and inside this layer molecular diffusion predominates Same conditions apply as for the thickness of the ow boundary Diffusion of nutrients in or harmful byproducts out can effect photosynthesis N V 26 L V BIOL 687 Spring 2005 Properties of water Size and shape can effect diffusion 1 Algae rely solely upon diffusion and convection to move materials unlike vascular plants ii The larger an alga the more dif cult it is for materials to diffuse to inner cells a Surface area to volume decreases as size increases Therefore we rarely see very large algae and the largest are in areas where ow is high intertidal or subtidal which increases the amount of material transport How does a large organism like a sh ever get oxygen to its tissue Why to some aquatic invertebrates from rapidly moving streams die if placed in still water Different situations where movement can alter concentration gradients 21 Swimming iii iv V b Falling c Attachment to a solid surface 27 BIOL 687 Spring 2005 Properties of water vi Recent studies of the marine sul de oxidizing bacteria show that veils of the bacteria can form immediately above the sediments These groups of bacteria attach to the sediment but use agella to move water past them Large groups of the bacteria can signi cantly increase the ux of oxygen into the surface of the sediment 40 times higher than molecular diffusion rates Fenchel and Glud 1998 Nature 394367369 vii Anabaena aggregates heterocysts to decrease inward diffusion of oxygen to harm nitrogenase in heterocysts Theoretical calculation with 10 heterocysts aggregate as follows Assume 3 pm cell diameter and 3 pm thick gel surrounding the heterocyst Indeed aggregated heterocysts x nitrogen at an elevated rate 28 BIOL 687 Spring 2005 Taxis motility growth Microbial Ecology Topic 6 Chemotaxis phototaxis motility behavior photosynthesis and growth I Motility A Bacterial agella 1 Built from the monopeptide agellin Macnab 2000 ASM News 66738745 N V Attached with a hook to a basal body the basal body is a spiral with units added on to the outside Engelhardt et al 1993 Science 26210461048 handout Hook is a single protein about 1700 angstroms long Hook anchors the lament and transmits rotation to the lament Not known exactly how the basal body works as a circular engine mum VVV 6 In E coli numerous agella rotate forming a bundle behind the cell and propelling the cell forward when they start rotating the other direction they tumble important in taXis V In Rhodopsuedomonas rubrum alteration of the rotation causes movement in the opposite direction 29 BIOL 687 Spring 2005 Taxis motility growth 8 Bi agellates can also have both agella on one side and alter direction by changing rotation and tumbling 9 Mono agellates change direction by altering rotational direction 10 Spirochetes have a thick envelope outside of the cytoplasmic membrane which contains 2 agella i When the agella rotate it causes helical waves to be transmitted to the outside of the envelope allowing for motion ii This motion is much more ef cient when viscosity increases ie close to solid substrates but remember viscosity already fairly high in open water 11 Motility can lead to cooperative behavior that enhances growth in Mycobacterium Velicer and Yu Nature 4257577 presentation B Eukaryotesciliates 1 Single cilia same as before 2 Multiple cilia beat in waves causes directional movement C Ameboid movement possible because of lack of a cell wall and a cytoskeleton which allows the organisms to change shape 30 11 111 D Gliding l O V V E O V V BIOL 687 Spring 2005 Taxis motility growth Organisms that require a solid substrate to move against are said to glide Cyanobacteria glide but the mechanisms is not well known i Initially it was proposed that they extruded slime to glide but it has been calculated that several body volumes per second would be needed to move them along They do leave a slime trail however ii The 39 39 contain trichomes but beneath an elastic outer membrane The rotation of cyanobacteria as they glide corresponds to the angle the micro brils are against the trichome Mycoplasmas and spiroplasmas have no cell wall but appear to glide anyway This gliding seems to be mediated by micro brils but there is not any explanation of how this works Desmids green algae and diatoms also glide This movement is accompanied by slime extrusion but still the mechanisms of movement have not been characterized The cost of motility Crawford 1992 Microbial Ecol 24110 Metabolic costs of motility can be calculated based on Stokes Law for a sphere P3 TE szn Ppower Ddiameter Vwelocity n Wiscosity see handout Cell sizes from 1100 um speeds from 1005000 umsec takes less energy to move smaller cells but respiration related to volume VD ratio increases as cells get smaller Generally cost is less than 1 respiration but for small cells it can be quite an experience to move rapidly see handout and discuss shape and sinking There is also an effect of size on metabolism larger cells can generate greater rates of metabolism with come constrainst of nutrient limitation Finkel et al 2004 Marine Ecol Prog Ser 273269279 Bacteria and Archeae don t swim all the time Grossart et al 2001 Aquat Microb Ecol 25247258 1 Marine study of La Jolla 40 swam 20 of the time and had bursts of motility 2 Winter 525 motle spring and summer 4070 motile 3 Particulate material encouraged motility Models of taxis rst described over 120 years ago for microbes It is almost certain there will be some form of taxis if you are a motile organism natural selection only favors motility when there is a signi cant bene t to motion taxis adds direction to the selection agent 1 If an organism moves into an area where metabolism slows down and motility is a strict function of metabolic rate the organisms will tend to concentrate in regions where things are bad 2 Thus increased motility when things are bad is necessary if there is motility at all Integration of environmental resources with time or space is a necessity 1 A comparison must be made with previous conditions for integration with time 2 For the most part microorganisms are small relative to the size of gradients around them so sensing gradients that vary from one side of the cell to another is not useful An exception may be long lamentous forms 3 Therefore it is a necessity to know if things are getting better or worse as you move in one direction eg integration over time The simplest model is to stop moving when things get good or start when they get bad or both above a certain threshold generally relative to previous environmental conditions 2 r u u L V 4 V 31 03 V 3 B BIOL 687 Spring 2005 Taxis motility growth A simple way to do this is to simply drop off your agella This may explain why E coli stops agellin biosynthesis in the presence of glucose In Caulobacter it is agellated until it enters a rich medium and then it divides and forms one cell which attaches with a stalk and one that retains agellation This is hedging bets if things get bad again it will be possible to get out again Step up response Only respond to increases in the substance or factor stop moving 5 Step down response Only respond to step down start moving Random walk model 1 Another strategy is to swim in one direction until things stop getting better if they get worse you stop and change direction randomly and go a set distance if they don t start getting better over this set distance then tumble again 2 E coli displays this behavior Note these responses have to be inverted for a phobic response For example in response to a toxin when it increases tumble otherwise swim straight for awhile Chemotaxis Five main steps Reception genes now being characterized Sourjik and Berg 2004 Nature 428437441 presentation Summation of information Adaptation to background levels of stimulation Initiation or repression of chemotactic signal 5 Increasing or decreasing rate of reversals Chemical responses are probably the most important mode of microbial interactions Example of bacteria isolated some where the toxic cyanobacterium Microcystis occurs and others not Those isolated from where the cyanobacterial was present tended to move toward compounds exuded by the cyanobacterium and did well with growth on those compounds Those not subjected to prior experience with the Microcystis were inhibited by the extracts from the cyanobacterium not stimulated Casamatta and Wickstrom 2000 Microb Ecol 41 6473 Cemotaxis allows bacteria to colonize organic particles where they can be very abundant Kiorboe et a1 2002 AEM 6839964006 Heterotrophic bacteria respond most quickly to low molecular weight organic compounds Fenchel 2002 Science 29610681071 Phototactic photophobic response Why might an organism display photophobic response 1 Photoinhibition 2 Obligate anaerobe Several reasons why phototaxis would occur 2 3 4 b N V V V 3 V 5 V 32 VI V11 0 V 3 03 V D V B BIOL 687 Spring 2005 Taxis motility growth 1 Photosynthetic organisms 2 Organisms which are obligate aerobes Oscillatoria During night it is not present upon sediments glides down Tends to have attraction to sul de at moderate concentrations but not to much Why it is toxic at high levels but can grow heterotrophically During morning moves up to surface of sediment positively phototaxis spreads out Phototaxis overrides the chemotactic response Under high light the oscillatoria forms clumps on the surface self shading avoids photoinhibition The cues for this aggregation are not known probably chemical Magnetotactic response Several years ago it was noticed that certain bacteria have a speci c response to magnetic elds Now it is known that many groups of bacteria could do this 1 Could reverse the direction of movement by changing the magnetic eld 2 Electron micrographs showed that these bacteria had magnetosomes iron rich particles in the cell Hypothesized that this was a way to stay on the bottom of the ocean as these were sedimentary bacteria streamline on transition between oxic and anoxic zones Bazylinski 1995 AEM News 61 337343 1 The earth s magnetic eld has a down component away from the equators N V 3 V 2 to prove this the same species of bacteria was isolated from the South Atlantic and showed the opposite response Since this magnetotaxis and magnetosomes have been described from several distinct lines of bacteria Most bacteria have iron oxide particles Fe304 but some have iron sul de Fe3S4 particles rRNA analysis shows that the iron oxide and iron sul de groups are distinct and that they evolved separately convergent evolution DeLong et al 1993 Science 259803806 Enzymes to produce microscopic magnetic particles have been isolated When these are linked to drugs it may be possible to target speci c sites Put drug in blood stream and put magnet on the site see handout Cyanobacterial gas vacuoles a sort of motility and taxis Many species of cyanobacteria synthesize protein vacuoles which contain gas Increase buoyancy allow them to oat in calm waters In extreme conditions such as Hartbeesport Lake in South Africa these algae used to form hyper scums before phosphorus control The algae oat and prevailing winds blow them to one end of the reservoir The resulting mass is several meters thick If the wind reverses large chunks break off and reenter the lake causing recreational users to gross out If the vesicles break down synthesis stops the algae sink Only just beginning to understand the factors involved in synthesis and collapse of gas vacuoles 1 Synthesis seems to be stimulated under high nutrients in the absence of light 2 33 VIII BIOL 687 Spring 2005 Taxis motility growth 2 Synthesis stops or collapse increases with higher rates of photosynthesis and with nutrient epletion 3 Adaptive because nutrients tend to be deeper in lakes where cyanobacteria exist so they sink down to get the nutrients 4 After they get the nutrients they can synthesize more vacuoles and rise to the surface where they can photosynthesize 5 Being able to get right to the surface is an advantage in many systems where cyanobacteria are present the light attenuation is so great because of all the algal biomass that most of the algae are light limited most of the time When do you stop moving Some cells move or at least do not attach at some point These organisms form bio lms A V B 1X 9393 X 3 B Important in biomedical situations because bio lms can lead to infections in both human tissue and implanted materials Speci c chemical interactions are necessary for microbes to attach to solid surfaces and form bio lms Ista et al 2004 7041514157 presentation cell to cell signaling may be necessary for these bio lms to form correctly Many species of bacteria are able to coaggregate with some species able to aggregate with many other species and may form bridge that builds bio lm Rickard et al 2002 AEM 6836443650 In this case mutants not able to from bio lms with channels that allow for passage of water in Pseudomonas aerugenosa Davies et al 1998 Science 280295298 How do you know about time Prokaryotes have circadian system Most studied in cyanobacteria Some parts unique relative to the eukaryotic systems Golden et al Current Opinion Microbiology 1669673 Not known what functional reasoning is for system probably has to do with positioning for light Light harvest of algae Photosynthesis harnesses light energy 1 Pigments absorb light 2 Light energy excites an electron 3 This electron is transferred by a complex series of proteins photosystemI and 11 i draw the typical Z scheme 4 The electron is eventually used in turning C02 to sugar or to form ATP a high energy chemical compound Different types of algae and plants have different pigments 1 Higher plants and most algae have at least some chlorophyll 34 BIOL 687 Spring 2005 Taxis motility growth i Show the absorption spectrum of chlorophyll a ii When chlorophyll is excited by light the electron will have no where to go if it is in solution so the electron will fall back to the low energy state in the chlorophyll molecule when it does this it released light this is called uorescence 2 Cyanobacteria have a special system to collect energy i These pigments are called phycobilins a Absorption b Arrangement ordered so as to drop the energy to the light harvesting core 35 BIOL 687 Spring 2005 Taxis motility growth c Demonstrate their uorescence 3 Different photosynthetic bacteria have different maximal absorption bands for photosynthesis 4 New photosynthetic microbes have been described from the open ocean Pennisi 2000 Science 2891869 Beja et al 2000 Science 28919021906 i Use bacterial rhodopsin to harness light energy to form ATP ii Not oxygenic iii May make up 1 of marine planktonic biomass C The speci c pigments can be important in competition for light 1 The absorption for phycobilins is complimentary Note the phycobilins are able to absorb light that the green algae let through 2 This allows for the blue green algae to inhabit deep areas in lakes which are very low in light so we may see populations as so as with many microbial patterns it can also be compressed in sediments 36 BIOL 687 Spring 2005 Taxis motility growth 3 In microbial mats a similar thing with complementary pigment absorption can occur with bacterial chlorophyll Needs anaerobic conditions for sul de to serve as an electron donor see sulfur cycle below and shallow depth because the infrared does not go deep in water It is absorbed rapidly XI Rates of Photosynthesis in the environment 1 Photosynthesis of an algal culture is related to light as diagramed 2 Show the zone of inhibition the zone of compensation different parameters 3 Show how algae adapt to low light B In lakes or microbial mats this causes the following conditions 1 Summer graph in full light shows the population of algae high but a dip in photosynthesis at the surface due to inhibition 37 BIOL 687 Spring 2005 Taxis motility growth XII Rate of photosynthesis versus temperature A Rate approximately doubles with every 10 C 1 Important parameters Q10 how much change over 10 C XIII This means it is not trivial to measure photosynthesis in the natural environment Need to estimate response to numerous variables light and temperature regimes before the overall response can be adequately modeled XIV Batch Culture laboratory growth with xed amount of medium A Cells in monoculture will grow in 4 phases B This is what you see if you inoculate bacteria into a ask containing a set amount of growth medium 1 Lag phase dormant phase before growth starts 2 Log phase or exponential phase i Cells grow exponentially a linear relationship between cell number and time ii In log phase all cells are growing at maximum rate iii No nutrient limits growth iv Cells have a uniform composition v Only time that physiologists can be certain that the cells will behave the same from 38 XV 3 O V 9 BIOL 687 Spring 2005 Taxis motility growth experiment to experiment Stationary phase i Cells start to run out of nutrients ii Some bacteria continue to grow others become dormant Death phase i Exponential decay of population ii Cells dye some cells release their nutrients iii Other cells pick up these nutrients and hang on iv Eventually all the cells in this phase would die v Some species are able to cannibalise members of their own species to survive such as Bacillus subtilis GonzalezPastor et al 2003 Science 301510512 presentation vi eContinuous culture chemostat Used to keep cells in log phase at all times 3 4 V As cells grow they are washed out 1 Rate of growth washout rate 2 If washout rate is higher than growth rate then all cells are washed out Technique is useful if cells are needed in log phase at all times Kept in log phase because nutrients are added at all times Batch cultures are only in log phase for very short periods of times Can test physiology of microorganisms under optimum growth conditions Also used to test ecology of mixed population under equilibrium conditions or responses to perturbation Synchronous culture Natural population of cells in culture has cells in all stages of development V 2 3 4 Since physiologist is looking at all stages of growth at one time in a culture this can present dif culties 1 Cells which are just about to diVide have 2 X the DNA as those which have just diVided 2 Cells that have just diVided need proteins to build up more cells so they have different nutri tional needs Selective and inducement methods can be used to synchronize cell growth 1 Inducement methods make use of repeated shifting temperature 2 Differential ltration can be used to separate out cells i cells which have just diVided are smaller Synchronized cultures can be used to show that different cells in a population grow at different rates they become non synchronous after some time 39 BIOL 687 Spring 2005 Taxis motility growth XVI Bacteria that compete well at low nutrient concentrations are called oligotrophs They are dif cult to culture in the lab because they grow slowly and will only compete well on media with extremely low substrate concentrations standard media have high nutrient concentrations Bacteria which compete well in nutrient rich environments are called eutrophs XVII Note read handouts on redox Important for the next lecture or try your own biochem or chem book 40 BIOL 687 Spring 2005 Redox and Oxygen Microbial Ecology Topic 7 Redox oxygen and controlling factors I H A V B Q 9939 Redoxcontrolling factor in most chemical cycles Reduction oxidation potential is a measure of the ability of the environment to supply or receive electrons The redox state of a solution is de ned by the number of free electrons present in the solution in a fashion similar to pH pH 10g PF pE 10ge39 l a high redox potential low pE means that few electrons are available 2 A low redox potential means that many electrons are available In the presence of oxygen redox is generally in excess of 400 mV in the absence of oxygen it will decrease Why is this The redox of the environment determines what chemical reactions will be exergonic and which will be endergonic See table draw gure and have handout from geochemistry book 1 Note what de nes 0 redox is arbitrary it is solely a relative scale Is oxygenic photosynthesis working with or against redox potential 2 Under what conditions may photosynthesis work with the redox potential Organisms must maintain internal redox as well a pH Even organisms in high or low pH environments usually maintain pH close to 7 Need to maintain electromotive force and proton gradients across membranes for the cells to function See gure 821 on page 317 of Atlas and Bartha If they are in oxidizing environments they tend to have a lower potential than their surroundings in extremely reducing environments they have a higher redox potential There is some variability possible in internal redox Obligate anaerobes have a low intracellular redox related to oxygenic photosynthesis Oxygen Oxygen is important because most higher animals require it to live Freshwater organisms use oxygen which is dissolved in water The cycling of major nutrients is also related to oxygen We mean molecular oxygen most of the time Oz Atmosphere 20 oxygen by volume or 300 mgl Water has much less 5 C l2mgl saturating l 20 C 9mgl saturation Sources and losses of oxygen in aquatic environments 1 Wind mixes oxygen from atmosphere increased turbulence causes more transfer to microbes in aquatic situations and in terrestrial situations 2 Photosynthesis supplies oxygen 3 Heterotrophs use up oxygen 41 111 F E 03 V O V BIOL 687 Spring 2005 Redox and Oxygen Strati cation and oxygen relationships in lakes and oceans 1 Terms de ne aerobic and anaerobic oxic and anoxic 2 Draw strati ed lake with temp gradation 3 Eutrophic lake show oxygen disappearing with time 4 Hypolimnion does not become anaerobic in oligotrophic 5 Rates of loss of oxygen in hypolinmion Oligotrophic lt 015 mglOzmonth Mesotrophic lt ll5 1 Eutrophic gt 15 If all oxygen in a lake disappears all sh die 1 During the winter ice and snow covers i Little light ii No atmospheric O2 in iii If BOD is high eutrophic sh will die iv Called winter sh kill 2 In shallow hyper eutrophic lakes massive sh kills i Large biomass of algae builds up ii One or two days of no wind and cloudy weather a Low photosynthesis b Little mixing iii This happens periodically in some of the large tropical lakes in Africa Lake George Tolerance limits for organisms 1 2 mgl most sh avoid will kill some trout 2 51 starts to kill cladocerans and copepods 3 0 lsome species of worms can tolerate this using a special form of respiration many bacteria some plants blue greens etc can handle it some bacteria require it Oxygen anomalies 1 draw lake pro le with high 02 peak at metalimnion 42 BIOL 687 Spring 2005 Redox and Oxygen 2 Lakes and Black Sea which are permanently strati ed have anaerobic hypolimnia even if they are oligotrophic Draw picture of lake Tanganika This is how the deeper ocean has lower oxygen even though the ocean is very oligotrophic E Oz free zones gt11 V 1 Inside animal guts 2 Sediments many anoxic below surface where respiration lowers Oz faster than it can diffuse it 3 In decaying particles soil sediment or aquatic see handout 4 Pockets in soil regions with high input and low Oz diffusion 5 Microbial aggregates marine snow algal colonies etc Kepkay 1994 Mar Ecol Prog Ser 109293304 Passow amp Aldredge 1994 Mar Ecol Prog Ser 113185198 6 Groundwater can be anoxic in entire system if high C input Can have regions eg near stream Oxygenation of the earth s atmosphere 1 Initially the earth s atmosphere was anaerobic and bacteria were the predominant life forms This is known because ancient rocks do not have much oxygen in their chemical makeup 2 Blue cyanobacterialike prokaryotes evolved which photosynthesized and produced oxygen 3 These prokaryotes produced the oldest known fossils 4 As photosynthesis proceeded most of the world became aerobic Oxygen forms ozone in the stratosphere shields UV light and makes light in the sun on land and near the surface of the water much more inhabitable 5 About 27 billion years ago there are massive banded iron deposits which were the rst place that the produced oxygen went to oxidize the chemical species that were present Drop the overall redox of the earth Carbon isotope signatures also suggest the appearance of 43 BIOL 687 Spring 2005 Redox and Oxygen photosynthesis on a global scale at this point Ratios of stable carbon isotopes are similar to those cause by the slight fractionation 6 Following and initial phase of oxidation of the reduced chemicals in the environment there was evolution of oxygen into the atmosphere IV Global 02 budget 44 687 Spring 2005 Carbon Microbial Ecology Topic 8 The carbon cycle I H A B Carbon Dioxide Needed for photosynthesis in atmosphere at less than 1 350 ppm 1 2 3 Bic 1 Carbon dioxide 1 Very soluble in water rarely becomes depleted First because respiration produces Water can store a lot of C02 as bicarbonate arbonate equilibrium C02 H20 ltgt H2C03 ltgt PF Hcog39 ltgt PF COf39 carbonic acid bicarbonate carbonate At any pH the concentration of the bicarbonate can be calculated with a series of equations C02 hydration CO2 air ltgt co2 Hzco2 co2 H20 ltgt HZCO3 H2003 ltgt in HCO339 HCO339ltgt in cogquot 2 V L V Photosynthetic organisms need to use it as C02 but many can assimilate bicarbonate and photosynthesize equilibrium feeds it in via a series of equations A lot of the carbon in the world is stored as bicarbonate in the oceans this is the most uncertain parameter in calculating the greenhouse effect how much C02 the oceans will take up depending upon the rate equation given above and the transfer coefficients with varied turbulence and surface organisms C is generally not a limiting nutrient in lakes we will talk more about this when we talk about acid rain and eutrophication Hypolimnetic C02 1 In most lakes the main source of excess inorganic carbon in the hypolimnion is from respiration of materials that are xed by primary production in the epilimnion and sink to the hypolimnion The primary production of a lake can be calculated using the total increase in inorganic carbon in the hypolimnion Lake Nyos in Africa Cameroon experienced an extreme enrichment of carbon dioxide in the hypolimnion i In 1986 about 1700 people and 3000 cattle were found dead in the valley below the lake Kling et al 1987 Science 236169175 ii A year earlier Lake Monoun caused a similar disaster where 37 people died iii It turned out that the lake caused the deaths iv The lake was permanently strati ed amictic for a long period of time v Volcanic activity in the region vented supersaturated solution of carbon dioxide into the hypolimnion of the lake causing a large store to build up vi Probably cool weather caused lake to mix Kling 1987 Science 23710221024 viiUpon release of the pressure below the lake released its supersaturated carbon dioxide Like a soda releasing its carbonation when the cap is removed The lake washed up 20 m on one side from the force of the CO2 release viii The carbon dioxide is heavier than air it displaced the oxygen and owed down the outlet valley 45 III E 687 Spring 2005 Carbon ix The people who died where asphyxiated by the lack of oxygen survivors reported odor of sul de then passed out for 30 hours x the lake is currently rebuilding an excess of carbon dioxide in the hypolimnion Within 20 years a similar release could occur Evans et al 1994 Geochem J 28139162 xi Currently a French team is placing tubes in hypolimnion causing water to cascade to the surface Carbon budget Processes 1 Photosynthesis 2 Fermentation yields acids 3 Methanogenesis i Under extremely anaerobic and low redox conditions 350450mV carbon dioxide can be reduced to yield energy sulfate in environment will shut down methanogenesis HCO339 PF 4H gt CH4 3HZO or 4H2 C02 gt2HZO CH or C0 3H2 gt CH4 HZO less efficient why or C0 2H2 gt CH3COOH even less efficient ii Otherwise acid byproducts of fermentation are converted to methane and C02 not enough electron acceptors to allow the processing of methane This source of methane can occurs at a higher redox than C02 reduction Thus a signi cant amount of methane is generated by fermentative processes not methanogenesis Segers 1998 Biogeochemistry 412351 See table for complete sequence Furthermore denitri cation byproducts nitrite NO N2 0 are inhibitory to methanogenesis Kluber and Contrad 1998 FEMS Microbiology Ecology 25331339 Methanogens are unusually susceptible to UV why 4 Some communities with methanogens produce acetate and H2 If partial pressure of H2 is low this is bioenergetically preferred Dol ng 2001 Microb Ecol 418389 Methylotrophyi bacteria that use methyl groups or carbon monoxide Methanotrophs utilize methane i live in aerobic areas near where methane is being produced ii SCH4 802 gt 2CHZO 3 CO2 8 H20 Some archea also live in anaerobic conditions and use sulfate as an oxident in concert with sulfate reducing bacteria Boetus et al 2000 Nature 407623626 iii Probably most important globally in deep sea sediments where methane is produced D Hondt et al 2002 Science 29520672070 6 Archeaa can also reverse methanogenesis in anoxic sediments Hallam et al 2004 Science 30514571462 presentation 7 Carbon monoxide oxidation can be oxidized by some microorganisms for example Pseudomonas carboxydo ava as a net energy source C0 H20 gt CO2 H2 8 Hydrogen bacteria can use the hydrogen chemolithotrophically and do so more ef ciently than CO utilization 6H2 202 CO2 gt CHZO 5 H20 9 Breakdown of complex organic molecules Lquot V 46 687 Spring 2005 Carbon i Dead organisms leave complex polysaccharides in the environment They can yield energy if they are broken down See handout ii Lignin and tannin are two examples of molecules which are hard to break down but microbes can do it iii Cellulose also dif cult for higher organisms to deal with they use gut microorganisms to do it for them B Diagram the processes of transformation note this diagram will hold in a lake which does not stratify and deoxygenate the anaerobic portion of the cycle happens in the sediments or in aerobic and anaerobic soils Also methanogenesis can occur in the open ocean Methane is produced in anoxic microzones in fecal pellets of zooplankton Bianchi et al1992 Mar Ecol Prog Ser 8855 60 or other aggregates called marine snow IV The global carbon budget 47 687 Spring 2005 Carbon A Pools versus uxes Ocean has largest pools calcitic desert soils have largest terrestrial pools but the ux rates associated with the pools is what matters Relatively high uxes associated with photosynthesis and respiration High uxes associated with destruction of forests Although anthropogenic loading is relatively small the net effect is an imbalance in the system and a net increase of CO2 see gure 4 Most recent estimates are net primary production of 1049 pentagrams of carbon per year Roughly equal contributions from land and oceans Field et al 1998 Science 281237240 Global primary production handout here Although there is only 15 ppm methane in the environment there is a greater net rate of increase 1 per year when compared to the rate of C02 increase see Figure p 56 Biogeochemistry but for some reason the methane accumulation rates are slowing Steele et al 1992 Science 358313316 may have to do with decreased losses of methane during petroleum operations 1 Global sinks and sources of methane after Schlesinger 1997 N p A V V L V 03 V Sources and Sinks Methane ux 1012 g Percenta CH4 y39l ge of total Sources Natural wetlands microbial l 15 21 Freshwaters microbial 5 l Termites microbial 20 4 Oceans and geological microbial and 20 4 abiotic Human activities burning land lls gas and coal mining 230 43 Rice paddies microbial 60 ll Grazing animals microbial 85 16 Total sources 535 Sinks Reaction with OH and loss in atmosphere 475 89 Soil microbes 30 6 Increase in atmosphere 30 6 2 CO 5 of total carbon released during combustion is CO which makes up about half of the global production Reacts with OH radicals to form ozone in the lower atmosphere bad Termites were thought to be an important source of methane but these estimates may be to high Methylotrophic bacteria reside in their mounds and eat the methane before is escapes The original estimates were made with measurements made on termites in jars not whole mounds 48 3 3 4 V 687 Spring 2005 Carbon A source that may be largely under estimated is shallow marine sediments Horland and Judd 1992 Coast ShelfRes 1212311238 handout For example massive microbial reefs 4 m high carbonate structures form in the black sea that oxidize methane Michaelis et al 2002 Science 29710131015 Methane seeps are ubiquitous the degree that methane oxidizing bacteria can intercept it may be crucial in determining escape rates to atmosphere The greenhouse effect COZ methane N20 and other gasses absorb heat 1 Heat of sun hits the earth and radiates back 2 Is trapped by greenhouse gasses 3 Will lead to eventual warming of the earth B Predictions are for a 25 to 5 C 45 9 F warming in the next 50 years C Lots of uncertainty in particulars not so much in general idea ultimately driven by microbial C cycling 1 When permafrost melts will there be a net release of methane and C02 resulting in a positive feedback loop 2 How much C02 will the ocean absorb An example of how complex this question is Global warming will decrease mixing of ocean and thus fewer nutrients will come in to upper layers lowering photosynthesis Thus even less CO2 will be taken up by phytoplankton a multiplier effectWoods amp Burkman 1993 J Plankt Res 1510531074 3 In southern oceans higher temperatures will lead to greater strati cation More stable strati cation can shift dominance from Phaeocystis to diatoms Diatoms sequester less COZ leading to greater buildup of atmospheric C02 Arrigo et al 1999 Science 283365367 4 Arctic sea may stop circulating to Atlantic ocean cooling northern Europe 5 Particles from atmospheric pollution may slow warming by re ecting heat back make more clouds Currently the back re ection of heat by pollution particulates is about equal to the increased absorption of heat that should be happening related to the increased C02 Currently there is a signi cant rise in mid continental night time temperatures consistent with the idea that daytime re ectance is greater Eventually the C02 will prevail because particulates are in the air for 12 weeks but the C02 turnover rates are 10100 years in the atmosphere The geritol solution 1 L V 3 V Mid oceanic species of plankton can be stimulated to increase photosynthetic rates by addition of iron and productivity of plankton highly correlated to iron concentration de Baar et al 1995 Nature 373 412415 particularly unicellular cyanobacteria in south Paci c Behrenfeld and Kolber 1999 Science 283840842 Led one oceanographer John Martin to comment give me a tanker full of iron and I will give you an ice age Why might this work Calculated it would take 1 billion dollars a year to have a signi cant effect but still may cost less than emissions controls What are potential problems i Species shifts with fertilization may damage marine food webs ii Fixed carbon may not sink if it is efficiently respired by higher community levels iii If it does sink the CO2 trapped in the deep ocean will eventually come up 100 s of years and exacerbate the problem in the future iv Decreased oxygen in the deep ocean why may lead to increased production of methane and N2 0 49 5 O V l V 687 Spring 2005 Carbon Led to the IronEXII Coale et al 1996 Nature 383495501 i Iron patch of 225 kg over 72 km2 was created ii Led to a phytoplankton bloom over 10 times ambient conc iii Con rmed idea that iron limits open ocean iv Led to large increase in dimethyl sul de an organicsulfur gas production as well Turner et al 1996 Nature 383513517 indicating a link between global iron and sulfur budgets Iron nitrogen and phosphorus may all be important in tropical North Atlantic rely upon dust from Africa Mills et al 2004 Nature 429292 presentation Most recent experiment in southern ocean SOFEXII presentation Bishop et al 2004 Science 304417 Coale et al 2004 Science 304408 Buesseler et al 2004 Science 304414 50 BIOL 687 Spring 2005 Nitrogen Microbial Ecology Topic 9 Nitrogen Cycling 1 Types A Nitrogen gas 80 of the atmosphere N2 B Combined nitrogen l Ammonium NH4 2 Nitrate NO 3 Organic nitrogen i With carbon compounds ii Such as protein and amino acids Nitrous oxide N20 gas 3 V H Transformations A Dissolved inorganic utilization l Dissolved inorganic nitrogen is made up of nitrate nitrite and ammonium i Ammonium is taken into organic compounds by the gogat pathway handout V ii Nitrate needs to be reduced to ammonium before use with nitrate and nitrite reductase This requires more energy than ammonium uptake consequently nitrate and nitrite less preferred than ammonium some organisms can not grow on nitrate or nitrite but these are an exception B Nitrogen xation N2 lots of ATP 6 at least 6 e39 gt 2 NH3 2 Some cyanobacteria Azotobacter and other prokaryotes are capable of using atmospheric N2 gas only prokaryotes possess this ability When other forms of nitrogen are unavailable this confers a competitive advantage to the organisms able to use this nitrogen source The enzyme that is used to x nitrogen is called nitrogenase nitrogenase requires at least 6 ATP to run for each N xed so it is energetically expensive to vis atmospheric nitrogen This is the least preferred source Nitrogenase is irreversibly denatured by oxygen so organisms that x nitrogen must take steps to protect their nitrogenase Note thermophilic bacterium Streptomyces thermoautotrophicus has been demonstrated to have a nitrogenase that is insensitive to Oz but different from that found in other organisms Robbe et al 1997 J Biol Chem 2722662726633 6 Some have protective membranes and very high intracellular respiration rates eg Azotobacter some clump up such as Aphanezomenon or T richodesmium others possess specialized structures called heterocysts i Azotobacter requires much organic carbon to actively x nitrogen as a heterotroph ii the rates of nitrogen xation are greater near the rhizosphere roots for Azotobacter than away iii Roots exude organic materials allowing for greater rates of heterotrophic activity lower oxygen in cells and increased energy supply Heterocysts see picture in handouts i V L V Jib VV l V 51 BIOL 687 Spring 2005 Nitrogen i Only found in lamentous cyanobacteria look clear and obviously differentiated from the rest of the cells ii Commonly every 8th cell is a heterocyst iii New heterocysts are not produced when there is ample nitrogen around the presence of ammonium stops their formation cells turn nitrate into ammonium However relatively high levels ie lmM are required a When trichomes chains of cells in cyanobacteria become nitrogen limited heterocyst synthesis begins b A number of new 39 are 39 39 39 39 39 39 the one in each region which matures fastest starts xing nitrogen transports it out and suppresses further development of adjacent heterocysts This explains why there are heterocysts about every 8th cell A new one can be synthesized when it is not close enough to a nitrogen xing cell Recently shown that signal is a polypeptide that inhibits heterocyst formation Yoon and Golden 1998 Science 282935938 This type of intercellular signaling mechanism has only been shown in Eukarya previously iv In the issue of Nature where it was rmly established that the heterocysts was the site of nitrogen xation isolated and retained their activity another paper was published that stated the heterocysts were just resting cells v Photosystem II is turned off in heterocysts a Do not produce oxygen b Photosystem I produces ATP via cyclic photophosphorilization for energy c Takes in organic carbon from the surrounding vegetative photosynthetic cells to respire away oxygen and to produce reductant for nitrogen xation The cells also produce oxygen consuming glycolipids which reduce the oxygen in cells d Heterocysts are surrounded by dense layers of gel which inhibit the inward diffusion of gasses oxygen nitrogen and carbon dioxide Daily pattern of nitrogen xation by photosynthetic heterocystous organisms often shows a peak in the early morning and after sunset Nitrogen xation is one reason that the cyanobacteria are still so successful even though they appear to have been around for a long period of time longer than any other known photosynthetic organisms 10 Nitrogen xation can be a signi cant source of nitrogen on ecosystem level If nitrogen inputs from other sources are low and ow through is low then nitrogen xation can be quite important A few so V O V 52 BIOL 687 Spring 2005 Nitrogen i In new soils the process of nitrogen xation can be important in soil formation a A n V In high arctic the land is still rebounding from the weight of the glaciers Land near the ocean was recently under water soils have not formed and Nostoc nitrogen xing cyanobacteria is the rst colonist and increases the levels of organic materials and nitrogen in the soils In Antarctica plants cannot survive for the most part Nostoc forms crusts on the surface These become freeze dried for most of the year In the summer when it thaws they resume photosynthesizing and xing nitrogen within 20 min of thawing g or becoming wet Adaptation to very intermittent growing conditions Nostoc or something similar was probably the rst plant to inhabit the terrestrial surface on earth It was probably what formed the earliest soils In Hawaii an introduced tree with nitrogen xing rhizobia is taking over In established stands of native vegetation it cannot become established As the volcanoes disturb and area they produce very low nitrogen growth surface The introduced tree comes in and is able to establish more rapidly than the other vegetation and then crowds out the other vegetation ii Rice paddies n xation important to maintain fertility in unfertilized rice paddies all of them up to 1960 s iii Open ocean a C 39 A 33 A amp f 1 r u iuiu T 39 common in open ocean areas Fixes nitrogen May be limited by temperature in tropical oceans Staal et al Nature 425504 507 report on this About half of the new N input into the N limited areas of open ocean may be from n xation Karl et al 1997 Nature 388533538 A large diatom Rhizoselem39a is also important N input because it takes up nitrogen deep and then releases it after it oats to the surface Villareal et al1999 Nature 397423425 Important in global N and C budgets C Organic nitrogen Many bacteria are able to use organic nitrogen especially urea and amino acids In addition cells that are able to engulf particles and other organisms predators may derive some of their nitrogenous nutrition from this source Ammoni cation Organic N gt NH4 COZ energy 1 In anaerobic situations not all of the carbon is converted to C02 and it is called putrifaction older term Some of the nitrogenous byproducts and ammonia smell bad hence the name Inorganic N oxidation nitri cation NH4 Oz gt NOZ39 energy ammonium oxidation i Nitrosomonas does this conversion the rst step NZO can also escape here to V gt11 V NOZ39 Oz gt NO339 energy Nitrobacter nitrite oxidation Requires aerobic conditions but also a source of ammonium Low dissolved carbon may be necessary since heterotrophs can outcompete for ammonium when they are active The process is sensitive to sul de Joye amp Hollibaugh 1995 Science 270623625 Anoxic zones produce ammonium but can also produce sul de Thus sulfur cycle in part can control N cycle Some additional bacteria have been found that can oxidize ammonium to nitrogen gas in a single step Called anammox 53 3 BIOL 687 Spring 2005 Nitrogen 1 These bacteria apparently use nitrite as an oxidant Kuenen and Jetten 2001 ASM news 67456463 A peculiar form of denitri cation nitri cation 2 Can also use N204 as an oxidant Schmidt et al 2002 AEM 6853515357 3 May be quite important up to 65 of N2 produced in a Baltic sea sediment was from this process Dalsgaard and Thamdrup 2002 AEM 6838023808 Kuypers et al 2003 Nature 422608611 Denitri cation 4 N03 C5H1206 gt 6COZ 6 H20 N2 1 An alternative form of respiration 2 Bacteria of many different types do it 3 Done primarily in anaerobic areas however many can continue to do it under aerobic conditions Patureau et al 2000 Microbial Ecology 39145152 4 Steps ofprocess are N05 gt N0239 gt NO gt N20 gt N2 5 Can alternatively result in nitrous oxide formation N2 0 which is lost as a gas important greenhouse gas or N2 in any case it is a net loss to the system Some interesting recent results on formation of organic chemicals When glycine is repeatedly cycled through hot and cold regions oligopeptides formed When copper ions were present hexglycine formed Under moderately reducing conditions and 100 C synthesis of all 20 amino acids is energetically favorable whereas it costs signi cant energy with Oz present at more moderate temperatures Amend and Shock 1998 Science 281 16541662 Under some conditions NH4 is formed instead of N20 or N2 This is called dissimilatory nitrate reduction and occurs under very low redox Data from oceans suggest that denitri cation may be happening there at rates that have been underestimated leading to major miscalculations of the global nitrogen budget Tyrrell and Law 1997 Nature 387793396 It has been further suggested that during glacial periods denitri cation rates decreased in ocean leading to increases in N more C xation in oceans and thus a feedback to keep the earth cool lower greenhouse C02 Used natural abundance of 15N14N in marine sediment cores to make these arguments since denitri cation alters these ratios Ganeshram et al 1995 Nature 376755758 Another big unknown in greenhouse effect If climate is changed enough for deep ocean to go anoxic as it has several times in earth s history may exacerbate greenhouse effect by increasing nitrous oxide ux rates Complete nitrogen cycle diagram 1 V N V L V 3 V 54 BIOL 687 Spring 2005 Sulfur Microbial Ecology Topic 10 Sulfur Cycling 1 Sulfur A Forms 1 Organic i Sulfur side groups in proteins ii dimethyl sul de gas arises from cyanobacteria and salt marsh plants such as Sparring Turns into methane sulfonic acid CH3S03H other forms also but less important a Inorganic Sul de SZ39 HS39I HZS depends upon pH valance 2 Sulfate SO4239 6 Sul te SO3239 4 Thiosulfate S203 6 2 Hyposul te S204 2 Elemental sulfur S 0 iii AAAAAA O Lquot J L N VVVVVV B Transformations 1 Assimilation SO439 gt SZ39 gt cysteine uptake for biosynthesis 2 Putri cation organic S to His 3 Sulfur reducing respiration bacteria ie Desulfovibrio Desulfomaculum 3 V 1 H2s04 2CHZO gt 2C02 2H20 HZS ATP or ii HZSO4 4H gt 4HZO HZS ATP or iii S20 CH3COO39 PF gt ZHS39 2COZ H20 iv This is why anaerobic muds stink of sul de v This process is responsible for 50 of the oxidation of carbon in coastal marine sediments One candidate for much of this is the organism Shewanella putrifaciens that can take thiosulfate sul te or elemental sulfur to sul de as a source of reductant for carbon utilization Found widespread in marine environments very important in anaerobic portion of the Black Sea Perry et al 1993 Science 259801804 Previously though to occur only in anaerobic situations More recently it has been shown to occur in aerobic situations Not known which organisms do it It is less ef cient than using oxygen but some organisms may have no choice Chemosynthetic sulfur oxidation i ie Beggiatoa and T hiothrix hot springs anaerobic muds sewage treatment a HSS 12 Oz gt Squot H20 ATP b Elemental sulfur deposited inside the cell and used after sul de is depleted c 15 02 Squot H20 gt HZSO4 ATP d Thiobacillus e 2 NaZSZO3 Oz gt 2S 2Na2SO4 ATP or i 5S 6NO339 2C0339 gt 5SO439 2COZ 3N ATP It has also been shown that T hioploca a common gliding marine and freshwater lamentous a Bacterium in sediments can glide up in sediments concentrate N03 up to 05 V i ii m b In a liquid vacuole glide to anoxic areas and oxidize sul de with it to yield ATP c This is another link between sulfur and nitrogen cycles Fossing et al 1995 55 BIOL 687 Spring 2005 Sulfur Nature 374713715 Jorgensen and Gallado 1999 FEMS Microbiology Ecology 28301313 A similar species Thiomargarita namibienses is large enough to be seen with the naked eye 02 mm diameter The previous largest bacteria Epulopiscum selsom39 from sturgeon guts is longer but not as large in terms of ofvolume Schulz et al 1999 Science 284493495 Form shining white beads Schulz 2002 ASM News 68122127 5 Photosynthetic sulfur bacteria 39 Green photosynthetic sulfur bacteria ii 2C02 ZHZO HZS Light gt 2CHZO HZSO4 or iii COZ HZS gt CHZO S iv Note in this case that HZS takes the same role as H20 in oxygenic photosynthesis This may have been the primitive form of photosynthesis Purple sulfur bacteria can also use thiosulfate the purple nonsulfur bacteria ie Rhodospirillum can grow photosynthetically or heterotrophically using sulfur or sul de 6 The main gaseous product of the sulfur cycle entering the atmosphere is dimethyl sul de This is given off by marine algae cyanobacteria and some plants Has been shown to be a chemical defense It is released when algal cells are grazed Hydrogen sul de is a gas as well but as we have seen this is often oxidized rapidly and does not have a chance to build up Yoch AEM 6858045815 presentation i Dimethyl sul de becomes methane sulfonic acid upon exposure to light ii Bacteria isolated from the soil can convert methane sulfonic acid into sulfate and carbon dioxide yielding energy Thus we do not have excessive buildup of methane sulfonic acid Disproportionation is an anaerobic process that yields energy i Thiosulfate S203 gt SO439 HS39 ii This process has been shown to be important in determining stable S isotope of marine sediments Tends to deplete S pool in 34S Have thus shown a global effect on S cycle Can eld amp Thamdrum 1994 Science 26619731978 in Can also work with elemental sulfur with continous removal of sul de janssen et al 1996 Arch Microbiol 166184192 presentation A amp 4 c 11 Metal Deposition A Pyrite forms with sulfur B sulfate reducing bacteria produce sul de leads to deposition of metal compounds such as iron pyrite zinc sul de arsenic sul de Can control heavy metal concentrations in groundwater Labrenz et al 2000 Science 29017441747 1H Diagram of the sulfur cycle 56 BIOL 687 Spring 2005 Sulfur IV Global sulfur budget 57 BIOL 687 Spring 2005 Phosphorus iron and others Microbial Ecology Topic 11 Phosphorus Iron and Minor Nutrient Cycling I II A 03 V O V 9 gt11 V 3 03 V O V Phosphate Needed for all cells for DNA RNA and ATP 1 ATP is the energy currency for the cell Unlike nitrogen in nature it is only found in one inorganic form 1 P04 2 No gaseous component to this nutrient cycle phosphene is very toxic so organisms cannot reduce phosphate In aquatic environments it is rapidly recycled so it does not sink out as fast as N In soils phosphate is bound and is less easily leached than nitrate In lakes and ocean bacteria and phytoplankton take up dissolved phosphate Bacteria can compete successfully for phosphate when there are low levels present In general bacteria need phosphate and compete for it in freshwaters Zooplankton eat these suspended particles Excrete the phosphate or die and sink Fish eat zooplankton and excrete phosphate Eventually phosphate sinks out of the epilinmion 5 It either ends up in the sediments or in deeper waters Phosphates are excreted into the environment or membrane bound These compounds break organic phosphorus compounds and make phosphate available rapidly They are associated with bacteria and phytoplankton Typical epilinmetic or ocean surface P cycle 2 3 4 Iron Essential nutrient for all cells I Needed in hemoglobin and other respiratory proteins Can be found in two ionic forms 1 FeH only in water in the absence of oxygen ferrous 2 Fe from which it takes in water with dissolved oxygen ferric Fe is not very soluble in water so much precipitates out Iron can easily become limiting in an epilinmion for this reason Chelators i Chelators are complex organics which can hold iron in solution ii Natural chelators are tannins and lignins a They leach out of wood b Dark waters are full of them iii Some algae also excrete chelators to keep iron available If there is to much chelator all the iron can be stuck to it 2 L V 58 III IV gt11 V Phosphorus iron and others i Iron is around but iron limitation occurs ii Lakes with lots of tannin and lignin have this a Brown color b Known as dystrophic lots of nutrients but don t have much production because they have little iron or the chelators are poison Iron can be oxidized chemoautotrophically DFWHm0 w mmOme 2 This mainly occurs at low pH at neutral and high pH this reaction occurs abiologically too rapidly 3 Ferrous iron can also be oxidized in the process of magnetite formation for making magnetosomes Gueriea and Blakemore 1992 AEM 5811021109 Iron can be oxidized in anaerobic conditions by anoxygenic photosynthetic organisms Straub et al 2001 FEMS Microbiol Ecol 34181186 Iron reduction Oxidized iron can be used to oxidize organic carbon 1 This can also mediate arsenic release in groundwaters Important in groundwater in India where arsenic poisoning is a severe problem Islam et al 2004 Nature 43068 presentation Anaerobically iron precipitates with sul de forming insoluble pyrite this takes some time Mn cycling Similar to the iron cycle manganese nodules form in deep sea and bottom of lakes It has been proposed that the nodules may be mined at some point in the future currently not economically feasible xx l4112 CB 92 Ei 12 Oz gt MnOz Interactions among phosphate iron and sulfur Fe and PO43 combine and precipitate FeH does not precipitate with phosphate In anaerobic eutrophic hypolimnion iron and phosphate dissolve easily from sediments because there is no oxygen In aerobic zones there is considerable PO4339 storage there is typically an oxidized microzone across which phosphorus will not pass An example of interactions between the cycle has practical applications Smolders amp Roelofs 1993 Aquat Bot 46247253 1 Wetland in Netherlands had eutrophication problems Decided to divert low phosphorus river water in Made problem worse More nuisance plants Water was high in sulfate and low in iron Sulfate converted to sul de by reducing bacteria sul de precipitated iron More phosphate released from sediment as a result Example of why understanding nutrient cycles and links is important 2 3 3 V 59 VI Phosphorus iron and others F Aquatic macrophytes or terrestrial plants can bring up PO4339 from below the oxidized zone and cause internal loading 1 When lakes or oceans mix lots of the iron precipitates out In aerobic hypolimnion oligotrophic lakes neither can dissolve 1 Lots of iron and phosphate stuck in oligotrophic sediments Iron may be the limiting factor in many areas of open ocean Iron precipitates out or sinks in algal cells As you move further from land there is less and less iron and other nutrients The main source of iron for cells in the upper waters becomes atmospheric deposition Talked about before with regard to the carbon cycle Nitrate interacts with arsenic through iron cycle Nitrate can be used to oxidize iron in anoxic conditions to form ferric oxides that sorb arsenic This can oxidize arsenic as well Leads to predominance of arsenic in the particulate form Senn and Hemmond 2002 Science 2962373 Silicon 3 E 1 V 2 3 3 orms 1 Silica SiOz 2 Silicic acids H4 SiO 3 Particulate i In diatoms and some other organisms ii Complexed with organic or inorganic particles Cycling in epilimnion is controlled by diatoms global importance in biogeochemistry 1 Can often take it up more rapidly than it comes in Sink out of the epilimnion more rabidly than the silica is replaced and then silica slowly remineralized Silica builds up in the hypolimnion or deep ocean sediments In high nitrate iron poor areas of the ocean iron fertilization leads to blooms of diatoms that are rapidly limited by silicon Cells tend to be high Si and low N and P Takeda 1998 Nature 393774777 This silicon sinks out of surface waters and this may be the main secondary limiting nutrient in vast areas of the open ocean Hutchins and Bruland 1998 Nature 393 561564 Silicon uptake buffems pH increased C02 availability in ocean so may give diatoms an advantage Milligan and Morel 2002 Science 29718481850 N V 5 60 Phosphorus iron and others 6 Typical silicon curves with season in a mesotrophic lake are as follows this can account for spiing blooms of diatoms see gure VII Hydrogen cycles probably important but mostly ignored We have seen hydrogen consumption by sulfur reducing bacteiia H2 transformation inescapable part of N2 xation hydrogen trophs use C02 carbon monoxide oxidizers provide hydrogen methanogens can use H2 H2 required for many fermentation pathways or they re more efficient when it is present Anaerobic microbiologists have paid attention to these interactions geologists have paid less attention VIII All elements have their cycles see handout p for links 3 003 VV 61 BIOL 687 Spring 2005 Nutrients Microbial Ecology Topic 12 Nutrient Uptake Utilization and Recycling I CB 92 3E 11 III 93 O V E2 93 How to build a microbial cell nutrient requirements Red eld ratios Each cell constructed of similar proportions of elements and require such table p These elements are required as dissolved ions or as cofactors in required vitamins Some require vitamins usually B vitamins but others are required as well 1 Oligotrophic species less likely to require Some nutrients are toxic if present in to high concentrations For example typical concentrations of algal growth media are like a highly emiched lake Many species of microorganisms found in oligotrophic lakes can not tolerate culture in this concentrated medium Red eld ratios 1 In the late 1950 s A Red eld noticed that the plankton in the ocean had approximately the same molar ratio of CNP as dissolved in the surrounding water Under balanced growth ample nutrients exponential growth no limitations plants display a CNP ratio of 106161 by moles Under nutrient stress the Red eld ratio changes i With N limitation the CN ratio increases and the NP ratio decreases ii With P limitation the CP ratio increases and the NP ratio increases iii This has been shown to be true in culture iv Some people feel that this can be used as a technique to determine nutrient limitation in lakes and the ocean v The problem with application of this method is that there is lots of other material oating around in a lake or the ocean which is not living algal material such as dead algae bacteria zooplankton unidenti ed organic detritus vi In the ocean the plankton appears to be at Red eld ratio so some people argue that nutrients do not limit productivity in the ocean this is a contested topic viiIn Flathead lake the NP ratio is always above 161 however when you correct for non algal material the NP ratio is close to 161 The CP ratio is always in excess of 1061 and the CN ratio is always in excess of 101 Red eld is 661 This suggests that both N and P limit productivity in Flathead Lake 4 Another approach is to look at nutrient uptake nitrogen and phosphorus appear to be the most likely nutrients to limit algal growth in aquatic systems Nitrogen Discussed in n cycle 2 L V Phosphorus Needed for ATP lipids and amino acids Cells have an incredible affinity for phosphorus able to take up and fray effectively even when it is below 1 nanomolar Many algae are able to store phosphorus as polyphosphate bodies they can store 10 to 100 cell divisions worth of phosphorus since relatively little per cell is needed Rapid uptake and subsequent storage is called luxury consumption This ability re ects the sporadic nature of phosphorus availability in aquatic environments Everything else Uptake of silicon is somewhat characterized Iron can be limiting 1 Some algae excrete compounds which chelate iron and keep it more available Inorganic carbon can be used as carbon dioxide or as bicarbonate Calci ed species more rare in freshwater Charophyceae are often calci ed often have high requirements Uptake kinetics 62 BIOL 687 Spring 2005 Nutrients A The rate that organisms take nutrients up from the environment is dependent upon the concentration in the environment the need for that nutrient and the ability to assimilate the nutrient once it enters the cell First the nutrient has to be taken across the cell wall If the internal concentration is higher than the external then the uptake has to maintain the concentration against any backleakage C MichaelisMenten equation describes the uptake of nutrients V vmax ssk 03 V Many species of algae and bacteria take up nutrients in a fashion which can be modeled with the MichaelisMenten model 2 Even more interesting and useful is that the uptake of populations of algae can be described with the model some problems with this mixed assemblages do not act exactly this way The advent of access to computers and nonlinear curve tting programs has made tting the equations easy to do Uptake capacity seems to be tied to cell size a lower value for Ks i Smaller cells are able to use lower concentrations more effectively ii This is probably because they have more surface area per unit volume than larger cells Assuming that cell membrane proteins are required to take up nutrients and a certain maximum number of these proteins can be packed in per unit area cell membrane a cell with a larger relative area of cell membrane per unit volume can take up more nutrient per unit volume at low concentrations D Monod equation is similar except it describes the growth of the organism If the concentration of the nutrient in question is low enough that nutrient controls growth then the growth is dependent upon uptake and we can write the equation u umax ssk V L V 3 V E The Droop equation forms links from uptake to growth VI u umax lQoQ where u max growth or maximum growth Q is the amount of nutrient in the algal cells and Q0 is the minimum concentration which will support growth 63 BIOL 687 Spring 2005 Nutrients A The uptake of nutrients can be affected by light It requires energy to take up nutrients and the uptake of nutrients can be modeled in a similar fashion that photosynthesis versus irradiance is The main modi cation that is necessary in this case is that dark uptake usually needs to be considered This means that measurements of nutrients uptake need not be only done in the day or erroneous results may result VII Nutrient limitation A Leibigs s Law assumptions Assume all cells have same requirements Assume that nutrients are added at a constant rate Assume that nutrients are well mixed in the system may not be an unreasonable assumption in lakes With these assumptions the nutrient that is present in the lowest relative supply will be limiting to all species in the community ie the one nutrient is in below the proportions more than any other B This leads to the paradox of the plankton which was posed by Hutchinson 1 The competitive exclusion principle Harding states that in an equilibrium environment with one limiting resource only the species which competes best will predominate 2 Hutchinson noted that lakes appear to be well mixed but there are often in excess of 20 species of algae present in the phytoplankton community This would appear to be in direct con ict with the competitive exclusion principle 3 Explained by deviations from assumptions i Heterogeneity it turns out that nutrients are not necessarily homogenous A large amount of the supply of nutrients is often by regenerated or from zooplankton excretion These fecal pellets create pulses of nutrients Also storm events mix nutrients from the bottom of lakes unevenly this may also cause patches Near rivers which are sources of nutrients there may be elevated patches as the water mixes in Also plankton may deplete nutrients around them resulting in patches 4 Variable requirements as we have seen there are variable requirements of algae Diatoms require more silicon Cyanobacteria to not necessarily require dissolved inorganic nitrogen Tilman s approach to explain the paradox of the plankton 1 Tilman noted that even if nutrients are well mixed there are differences in competitive ability for nutrients For example two species can coexits if they have different competitive abilities Tends to be a trade off between Ks and Vmax This has been proven in chemostats but extension to natural phytoplankton populations remains somewhat controversial WNH VVV 3 V O V 64 Q m V gt11 V H BIOL 687 Spring 2005 Nutrients 2 The resource approach also implies that there will be more diversity in more oligotrophic environments See handout Application of Leibig s law also ignores the potential contribution of grazing A very successful competitor may also be grazed and not be able to gain competitive dominance Also there may be facilitation between species which relieves the lack of competitive ability Viruses may also explain the paradox 1 ultra ltered water from the ocean contains numerous viruses and when these concentrated viruses are added to marine algal assemblages they can lower productivity by 80 by causing infection and lysis of cells It turns out that if any algal population gets very large the viruses will be transmitted there is a certain critically concentration of host cells ie distance between cells or probability of infection as this threshold in exceeded the disease spreads throughout the population 3 This mechanism would keep any one species from dominating 4 It is interesting to note that all microbial species have their viruses The role of viruses in microbial ecology is ill de ned will discuss a little later under section on microbial loop The competitive exclusion works well in a chemostat a chemostat is a ow through systems with well de ned nutrients and well mixed but in natural communities it may be tenuous to apply Liebig s Law Consequently more than one nutrient may limit primary productivity What nutrients are limiting in the ocean 1 In many coastal areas there is a stimulation of primary production by nitrogen Upwelling gyres form and spin off into the less productive areas These gyres contain nitrate and they loose nitrate productivity decreases Recently nitrate in acid precipitation stimulated coastal productivity Paerl 1993 Can J Fish Aquat Sci 5022542269 The question is why don t nitrogen xing organisms dominate and rectify this condition Heterocystous cyanobacteria are rare in the open ocean and there is not enough organic carbon for the aerobic nitrogen xing bacteria to be successful Nitrogen xation in the sea In many areas T richodesmium is as cyanobacterium that forms bundles of laments These bundles exhibit low but signi cant levels of nitrogen xation Can have some low oxygen in middle and can x nitrogen when oxygen consumption exceeds production BermanFrank et al 2001 Science 29415341537 These cyanobacteria are apparently P limited at times SanudoWilhelmy et al 2001 Nature 4116669 Some diatoms have endosymbiotic unicellular cyanobacteria which actively x nitrogen These endosymbionts seems to have a full pigment compliment and actively photosynthesize so it is uncertain how they protect nitrogenase It could be that the diatom provides enough extra carbon that they can respire rapidly enough to lower oxygen tension These diatoms are common in many parts of the ocean in Some bacteria sized cyanobacteria have been shown to x nitrogen Gleothece These also have no heterocysts and are actively photosynthetic so how do they x nitrogen protect nitrogenase They seem to have densely folded membranes inside of the cells so they may partition nitrogenase inside areas where little photosynthetic oxygen production is occurring iv Some suggest that the ratio of molybdenum to S04 239 is to low Molybdenum is required for nitrogen xation it is a cofactor for nitrogenase and sulfate competes for the sites of molybdenum uptake If there is too much sulfate the cells will never get enough molybdenum to x nitrogen N V N V L V G n H 65 VIII 3 BIOL 687 Spring 2005 Nutrients v All things considered the rates of nitrogen xation are not high enough to alleviate nitrogen limitation in many parts of the ocean But P or iron may limit in some areas SanudoWilhelmy et al 2001 Nature 4116669 Wu et al 2001 Science 293847849 4 As mentioned before the CNP ratios are similar to cells growing at maximal rates in cultures This suggests that neither N nor P limits production and that grazing or some other nutrient is limiting How is y39 l 39 39 r in very 39 39 waters Photosynthetic rates often exceed the measured inputs of nutrients in to the system in oligotrophic waters There was some question as to how to explain this incongruity in marine systems Nutrient regeneration In oligotrophic waters nutrients are very low near detection often less that 01 umolar Given the observed uptake rates of nutrients the dissolved pools of nutrients would be used up rapidly in minutes to days in many systems David Lean and Frank Rigler both noted that phosphorus pools turn over in a matter of minutes in oligotrophic lakes so something is supplying nutrients in the water column Similar observations have been made for the oligotrophic ocean Heterotrophic organisms recycle nutrients rapidly i Algae die and release the contents of their cells ii Algae continually leak organic nutrients in Algae and other organisms are eaten and nutrients are released iv Assuming organic material either excreted or prey is approximately at the Red eld ratio the organisms which take it up are going to need only about 1 10th of the nitrogen and phosphorus in the material This is because they need to oxidize about 10 carbons for every one that is assimilated Most heterotrophic organisms as a consequence excrete nitrogen and phosphorus and other trace elements Pulses Zooplankton such as Daphnia and Copepods crustacean zooplankton release their excreta in pulses These make large to algae patches of nutrients John Lehman and Don Scavia have shown in elegant experiments in culture that if you label zooplankton with radioactive phosphorus and put them in with unlabeled algal cells some algal cells take up much more nutrients than would be expected on the basis of chance This is direct evidence for the importance of pulses on the basis laboratory experiments Some algae exhibit elevated uptake rates for phosphorus and nitrogen over the short term which has been taken to mean that they are adapted to take up nutrients like mad when they enter a patch Recent evidence in oligotrophic lakes and marine systems suggest that regeneration by small organisms is lt 10 pm is extremely important These organisms are so small that nutrients do not tend to pulse but are rapidly dispersed by diffusion Therefore patches caused by zooplankton are probably relatively unimportant The main cause of patches is probably patchiness of organisms that take up the nutrients and those which regenerate it u 2 I V N V L V 3 V Lquot V 66 BIOL 687 Spring 2005 Bactivory Microbial Ecology Topic 13 Bactivory I 11 A 03 V 9 Functional and numerical response of predators How do predators respond to an increase in populations 1 Numerical response increase number of predators 2 Functional response change rate of prey consumption Holings functional response curves 1 Type I strict linear increase in prey eaten levels off at high density 2 Type 11 very rapid increase in prey taken with a gradual saturation note similarity to MichaelisMenten uptake curves 3 Type III slow start picks up and then levels off Strategies for eating bacteria Engulfment Scraping off or eating entire microbial communities common with larger animals i Snails rasp and scrape periphyton ii Earthworrns other oligocheates eat large amounts of soil and digest the microbes Eating detritus engulfment i Oftentimes microorganisms must condition detritus in soil and water before it is consumed ii The microbes make the detritus more easily digestible by breaking down lignin and cellulose among other products iii Deer mice store seeds until they reach an optimum degree of rot iv Aquatic invertebrates often prefer microbially processed detritus or cannot live on sterile detritus Penetration Bdellovibrio This is probably what Cytophaga does in marine systems to phytoplankton Will kill all species of phytoplankton tested May control algal blooms in marine systems Imai et al 1993 Mar Biol 116527532 Bdellovibrio are probably speci cally associated with certain surfaces In Chesapeake Bay they occurred in 79 of water samples 44 of sediment samples and 100 of oyster shell bio lm samples Williams et al 1995 Microb Ecol 293948 Bacterium Alcaligenes denitrificans causes cell lyses and death of Microcystis Manage et al 2000 Aquat Microb Ecol 22111117 Other bacteria penetrate cyanobacteria Rahsidan and Bird 2001 Microb Ecol 4197105 i V N V 1 Filtration 1 How to measure i Inert uorescent particles Available in a wide variety of sizes can be counted inside 67 BIOL 687 Spring 2005 Bactivory organisms with uorescent microscopy ii Labeled bacteria Use uorescent molecules or radioactivity to label bacteria This is not as controlled as using inert uorescent particles However lter feeders distinguish between particle types and rates from plastic particles can be signi cantly lower than those for labeled bacteria Hydrodynamic problems i Smaller size lters are harder to push through water bacteria sized lters have very viscous ow between pores Puts lower limit on lter feeders ii Mainly rely upon impaction 3 Complex ltration apparatus in many organisms 4 Filter feeding in Daphm a i Legs that move water up along brushes near mouth ii Brush food into mouth iii Have an optimum lter size 21 Bacteria can be captured but not ef ciently b Particles greater than 100 um can not be handled efficiently iv Large Daphnia are more efficient grazers than small species of Daphnia This results in the top down control of productivity in some aquatic systems Small sh eat large Daphnia causing a shift to smaller species These species eat microalgae less ef ciently and algal biomass can increase with the small s If you add large sh they eat the small sh Daphnia size increases and the biomass of algae goes down An example of the higher trophic levels controlling the microbial biomass There are probably analogues for terrestrial microbial communities Elongate cells are less likely to be collected laments are rejected most frequently spherical cells are the best to eat Hartmann and Kunkel 1991 Hydrobiol 225129 154 111 Functional response of organisms which tler microorganisms A Ciliates and Daphnia 1 Effect of particle size i Mouth size a primary determinate of the range which can be successfully utilized a Holotrich ciliates can retain particles as small as 01 pm most ef cient in range from 03 to 1 pm b Spirotroch ciliates take no particles smaller than 12 pm ii Functional response can often be modeled with MichaelisMenten equation this only works for particles smaller than a threshold number High half saturation constants are impossible with large particle sizes p iii Similar to the particle size response seen in Daphnia N V A 99 9 VVV a Vow N V Effect of particle concentration i MichaelisMenten models can be used to describe the relationships between particle capture and use in ciliates Values of K5 are dependent upon the size of the organism and the size of the particle consumed The shape of these curves can depend on temperature Iriberri et al 1995 Freshwater Biol 33223231 68 3 V BIOL 687 Spring 2005 Bactivory ii Daphnia shows an increase and then a leveling off eventually there is a decline again as the ltering apparatus becomes clogged Curves are generally most consistent with the Type III functional response low rates at low conc then a rapid increase ChowFraser and Sprules 1992 Hydrobiologia 232175191 Protection from grazing Shape 1 Larger diatoms have spines that protrude probably keep predation from rotifers and Daphnia to a minimum 2 A green alga Scenedesmus forms larger colonies when exposed to water that Daphnia has been grown in 3 Large aggregations of cells are often more dif cult for small grazers to handle 4 Cells that are tightly appressed to solid surfaces are often dif cult to get 5 Cells colonize cracks and pits in soils sediments and rocks to keep from being grazed i There may be increased resistance to diffusion in such habitats ii Often all of the microorganisms on the raised surfaces are removed by grazing so it is the only choice Chemical defense 1 Two types allelochemicals toxins to stop grazing and low nutritional content 2 Both types require dominance i If the grazer takes in a large number of cells and only a few are toxic then it is of limited effectiveness to have toxicity because it will be diluted out by nontoxic forms or forms with other toxins so there is no cumulative effect ii The same arguments apply for being of low nutritional quality if the other microorganisms can supply the remainder of the nutrition you may be considered a tasty appetizer or a piece ofjunk food 3 Algal toxins i Cyanobacteria often bloom and become the dominant species of algae present and sometimes these blooms are toxic ii Contain several types of toxin Anatoxin which is often known as very rapid death factor and hepatotoxins Both peptide based Coded for on a plasmid which can be passed through populations Other toxins are based on lipipopolysacharides or alkaloids Kaebemick and Neilan 2001 Fems Micrbiol Ecol 3519 iii The blooms kill livestock and occasionally make people sick Hepatotoxins are linked to increased levels of liver cancer commonly occur in Kansas waters iv Probably produced to decrease grazing a Only released when cells die or are broken why not release a toxin into the water b Can kill Daphnia and other microcrustaceans proposed as a rapid bioassay for cyanobacterial toxicity 69 Q m V gt11 V BIOL 687 Spring 2005 Bactivory v It is not certain which populations will be toxic and which will not Klamath Lake story vi Dimethyl sul de is a byproduct of a chemical defense of phytoplankton Toxic acrylate is produced when cells are broken and dimethyl sul de released This enters the atmosphere and serves as a greenhouse gas Has global importance viiDino agellates also produce saxitoxin a neurotoxin responsible for redtide Positive feedback problem Ciguarotoxin taken up by reef sh magni ed in food web Diatoms make domoic acid May be responsible for some of the seal die offs in northern Europe Mechanical defense 1 Oftentimes large amounts of mucilage make algae difficult to handle or digest 2 some species of green algae can pass through the gut of Daphnia unharmed 3 Actually these species bene t from being eaten because there are high levels of dissolved nutrients in the gut from the other busted algae They take it up as fast as they can and are fertilized and ready to go upon release Behavioral defense 1 In properties of water talked about microbes that use ow shear to indicate lter feeding and this initiates an escape response Data for the control of microbial populations in nature by bactivory Top down versus bottom up controversy is still alive and well in ecological literature Not much evidence directly for control of bacteria by predators protozoan grazing in soils lowers heterotrophic bacteria but excrete NHX They stimulate nitrifying bacteria by lower competition and increasing availability of NH4 Verhager et al 1993 AEM 5920992106 Bactivory increases when dead roots are added to soil in the area of the roots This is a direct example of bottom up control Christensen et al 1992 FEMS Microbiology Ecology 86303 310 The heterotrophic dino agelletes Gyrodinium dominans and Gyrodim39um spirale can control blooms of the red tide dino agellate Gymnodium mikimoto Nakamura et al 1995 Mar Ecol Prog Ser 125269277 Nakamura et al 1995 Aquat Microb Ecol 9157164 There are more examples but no general consensus of the importance of consumption We will talk about this more in the section on microbial loops 70 BIOL 687 Spring 2005 Microbial loop Microbial Ecology Topic 14 Microbial Loop and Microbial Diversity I Description of the microbial loop A Classic idea of the aquatic foodweb is that algae photosynthesize are eaten by microcrustaceans eaten by larger crustaceans or small sh on up to large sh and humans B A certain amount of the material xed by photosynthesis is leaked or lost upon death averaging 30 lo50 and grazers often feed inef ciently or sloppily releasing dissolved organic materials and the bacteria are very ef cient at scavenging this material In some systems runoff of organic material from land is greater than primary production so this becomes the major pathway of energy de ne autochthonous and allochthonous 1 Azam and others suggested that this organic material is recycled into the food chain because ciliates and rotifers graze upon the bacteria and each other and they in turn are grazed upon by the microcrustaceans see handout V This increases the ef ciency of freshwater and marine food webs signi cantly Commonly bacterial populations in lakes and oceans are grazed rather heavily i Small protozoa are usually the most important grazers ii It has also been shown that copepod nauplii can be important since they can require bacteriasized particles Roff et al 1995 Aquat Microb Ecol 9165175 iii This lowers the ef ciency of energy transfer since about 90 actually less many invertebrates are more ef cient is lost at each level iv 5080 of the bacterial production is grazed in fresh and salt waters what happens to the rest 4 Grazing on bacteria increases production of dissolved materials in water including DNA release rates Kawabata et al 1998 Hydrobiologia 3857176 Ishii et al 1998 Hydrbobiologia 3806776 Flagellates and ciliates may graze a substantial portion of the autotrophic picoplankton in Baltic sea Samuelsson and Andersson 2003 Aquat Microb Ecol 30239250 Overall average from fresh and salt waters bacteria utilize carbon at approximately 20 of the rate it is xed by photosynthesis whereas zooplankton only utilize it at approximately 10 of the rate This means that in terms of net material processing the bacteria are eXtremely important Rotifers are probably not controlling agellate ciliate populations or bacteria because there measured grazing rates are generally much less than the growth rates of these groups in aquatic systems Amdt 1993 Hydrobiologia 255256 231246 In Antarctic lakes there are no large consumers Heterotrophic nano agellates consume 01 97 ofbacteria production per day Parry et al 1995 J Plankt Res 918351850 C In addition to bacteria there are bacteria sized photosynthetic algae often called picoplankton WM VV Lquot V O V l V 8 V 71 II V m V O V BIOL 687 Spring 2005 Microbial loop 1 In the open ocean these cyanobacterial and eukaryotic bacteria sized particles can be responsible for up to 50 of the primary production An example in equatorial Paci c P a 39 519 of 39 39 Vaulot et al 1995 Science 26814801482 2 The organisms that graze bacteria also graze these particles see handout Sherr amp Sherr 1994 Microb Ecol 28223235 3 A study on a transect across central North Atlantic Li 1995 Mar Ecol Prog Ser 12218 Identi ed cells lt 15 m with cell cytometry layer uorescence Sorted 14C labeled cells with a cell sorter to get production Cell Type biomass 39 Prochlorococcus 78 11 19 Synechococcus 12 2 13 Eukaryotic 10 87 68 4 It may the main way in which this energy enters the food web A microbial loop also is formed in soil microbial communities of bacteria protozoa and nematodes Remineralization resulting from such loops is important Mikola and Setala 1998 Ecology 79153164 Freshwater sediments also contain an active microbial loop One study suggested that oligocheates copepods and cladocerans Wieltschnig et al 2003 Freshwater Biol 4818401849 The importance of viruses in microbial communities Electron microscopy has shown that viruses are common in lakes often there are 10 times as many viral particles as bacteria Borsheim 1993 FEMS Microbiol Ecol 102141159 what these viruses infect is unknown PCR primers to known algal viruses have demonstrated that there are numerous strains of closely related viruses in natural waters Short and Suttle 1999 Hydrobiologia 4011932 The number of viruses present in marine and freshwaters has been shown to change diumally with the numbers of bacteria 1 Between 216 of natural bacteria are infected with viruses in some studies In another study bacteria isolated from river sediments 80 of the isolated strains harbored virus Lammers 1992 Hydrobiologia 235236261265 Some Viruses decay rapidly i About 100 lost per hour in some natural waters Added cyanide which inhibits virus production but not the number of active viruses present in solution Showed a rapid decline in the numbers of viruses ii In another study marine viruses decayed at about 928 h391 in the dark but at 4080amp in light When UVB was shielded from light rates were maX of 17 h391 Suttle and Chen 1992 AEM 5837213729 iii Viruses may be harmed by uv but some may actually carry genes that protect their hosts from uv damage Jacquet and Bratbak 2003 FEMS Microbiol Ecol 44279289 iv Sunscreen may alter viral survival and production could be important in marine waters Danovaro and Corinaldesi 2003 4510911 presentation v Another factor that may be important is grazing of viruses by nano agellates Gonzalez and Suttle 1993 Mar Ecol Prog Ser 94110 Merchant and Scott 1993 Mar Ecol Prog Ser 925964 vi Solid particles can protect viruses from degradation but also the viruses may stick to them irreversibly Some cells apparently put decoy proteins into the water to bind to viruses the target the virus goes for viiIn addition the more bacteria the shorter the life of viruses Murray and Jackson 1992 2 72 Q BIOL 687 Spring 2005 Microbial loop Mar Ecol Prog Ser Why this correlation viii However one group looked at Norwalk like Viruses in bottled mineral waters found DNA sequences in 53 of 159 samples analyzed from 3 brands many survived over a year Beuret et a1 2002 AEM 6819251931 About 50 Viruses released per lysed bacteria Suggest that 224 of the natural aquatic of some bacterial populations may be lysed per hour Such release may be synchronous with surges over several fold in less than an hour Bratbak et al 1996 FEMS Microbiol Ecol 19263269 In marine phytoplankton studies ultra ltration removed particles from 02 to 001 mm diameter When these concentrated particles were added to the algae there was a rapid decrease in productivity related to cell lysis by the Viruses The most eXtreme effect noted was in one case where Viral density was increased by 20 ne particulates added and productivity dropped by 50 Suttle 1992 Mar Ecol Prog Ser 87105112 The small particles were Viewed under electron microscopy and shown to be Viruslike Arti cial concentration of the Viruses de nitely increases the infection rate May control algal populations At natural concentrations of algae not very high infection rates as numbers of large increase above 104 per ml the infection rates go way up contagious infection is possible Thus Viruses may act as natural checks on single types of microorganisms and disallow the dominance of one type promoting diversity of microbial communities In a hypereutrophic lake Viral control of bacterial production was substantial Fisher and Belimirov 2002 Aquat Microbial Ecol 27112 About 5562 of bacterial production was controlled Occasionally 16 per h of bacteria were removed from water column About 46 of bacterial carbon production growth was released to water column by lysis Loss to Viral infections was about 11 times higher than loss to grazing by heterotrophic nano agellates Rates may vary between lakes so infection rates are habitatspeci c Vrede et al Micro ecol 2003 46 4064 1 5 presentation It has been suggested that bactivory not Viral lysis controls most bacterial population Pedros Alio et al 2000 FEMS Micrbiol Ecol 32157165 Some types of Virus are widely distributed Wolf et al 2003 AEM 6923952398 presentation Nutrient cycling and the microbial food web in aquatic systems algae take up nutrients at such a great rate that the limiting nutrients nitrogen or phosphorus would disappear in less than a day or even within minutes if they are not replenis e Studies have shown that nutrients are recycled rapidly by the community Small organisms lt 3 mm are generally responsible for this regeneration How does this work Response of phytoplankton stoichiometry to reduced nutrient concentrations 1 CNP ratio with all the nutrients that can be used is generally 106161 as discussed before this is the Red eld ratio 2 As nutrients become limiting there is a increase in the relative proportion of C eg for N limitation you may see CNP of 50021 or for P limitation 500301 3 The material released from the algae when they die or leaked is depleted in nutrient in nutrient limited situations Effects of altered phytoplankton stoichiometry on grazers of algae All heterotrophs need food at a considerably higher C availability that 106161 Why is this Probably need about 1006161 ie 10 fold eXtra carbon What do these grazers do with the excess nutrient P often excreted as phosphate N and ammonium in aquatic organisms Why not urea as in many terrestrial organisms As nutrient limitation increases the carbon content of the increases and more and more of the i V 2 3 4 N p A V V L V 73 BIOL 687 Spring 2005 Microbial loop N or P are retained ef ciency increases regeneration decreases Bacteria have about 40101 CNP ratios because of relatively high nucleic acid content therefore they have a relatively large demand for phosphorus and nitrogen handout p i under N and P limitation bacteria compete successfully for nutrients ii they are small and have a large relative surface area thus are more able to compete for nutrients handout p Further up the food chain the ratios of organisms are similar to the Red eld ratio Therefore they tend to regenerate more nutrients However it has been demonstrated that Daphnia has low P requirements lower P composition and tend to preferentially regenerate P whereas copepods have high P requirements and have low P regeneration Small organisms dominate nutrient dynamics in both terrestrial and aquatic ecosystems The larger algae utilize the little nutrient that spills out This is functionally important because the more nutrients are tied up in large biomass the more they tend to sink out when the larger organisms sink out The smaller organisms are less likely to sink and thus keep nutrients in the upper layers where photosynthesis can occur This ef cient recycling of nutrients allows for a much greater system production per unit of nutrient coming into the system Why is this Presumably the microbial loop or something like it operates in all systems Microbial food web in a stream Bott 1995 ASM News 61580585 Microbial loop in soils Coleman 1994 Microb Ecol 28245250 Many complex interactions we are only beginning to understand Bio lm bacteria have more antagonistic interactions than others Long and Azam 2001 AEM 6749754983 Diversity of bacteria in different habitats how this is measured will be discussed in methods Are all lakes and rivers the same 1 It is clear that types of bacteria found in rivers and lakes differ from those in soils or marine habitats Zwart et al 2002 Aquat Microbial Ecol28141155 2 Studied 12 Scandinavian lakes some fairly similar in characteristics but only a few had very similar bacterial assemblages Lindtstrom and Leskinene 2002 Microb Ecol 44 1 19 Bacteria in ocean 1 Aerobic anoxygenic phototrophs are very diverse Beja et al 2002 Nature 415630633 Diversity of bacteria greatest in soil and sediments less in ocean and lakes How many species 1 Probably about 2 million species in oceans about 4 million species in each ton of soil Curtis et a1 2002 PNAS 991049410499 3 V Lquot V 74 BIOL 687 Spring 2005 Microbe Microbe Interactions Microbial Ecology Topic 15 Microorganism Microorganism Interactions and Ecological Principles I H A B 9359 General synopsis of types of interactions competition Resource competition a number of organisms utilize the same resource that is in short supply Interference competition when organisms seeking the same resource harm each other even when the resource is not in short supply Competition has been postulated by many especially from the mid 1960 s to the 1980 s to be the dominant mechanism structuring ecological communities Subsequently it is very dif cult to prove competition is occurring This may be an artifact of natural selection and competitive exclusion If two species compete either they evolve to use slightly different resources or one competes the other out of existence Competition may be an important factor in determining the structure even if it does not occur at the particular time of observation Competition must be important some times For example cyanobacteria were isolated and their extracts all had anti bacterial activity Competition is the best reason in this case for organisms to synthesize costly inhibitory compounds Ustervick et al J Appl Microbiol 8411171124 predation may include parasitism and other interactions exploitation is a better general term it is fairly obvious if one organism eats another then it a interaction Parasites may have limited effects There is certainly some costs to harboring parasites but it is in the parasites best interest to have as healthy a host as possible so over time virulence or damage seems to be selected out Other examples of for example a tree with rhizobia is able to x nitrogen and leaks some of it It is helpful to the surrounding plants The surrounding plants compete for light and thus have a negative effect on the nitrogen xing tree A de nite interaction but not a predator prey relationship mutualism is mistakenly called symbiosis by some A reciprocal positive interaction between one or more species Symbiosis mainly means organisms that live closely together Atlas and Bartha call mutualism protocooperation or synergism an ok term but not standard ecological usage Atlas and Bartha mistakenly call mutualism an obligate interaction more correctly referred to as obligate mutualism As long as both species bene t the mutualism is the correct ecological term Amensalism 0 book says it can be but this is different again non standard terminology Commensalism 0 only one bene ts Neutralism no interaction between types Indirect interactions may also be important but little is known of these from microbial communities Such interactions include two organisms that do not compete but compete with a third they are indirect mutualists Or two prey species They are both prey but the predator stops eating one and switches to the other when the rst gets to rare Again the two prey species are mutualists Interactions such as these have been established for communities of protozoa grown in various compositions in laboratory experiments Lawler 1993 Oecologia 93184190 Why algal and ciliate microbial communities are often used for examples of community level NH VV L V 3 V I NH VV L V studies of species interactions between microbes A B C They can be identi ed to species This can be done microscopically Bacteria have few general morphologies presently there is no good way to go into an environment and identify all of the bacterial species The scale at which interactions occur may be important as we saw with microscale physics 75 BIOL 687 Spring 2005 Microbe Microbe Interactions lectures Therefore the arena of algae and ciliates is at least somewhat similar to that of bacteria and lets us explore if general ecological principles can be extended from large scale terrestrial and marine studies down to the microbial domain III Succession in Microbial communities it may be important in bioremediation release of genetically engineered organisms A Several ideas surround the concept of succession 1 The rst idea has to do with population growth curve dNdt rN1NK 2 Where N population size r growth rate and K carrying capacity Graph of growth is L V 3 V Lquot V O V l V as follows Initial part of graph shows the increase is dominated by r later on the population is dominated by K Organisms which are able to grow quickly are said to be r selected species those which are able to compete well when density dependent are said to be K species Note similar to log log stationary phase in batch culture Early in succession R species are said to prevail Tend to have high rates of growth and wide dispersal In the case of microorganisms these species often have desiccation resistant spores or the like Later in succession the species tend to be able to be good competitors and less good dispersers Community then evolves on an r to K continuum There are data that suggest that some bacterial species are generalists and others specialists i Bacteria isolated from streams upstream and downstream ii Upstream are generalists can use most any type of leaf material for growth in Those further down tend to specialize resources more stable competition more erce and habitat must be partitioned more fully Koetsier et al Freshwater Biol 377989 Studies on microbial community formation show that when microcosms are placed in the eld the rst species are those that have resistant forms In enclosed containers denitri cation leads to nitrogen depletion phosphorus continues to recycle and nitrogen Xing cyanobacteria take over K selected with regard to competitive ability for nitrogen In addition crustacean grazers come in and the cyanobacteria seem to be more resistant to grazing than are many other species of microalgae Succession can be important when considering sewage treatment Active sludge formed when part of the downstream community is reinoculated upstream The organisms then do not need to be so r selected can reach higher densities and more efficiently processes organic carbon See the handout on microbial populations in sewage p i 76 B 3 BIOL 687 Spring 2005 Microbe Microbe Interactions The idea of facilitation Often times after disturbance there is a time lag before organisms can come in and become established This time period is related to the degree of conditioning In intertidal communities solid surfaces must be colonized by bacteria and diatoms before the large macroalgae and attached invertebrates come in In sterile soils establishment of a fungal community often is required before some types of plants can become established In the high Arctic the land is still rebounding from the ice age and new land is coming out form the ocean This land is colonized by Nostoc cyanobacteria containing lichens that develop the soils 6 This facilitation can lead to distinct sequences of observed microorganisms that are present Patterns of diversity during succession r V WM VV 3 V Lquot V V 1 What is diversity i Species richness ii Eveness iii Within and between habitat diversity 2 Initially there are few species because only the lucky ones have invaded a new habitat there are few well adapted r specialists 3 At intermediate levels there is maXimal diversity some r species left some K species established 4 At later levels of succession diversity may again decreases This is because the good competitors are able to eliminate all but a few other species 5 Denaturing gel electrophoresis of 16S rDNA showed bacteria attaching to glass slides increased diversity quickly for a week and then slowly over the neXt two or three months Jackson et al 2001 Ecology 82555566 6 Colonization sequences in arti cial streams suggest that these patterns hold for microbial communities as well as larger scale communities Initially small diatoms and bacteria are found on the substrates NeXt larger forms of algae colonize including greens and larger attached diatoms lamentous algae start to colonize at this point and diversity is maXimum Finally the lamentous green algae compete for light and become the dominants the total number of species decreases at this point 7 This has been demonstrated for a number of bacterial species following injury in cactus tissue Foster amp Fogelman 1994 AEM 60619625 8 Colonization by protozoa in plastic pools shows that there is a distinct successional sequence in the types of ciliates found at each successional stage 9 See handout for periphyton succession Founder effects Sometimes the species that are in a community rst can have a strong effect on the community that develops later Soybean seeds when planted with a bacterium that lowers rate of fungal infection Bacillus cereus Communities of bacteria signi cantly different after time than seeds planted without the bacteria This happened even when the original bacterium was not still present Gilbert et al 1993 Ecology 74840854 Intermediate disturbance hypothesis and microbial communities Intermediate disturbance hypothesis suggests that there is an maXimum diversity at intermediate levels of disturbance 77 B gt11 BIOL 687 Spring 2005 Microbe Microbe Interactions This idea is tied to the concept of succession At high levels of disturbance there is essentially all new habitat everything is kept at early successional states At intermediate levels succession is at the transitional state At low levels of disturbance the competitive dominants take over and lower the community diversity Microcosms 200 ml water were placed outside and allowed to colonize Disturbance was a 50 dilution of the community with deionized water Given various rates of dilution at high and low dilution rates diversity was lower than at intermediate dilution rates ie about 1 week Arti cial stream experiments Grazing by snails can be thought of as disturbance At high populations of snails the diversity of the algae was low as was biomass the biomass there was highly productive but not enough to have high overall system productivity At intermediate levels of grazing biomass was intermediate and diversity and productivity were at a maximum With low grazing biomass was maximum production was high but ef ciency was low and diversity was lower than at intermediate levels of grazing Grazing and wind disturbance have both been demonstrated to cause an increase in phytoplankton diversity in lakes Holzmann 1993 Hydrobiologia 249101109 In another study microbial communities were disturbed with 245T1ichlorophenoxyacetic acid oil and gasoline Used the amount of DNA reassociation to estimate diversity Dissomate DNA Label 12 bind other half to a lter pass the labeled half through lter label that sticks to the lter is the reassociated DNA These disturbances lowered diversity but the organism that were left were more resistant to future disturbances Atlas et al 1991 Microb Ecol 22 249 256 Not all disturbances increase diversity Bej et al 1991 FEMS Microbiology Ecology 86169176 altered soil microcosms by adding genetically engineered Psuedomonas capacia Used same technique DNA reannealment and showed this disturbance increased diversity Similar patterns with production Highest diversity at moderately enriched habitats Kassen et al 2000 Nature 406508512 Island BioGeography in microbial communities MacArthur and Wilson published ideas of island biogeography early in the 1960 s Have been subsequently studied heavily in macroscale ecological communities The idea has been extended to apply to islands of habitat ie ponds in land forest in prairie etc in addition to true islands 1 Initial observation As the size of the island increases the number of species increases SCA2 S species C constant 2 The number of species present is a function of immigration and extinction Far away islands have a lower immigration rate and thus less species 78 VI B BIOL 687 Spring 2005 Microbe Microbe Interactions 3 Small islands have greater extinction and less immigration thus less species Does island biogeography work in microbial communities Some investigators have the point of View that all microbes can be found everywhere you only need the appropriate enrichment medium to get them to grow Many support this view in that huge numbers of microbes make it so new colonists are always available Finlay 2002 Science 29610611063 They are not common except in hot springs and cannot be grown with the appropriate medium unless the environmental sample was taken from the hot spring Alternatively there are huge numbers of bacteria and the common types are probably widely distributed It is probably always possible to isolate a denitrifying organism from most any environment Fenchel and Finlay 2004 Bioscience 54777784 Whitaker et al 2003 Science 301 976978 presentation If everything if everywhere then there is no chance for the principles to extend to microbial communities Ruth Patrick has put glass plates of various sizes in streams in different parts of the world and has shown that there is a de nite relationship between number of diatom species and size of the substrate Paul McCormick used wading pools with various barriers to dispersal of protozoa different sized meshes which kept out large mammals all animals or animals and insects and showed that lowering the numbers of these dispersal agents lowered the number of species Basically made the islands farther away Also there were more r species that stayed competitive in the systems where most all dispersal agents were excluded Organism interactions became most important in the pools where all dispersers were allowed to enter suggesting a later successional stage N V L V 3 V Endosymbiosis of protozoa may be mutualistic may not Algal endosymbiosis 1 Paramecium bursaria often contains algal endosymbionts 2 The alga provides photosynthate the ciliate protection and nutrients from ingested particles 3 In dark the algal endosymbiont is often not present but reinfection will reestablish when light increases again 4 The Paramecium feeds faster in the light than in the dark see handout This is probably because there is more energy available The endosymbiont free Paramecium cells feeding rates were independent of the light 5 Perspira ovum a ciliate eats Eugleria It sequesters chloroplasts mitochondria and paramylon Occurs in low enrichment Oz from photosynthesis may stimulate metabolism 6 Dino agellates are always taking up small photosynthetic organisms They have coopted a number of different types of what are functionally chloroplasts and exhibit almost all known pigment systems of algae This is a nightmare for taxonomists who rely upon pigments 7 The diatom Epipihemia often harbors cyanobacteria that x nitrogen why low oxygen I don t know The ciliate M eiopus papaeformis harbors methanogens 1 This is an facultative anaerobic ciliate found in land lls 2 When water is not present it spends much of its time encysted The methanogenic bacteria 79 O V 9 V BIOL 687 Spring 2005 Microbe Microbe Interactions remain viable inside the cyst These ciliates may be responsible for a sizeable proportion of methane generation in land lls The ciliate receives no measurable energetic advantage from the endosymbiont The methanogenic bacteria appear to be protected from exposure to oxygen inside the ciliate whereas they would die rapidly upon exposure to oxygen Oz levels in the ciliate are presumably low The ciliate may represent a signi cant dispersal advantage to the methanogenic bacteria inside A gutless marine oligocheate has sulfate reducing bacteria that apparently use organic C or H2 that the worms can t to and respire producing sul de The sul de is used by other sulfur oxidizing bacteria to produce energy and organic C that the worms can use Dublilier et al 2001 Nature 411298302 Many new protozoa were found in the Santa Barbara Basin an anoxic depression at about 500 m off the coast of California 1 Many of there protozoa have Endosymbiotic or exosymbiotic bacteria or other organisms 2 of 28 species described half were new species and only one did not have symbionts Bernhard et al 2000 Nature 4037780 Legionella pneumophila and 30 related species infect 15 or more species of protozoa and replicate there The bacterium is dormant under nutrient rich conditions but causes cell deaths under low nutrient conditions Allows survival and growth in air conditioners cause aerosols to form which leads to human infection Harb and Kwaik 2000 66609616 3 4 5 6 V 80 BIOL 687 Spring 2005 Interactions with Plants Microbial Ecology Topic 16 Interactions with Plants I A V 9 Mycorrhizae relationships between plant roots and fungi Most plants have some type of mycorrhizial association For one grassland it was estimated that 96 of effective root length was mycorrhizial For a tropical forest 97 of the plants had mycorrhizial fungi associated with their roots May consist of the second largest pool of biomass in many terrestrial systems plants number 1 However since biomass doesn t equal productivity they may processes more carbon per unit time than plants Found in all habitats where there are plants including aquatic desert Arctic rain forests Biogeographical and fossil evidence suggest that mycorrhizae were associated with some of the earliest land plants thus this relationship may have helped plants successfully colonize the land Two general types ectomycorrhizae and endomycorhizae 1 2 Ectomycorrhizae surround the roots when the root is completely ensheathed it is called erecoid ie the ericaceae Endo mycorrhizae are also called vesicular arbuscular VA Penetrate the cell wall but not the plasmalemma See handout for distribution across landscape Some species of fungi are generalist others are only associated with one species or genus of plants Bene ts of the interaction for the plant 2 l V so V Increases root area of the plant Can transport nutrients to the plant primarily N and P have been studied 39 Thought to be most important in P transport Even species with Rhizobium have been shown to have mycorrhizae and in N poor environments the nitrogen xing bacteria associated with the roots are not able to provide all the needed nitrogen That the fungi can provide more by stripping it more ef ciently from the soil This makes sense because it may take quite a bit of energy to maintain active nitrogen xation and additional nitrogen that does not need to be xed may result in energy savings iii May transport nutrients from plant to plant If one plant is labeled with 32P that the label shows up in the neighboring plant much more rapidly than can be accounted for by diffusion through the soil Strange situation By increasing root area increases the ability to compete for water in dry habitats Increasing the root area increases the stability of the substrate Certain dune grasses cannot stabilize dunes Without their mycorrhizae May make antibiotics which discourage pathogenic bacteria May improve competitive ability of plants i it has been 39 39 that 39 39 1 between plants which are done in a greenhouse Without mycorrhizae can give an opposite result than those using the same plant species in the eld with their mycorrhizae ii Mycorrhizal fungi associated with heather inhibit growth of pine and birch This may partially explain Why the heat moors ie Scotland do not become forested over time Some saprophytic plants heterotrophic such as certain species of orchids rely upon the fungal partner to scavenge carbon Transplanting operations in many parts of the world have long realized that introduced species of plants may not be successful Without concurrent inoculation with mycorrhizae i Orchid culturists have long since early 1900 s been aware of this ii Planting fast growing hardwoods in Australia Rhodesia and Caribbean has been made possible by inoculating with plant s mycorrhizae 30 a H 81 m V gt11 V 53 3C V BIOL 687 Spring 2005 Interactions with Plants iii Trees planted as windbreaks in grasslands in North America and Ukraine have required their mycorrhizae iv Needed for trees to recolonize mount St Helens and during strip mine reclamation Bene ts to the fungi 1 Mainly increased carbon The plant translocates carbon to the roots and the fungi lives on t 39s Succession occurs with mycorrhizal communities 1 Early on facultative species are only one presents 2 Later the obligate species move in Transport of the mycorrhizae 1 Hyphal growth quite slow and can account for little dispersal 2 Some produce sporocarps mushrooms truf es which are eaten by animals and the spores are spread this way If a squirrel takes a mushroom to a tree to eat then the spores are quite likely to be dispersed a greater distance by wind than if they had just been released on the ground Some spores are released but wont go far inside the air ow boundary Some spores can also survive passage through guts When reforesting Douglas r stands in the Paci c Northwest the old practice was to poison the rodents so they would not eat the new r seedlings It turned out that growth was slower over all with this practice because the chipmunks were reinoculating the disturbed soils with mycorrhizae handout p Helper bacteria and mycorrhizal interactions Garbaye 1994 New Phytol 128197210 1 Some bacteria promote formation of mycorrhizal association Douglas r documented example 2 Mechanisms not known in some cases ltrate from bacterial culture medium can have the same effect 3 Other bacteria inhibit the mycorrhizal fungi Association with above ground structures Complex aggregate containing bacteria Archaea and Eukarya Andrew and Harris 2000 Ann Rev Phytopathol 38145180 Microbial association with leaf surfaces 1 Requirements i Survive in a low nitrogen environment ii Need holdfasts or slimes to stay on iii Extracellular enzymes common for breaking down the leaf cuticle 2 Some may hang out there waiting for the leaf to die or be injured and then take advantage of the newly available carbon 3 Pseudomonas common on many leaf surfaces i Contains a protein which enhances the formation of ice crystals at higher temperature ii Probably is bene cial to the bacterium because the plant becomes damaged more easily and can then provide more food ie leaks organics etc iii Genetically engineered a strain of this bacteria which lacks the ice nucleating gene When the new strain is put on plant leaves they are more resistant to frost damage Thought this would be a good way to project plants One of the rst controversies over release of a genetically engineered microorganism into the natural environment After much ghting the bacterium was released in controlled eld trials The bacterium did what it is supposed to except it was outcompeted by the wild organisms rather rapidly iv Longterm environmental effects of releasing massive number of iceminus mutants are unknown 21 Large numbers of dried up Pseudomonas is oating around in the atmosphere some A A 333 A amp e 82 H BIOL 687 Spring 2005 Interactions with Plants content that it is an important site for ice particle nucleation in clouds and plays a vital role in encouraging precipitation Will vast amounts of applied bacteria change this balance b If the organisms are routinely applied there may be a northward extension of weedy coldintolerant species as we K Cactus and yeast A l V N V L V Lquot V 6 species of cactus in the Sonoran desert are involved 5 species of Drosophila that feed upon yeasts in the cacti 1 on 1 cactus 2 on 2 other species and 2 on the remaining three Each species of y has its preferred species of yeast and several species of yeast are endemic to a single species of cactus The ies inoculate the yeast to the cacti and then eat them The species of yeast have different abilities to break down different compounds and are thus speci c to certain parts of each cactus In this way several species of ies can rely upon the yeasts found on the same cactus without actually competing with each other Gives an idea of the degree of evolution which can occur as a relationship of plantmicrobe animal interactions To make this even more complicated some of the species of yeast are toxic to others This is to avoid competition between them so they can not cooccur Ganter and Stamer 1992 Ecology 735467 Asymptomatic infections of plant leaves by fungi l 2 3 V 3 V Many species of plants have fungi growing in the leaves When Douglas r leaves are cured of their fungi they show no different growth rates than those seedlings infected with the fungi Health of the plants does not necessarily correlate with the presence of the fungi they are just there Probably live upon the organic material which would leak out between the cells anyway Produce some antibiotics so they may prevent infection by pathogenic bacteria or by other disease causing fungi Association with nitrogen xing microorganisms Legumes and Rhizobia 1 L V 3 V A number of economically important and ecosystem level important legumes have a mutualistic interaction with Rhizobia ie peas alfalfa This is an interaction where the plant provides nutrients and xed carbon and the bacterium xes nitrogen for the plant Free living Rhizobia occur in soils and are not numerically dominant generally also can not x nitrogen under aerobic conditions When they infect plant roots they induce formation of a nodule and are able to x nitrogen i There is a speci c chemical interaction between the Rhizobia and the root hair with chemotaxis to the root before infection and speci c chemicals excreted by the root are metabolized by the Rhizobia that initiate root binding to the bacterium and nodule formation The gene for this protein has been identi ed Mylona et al 1995 The Plant Cell 7869885 May be effective at 10quot12 M ii A minimum number of Rhizobia need to be present in the soil for nodulation to occur iii Different species of Rhizobia are found in different types of plants iv Plants supply leghaemoglobin to the nodule This molecule has a relatively high affinity for Oz so there are low enough oxygen tensions to allow for nitrogen xation but sufficient oxygen present to allow for respiration to occur in the Rhizobia cells Rhizobia has Oz detector protein on cell membrane When Oz concentration is low enough if gene expressed 83 111 B C BIOL 687 Spring 2005 Interactions with Plants Actinomycetes 1 Interaction which occurs between the prokaryotic F rankia and several genera of plants including alder and bayberry 2 The bacterium forms mycelial like structures so was rst thought to be a fungus later shown to be a bacterium 3 Nodules develop but not as advanced as in the legumes 4 Alder also provides a low oxygen environment with chemicals similar to leghaemoglobins 5 Quite important source of soil nitrogen in many high latitude areas 6 Castle Lake in California showed an interesting pattern When ammonium was added there was stimulation of productivity but when nitrate was added there was none Figured out that molybdenum was limiting and nitrate reductase could not be synthesized requires Mo as a cofactor Hypothesized that the extensive alder populations in the watershed had Frankia that successfully removed much of the Mo before it reached the lake Cyanobacteria 1 Anabaena Azolla symbiosis i The water femAzolla entraps Anabaena azollae in small pouches as it grows ii Alga provides xed nitrogen plant provides xed carbon sucrose to the alga iii This is an agriculturally important interaction The fern is cultivated in some rice elds in Asia As it grows it xes nitrogen and before the eld is planted it is drained and the deadAzolla emiches the soil with nitrogen This may be functionally equivalent to rotating legumes into elds in temperate agriculture 2 Cyanobacteria in rice paddies also associate with the green alga Chara and this may be important as well Ariosa et al 2004 AEM 7053915397 presentation 3 Wheat roots associate with Nostoc it is attracted to the roots and the plants grow better with it than without No real specialized structures work by Gantar et al New Phytol 4 Curious story about brain disease of old men in Guam The degenerative brain disease was traced to the practice of eating whole fruit bats The fruit bats feed on the fruits of cycads The cycads have endosymbiotic Nostoc The Nostoc produces a toxin that is concentrated by he fruit and even more by the bat Now that eating the bats is banned the disease is not occurring Holzman 2004 ASM news 70354355 Lichens A very ecologically important association between fungi and algae These are the rst colonizers of harsh terrestrial environment but they are also found in some aquatic environments Form 17000 or more species 1 Only several species groups of algae most of the differentiation appears to be a result of different species of fungi 2 The fungi called the mycobiont and alga called the phycobiont 3 Classi cation based on super cial morphological characteristics Foliose lichens 84 BIOL 687 Spring 2005 Interactions with Plants D Crustose lichens E Fruticose lichens F Only several genera of algae associated with lichens Green algae most common with 50 from the genera T rebouxia and Pseudotrebouxia Also T rentapholia and the Cyanobacteria Nostoc G Bene ts to fungi 1 Algae r39 39 N V Transfer r39 39 to fungi ribitol sorbitol or erythritol acyclic polyhydrly alcohols also called polyols Fungi convert to mannitol The alga excretes from 2040 of its photosynthate to the lichen This excretion stops as soon as the alga is removed from the lichen i Either connect to the alga by closely apressing hyphae to the algae cells apressoria or invading the algal cells haustoria Fungi which contain Nostoc get nitrogen from the alga which may be useful in low nitrogen environments i Lichens contain Nostoc in specialized structures called cephalodia these lichens have green algae as well ii Relatively little photosynthesis occurs in cephalodia lichen only uses these Nostoc as a source of nitrogen iii In a few species of lichen there is only Nostoc in these species both nitrogen and carbon is xed by the Nostoc iv The Nostoc are induced to from more heterocysts heterocysts frequency up from 4 top about 20 v Glutamate synthetase activity is inhibited in the Nostoc and they thus can not assimilate ammonium well They leak the ammonium and the fungi assimilates it H Bene ts to alga 1 Receive protection from desiccation Can survive in environments where they would not 85 VI gt lt V lt3 9393 Q m V BIOL 687 Spring 2005 Interactions with Plants make it in otherwise The excreted polyols are stored in the lichen thallus up to 10 of the weight can be polyols These polyols seem to protect the lichen from desiccation Large amounts of them leach out when they are rained upon 2 The lichens are able to take up nutrients from the atmosphere very effectively Tend to concentrate all of the incoming elements May be able to nd nutrition more readily than the freeliving algal cells would i This is bad for the lichen in places where there is air pollution ii Concentrates lead and other heavy metals the diversity of lichens is inversely related to the population and pollution levels in an area May be used as an indicator of pollution Scientists have also used them to collect pollution to prove that heavy metal contamination is occurring 3 Lichens often produce antimicrobial agents there may be some protection from negative interactions with bacteria but this has not been established 4 Fungi may produce vitamins Gimmler 2001 Progress in Botany 62194214 5 Lichens may aid in dispersal of the alga The nitrogen xing containing fungi can be very important in terrestrial nutrient cycles 1 The lichen Lobaria oregania reaches high biomass on old growth Douglas r trees A large portion of the nitrogen income to the forest oor for the growth of the trees comes from nitrogen leached from the foliose lichen which contains Nostoc 2 Home has shown that lichens in the Antarctica may be very important in initial soil formation because nitrogen is very limiting in this environment Experimentation with the lichens has proved to be very dif cult The phycobiont and the mycobiont can be cultured separately but the lichen itself grows very slowly 1 Growth rates are on the order of less than one doubling per year 2 People that reconstitute lichens in the laboratory have to run their cultures for years before they get a reasonable amount of growth Lichen reproduction 1 Vegetative reproduction occurs through soredia The thallus of the lichens and gives of balls with the mycobiont wrapped around the phycobiont Gene transfer with crown gall formation A grobacterium Interesting interaction from a biotechnological point of view Bacterium enters the host plant through the roots and causes formation of a tumorous gall This tumor is formed because the bacterium transfers a plasmid to the plant roots 1 The plasmid induces the plant to synthesize the amino acid derivatives octopine and nopaline which are then can be utilized by the bacterium as a sole carbon source 2 Also make the plants auxin de cient so they form tumors 3 May have other effects Currently a large effort to see if the bacterium can be used to insert economically useful genes into plants Data indicate that this mechanism was involved early in the evolution of the genus Nicotina bacterial genes are common in many members of the genus Nutrient remineralization In most systems the microbial community regenerates nutrients for plants to rely upon In terrestrial systems all nutrients would be buried in the form of complex organic molecules if it were not for remineralization In aquatic systems it would all be buried in the sediments or present as dissolved organics in the water column Disease of bacteria and fungi is extremely important in many plant communities Mainly studied 86 BIOL 687 Spring 2005 Interactions with Plants in the context of plant pathology and with domestic plant species See readings in teXt on this subject 87 BIOL 687 Spring 2005 Interactions with animals Microbial Ecology Lecture 17 Microbe Animal Interactions I Intestinal microorganisms A Rumen note course on this N V L V Lquot V Numerous types of large animals rely upon intestinal microorganisms in a special invagination off of their digestive system to help processes plant materials No mammals are known to produce cellulase Bacteria handout p i Mostly strict anaerobes ii Up to 1011 per ml 106 is typical of natural waters iii Bacterial isolates include gram gram bacteria and archaebacteria iv Processes by bacteria include methanogenesis plant cell wall digestion nitrogen xation ethanol utilization sulfate reduction proteolysis and lactic acid utilization v One of most common Bacteroides rumim cola 16 mm long extremely complex nutritional chemistry Ciliate protozoa i Mostly strict anaerobes most never cultured ii Can take up soluble compounds but primarily prey upon bacteria iii Have cellulase and can process particles of plant material but bacteria probably generally more important iv Many species present that differ with time and type of organisms v It has been controversial if the ciliate protozoa are bene cial or not a Organisms can be cured of their protozoa with chemicals or grown isolated so they get little gut fauna b in some studies there is a positive effect on animal growth in others there is a negative effect Appears to be a function of the animal diet etc Fungi i Yeast and aerobic fungi have been isolated but are probably transient and not important ii Anaerobic fungi have been cultured recently which may be of importance a Strictly anaerobic h Only found in ruminants never in other habitats c Found so far in sheep cattle deer impala musk ox kangaroo goat reindeer elephant horse and rhinoceros iii Originally thought to be agellates because much of the population occurs in the form of quadri agellate zoospores Upon culture these organisms assume a growth form similar to the aquatic hyphomycetes iv The number of these fungi seems to increase with high ber diet falling off to near 0 with low ber diets Establishment of a gut ora i Experiments with sheep show that a strictly anaerobic ora starts to appear by 2 days but there are abundant facultative anaerobes present at this time The facultative anaerobes become less important over the next 4 months By day 4 cellulitic bacteria are present a form of succession ii Lots of different routes Mothers cleaning young ingesting material which has saliva or fecal material on it airborne water droplets iii No resistant forms or cysts have been reported for the protozoa bacteria seem more resistant and to be passed more easily iv Diet determines what the nal stable composition is v An experiment was done with bacteriafree mice and Bacterioides thetaiotaomicmn Gene expression in mice was determined by analyzing mRNA 10 days after addition and 88 BIOL 687 Spring 2005 Interactions with animals comparing to uncolonized mice The bacteria changed expression of genes involved with nutrient absorption mucossal barrier forti cation and potential intestinal mutation Suggests evolutionary adaptation to interaction Hooper et al 2001 Science 291 881 884 6 Diurnal variation in gut ora i Protozoa in sheep fed one time per day show a peak at feeding time then gradually decrease until 4 h before the next feeding time ii Different animals show different cycles for bacteria and protozoa iii Appears to be a function of dilution rate saliva drinking and nutrient input eating Compartmentation within the rumen i Not just a homogenous bag there are several distinct habitats within the rumen ii The liquid contents form the rst type of environment Most studied but with a lower relative biomass than the other habitats The liquid functions to move the solids around and as a source of inocula for newly entered solids iii Large particles and tightly attached microbes make up the next type of habitat The microbes can actually be attached Mean residence of solid food in rumen is probably 2 3 days iv Loosely attached microbes shuttle between the solid and liquid phases v Along the rumen wall there is a small oxygenated habitat with a very distinct bacterial ora vi Foam at the top of the rumen may comprise yet another distinct habitat 8 The fact that we feed cattle berpoor food may make them more susceptible to diseases N V 3 V V because it disrupts their normal gut ora Then there is more of a need to use antibiotics in feed which causes its own problems Russell and Rychlik 2001 Science 292 1 1191 131 Termite guts Termites are probably the largest single consumers of plant biomass especially in tropical areas where their activities can dominate nutrient cycling and decomposition Complex populations of internal gut organisms Have an enlarged hindgut with large microbial populations has high pH and very low redox 230 to 270 mV Many species of the hindgut are obligate anaerobes i Some species of bacteria ii Some species of protozoa are found which are found no where else in nature these species are able to ingest and digest wood particles iii Higher orders of termites contain bacteria alone recent evidence suggests that fungi that degrade lignin are also present Hyodo et al 2000 Soil Biol Biochemistry 32653658 Complex series of microhabitats occur in termite guts directly related to carbon ow Brune and Friedrich 2000 Current Opinion in Microbiology 3262269 Contain protozoa that excrete cellulase and allow for the digestion of wood The protozoa seem to rely upon the bacteria i When the termites are cured of their bacteria by antibiotics so only protozoa remain the protozoa gradually die out 89 11 gt11 V BIOL 687 Spring 2005 Interactions with animals ii If the cured termites are reinoculated with their native bacterial ora the protozoa can stay alive iii It appears that the protozoa are excreting partially digested cellulose the bacteria live on these compounds and the protozoa in turn eat the bacteria Not only do the bacteria provide vital growth factors but their high respiration rates insure anaerobic conditions for the oxygen sensitive protozoa Fermentative processes in the hindgut result in formation of C02 H2 and acetate The termites utilize the acetate Spirochetes use H2 COZ and form acetate This competes well for the H2 that would be used by methanogens Important because methane is not useful to the termites but acetate is Ledbetter et al 1999 Science 283686689 Methane is emitted from termites Methanogens do quite well in the highly anaerobic conditions As discussed methanogens do quite well and the typical termite releases methane but methylotrophic bacteria living in the mounds trap much of the methane The hydrogen emitted form the ferrnentative processes will promote the methanogenesis 7 Some contain nitrogen xing microorganisms spirochetes which provide nitrogen Wood is a fairly nitrogen poor food source 00301 N this allows the termites to grow more efficiently Lilbum et al 2001 Science 29224952498 Insect guts have a large and varied nonpathogenic assemblage of bacteria Dillon and Dillon 2004 Ann rev Entom 497192 presentation Cockroach guts 1 Contain diverse microbial populations 2 Estimated that acetate and lactate production by the microbes accounted for up to 14 of the carbon taken from food by cockroaches 3 When fed high ber diets methanogens produce large amounts of methane in the gut causing atulent cockroaches 4 The bacterial species present have been shown to change with the diet feed to the insects Flea guts and Y ersinia pestis 1 Bubonic plague 2 Has a plasmid encoded phospholipase 3 Allows it to survive in ea guts without being digested Hinnebusch et al 2002 Science 296733739 4 Probably many endosymbionts have similar adaptations Earthworm guts have N20 producing bacteria and might be an important source of nitrous oxide Horn et al 2003 AEM 6916621669 presentation Insects are the most diverse group of organisms on earth supposedly However if each insect has Lquot V O V at least one unique endosymbiont there are more species of microbes A B C V Likely important in guts About 10 of insect species have speci c parts of their bodies that appear to function as places to house bacteria Such endosymbionts may be mutualists and allow the hosts to utilize specialized narrow diets such as plant sap from one species or blood of one vertebrate Moran and Telang 1998 Biosience 48295304 Analyses of such symbionts suggest that they diverge evolutionarily from related species Furthermore since the strains are descended from few individuals and passed to offspring a few individual bacteria at a time they diverge more rapidly than many other bacterial species Lamber and Moran 1998 PNAS 9544584462 However extreme evolutionary stasis 2000 times less than E 001139 has been documented in an aphid endosymbiont Tamas et al 2002 Science 2962376 90 III A B V O V m V gt11 V 53 BIOL 687 Spring 2005 Interactions with animals Corals The largest biogenic structure present in the world today the great barrier reel is a result of corals Dino agellates primarily Gymnodinium microadriaticum are the algal part of the mutualism zooxanthella There is some controversy if it is one species or a number of species since it occurs in a wide variety of different corals from throughout the world Chemical analysis by electrophoresis reveals several different types of zooxanthallae so morphology is not the only criteria for species Structure general as follow Note that many different species of animals are called corals so the structure varies considerably Algae photosynthesize and provide a wide range of carbon compounds The rate of photosynthesis is directly related to the deposition of calcium carbonate for structural properties The alga has also been shown to take up nutrients form the surrounding water The polyp extends toward light to maximize photosynthesis The coral can take up particles from the surrounding water providing organic carbon and nutrient Synthesis of the calcium bicarbonate wall also provides protection from grazing Very few organisms are able to graze corals Archaea are abundant and widespread on corals Wegley et al 2004 Mar Ecol Prog Series 2738996 Kellog 2004 Mar Ecol Prog Series 2738188 Recently been determined that there are also symbiotic cyanobacteria associated with some corals that x nitrogen Lesser et al 2004 Science 3059971000 presentation Sponges often part of coral reefs One species T ethya seychellensis has green algae deep in its body It has siliceous spicules that serve as light pipes to the algae in the sponge Gaino amp Sara 1994 Mar Ecol Prog Ser 108147151 Disease important but not very interesting to me unless I get one Fungal insect interactions Harvester ants Dixon 2004 ASM news 70388389 Take leaves off of trees and return to burrow Chew up leaves and inoculate with fungi from a sac near the anus When they eat the fungi they do not digest their enzymes that break down plant tissue so when they inoculate new leaves the enzymes quickly start breaking down the leaves Wait unit the fungal hyphae grow and eat the fungi Tend the gardens in their den to keep other species of bacteria and fungi from growing Encourage Streptomyces that produce antibiotics that lower the growth of undesirable fungi Queens take a ball of fungus to start a new colony 8 If the ants are removed the other species rapidly overgrow the harvested leaves Beetles 1 Ambrosia beetles live in live wood and cultivate fungi i Each species usually only cultivates a single species of fungus there are several fungal general involve IQMb VVVV 91 VI BIOL 687 Spring 2005 Interactions with animals ii Sequesters fungi in a specialized organ and inoculates wood from this organ as it bores a hole in the wood iii The beetles keep the holes clean and open and close the hole to maintain humidity close to the optimum for the fungi iv The ambrosia beetles also secrete an antimicrobial compound which keeps competing fungi down Without the beetle the fungi are rapidly overgrown v The beetle eats the fungi it is not able to break down cellulose on its own 2 Bark beetles also move blue stain fungi into pine trees Can cause massive mortality in Rocky Mountain pine forests i The fungi not only can break down cellulose and may be eaten by the beetle but it also kills the tree ii The rapid senescence of the tree wilting makes it so it cannot produce very much sap Beetles cannot establish in a healthy tree because the sap ushes them out The fungal infection makes the tree weak and allows the beetles to colonize the entire tree Some termites also cultivate fungi 1 Break down cellulose and tannins 2 Inoculate wood 3 Generally do not have the complex gut protozoan populations associated with some of the less evolutionarily advanced species of termites Scale insects The fungi Septobasidium and the scale insects are often found in association infection rates of the insect by the fungi run about 50 The insects derive nutrition by sucking plant juices the fungi get bene t from eating some of the scale insects The young insects are motile as they mature they become sedentary and loose everything but genitals and sucking apparatus straws with genitalia The scale insects suffer heavy predation by parasitoid wasps The fungal mycelia form an dense mat over the scale insects so the insects that are not infected are protected from predation by the fungi The fungi probably also lowers rates of predation by birds and frost damage Young infected insects disperse the fungi to different trees Infected individuals become females those without the fungi become males The method of control is not known Predatory fungi Fungi can trap nematodes or rotifers 1 Nematodes i Many species eat yeasts attracted to organic esters released by the yeasts the nematode trapping fungi release these compounds to attract the nematodes ii When grown in the absence of the nematodes some species of fungi do not make traps so they appear to be able to sense the presence of nematodes iii Adhesive branches stick to them as they crawl by b N V V V 3 V 5 6 iv Lethal lollipop knob with adhesive end 92 V11 3 BIOL 687 Spring 2005 Interactions with animals v Ring when the nematode crawls in there is a sudden osmotic swelling and the nematode is trapped vi With all of these predators the fungi then proceed to grow inside of the nematode and kill it getting nutrient from the nematode 2 Similar adhesive structures not rings trap rotifers Light producing bacteria Four genera responsible for most bacterial luminescence Beneckea Photobacterium Vibrio and X enorhab dus 1 Produce light by a luciferase catalyzed oxidation of reduced avin mononucleotide FMNHZ Oxidized using molecular oxygen and concomitant oxidation of a long chain aliphatic aldehyde 2 May be useful to free living species because the enzyme has an extremely strong af nity for oxygen can be used to shunt electrons from reduced substrates via avin to oxygen Under low Oz conditions can be a useful metabolic characteristic need to obtain an electron balance during fermentative metabolism 3 Not clear why freeliving bacteria would emit light however may be related to ecological roles as decomposers use to attract scavengers to a rotting food source and subsequently disperse the organisms 4 For very bright luminescence to occur cell concentration needs to be quit high Induction of luminescence probably involves an inducer which accumulates in the medium autoinduction For the most part freeliving bacteria only luminesce at low levels 5 Freeliving luminescent bacteria can occur in ocean at ranges from 103 to 106 per L 6 Requires about 104 to 105 bacteria per mm2 to visualize them 7 Numbers are far greater associated with the surface of most marine organisms A swap from the surface of most organisms will yield signi cant numbers of colonies 8 Meat is susceptible to overgrowth by luminous bacteria although the number of reports of this is less than it used to be probably because refrigerators have lights on them Common in the guts of many marine animals probably quantitatively the most important habitat 1 Found in shrimp mussels craps scallops sh and squid among others Found in many coelenterates including y39 39 39 and l 1 May assist in protection from predation light scares away predators 2 All species of marine luminous bacteria produce extracellular chitinase and thus probably aid the digestion of food 3 Species have been shown to be able to pass unharmed through sh guts 4 Cod and shrimp can produce luminous fecal pellets Luminous bacteria have been shown to infect the tissues of many marine organisms as parasites and also mole crickets hay ies ants woodlice and millipedes 1 Suggest that the colonies form on the rotting carcasses of these organisms at high enough densities to glow attract carrion eaters and allow for dispersal by or infection of the scavenger Have been found to be symbiotic in the guts of a parasitic nematode 1 As a juvenile the nematode is accidentally ingested by a caterpillar and bores through the gut wall 2 The X enorhabdus then infects the caterpillar 3 The bacteria produces a compound which inhibits rotting of the caterpillar and allows the nematode to eat more the carcass 4 The carcass of the caterpillar may glow and be attractive to other organisms thus aiding dispersal of both of the nematode and bacteria 93 BIOL 687 Spring 2005 Interactions with animals 5 The nematode cannot successfully parasitize and kill a caterpillar without the bacteria and the bacteria cannot breach the gut wall without the nematode E Some species of squid sequester luminous bacteria in a light producing mantle Will only enter into relationships with speci c strains of Vibriofischeri as a juvenile Before infection the organ has a elaborate epithelial structure covered with cilia and microvilli to transfer the bacteria into the organ After the bacteria are established for only 12 h Boino and McFallNgai 1995 Biol Bull 189347355 the structure regresses and disappears in about 18 days In squid not exposed to suitable bacteria the structure stays Establishing the symbiosis is part of the normal developmental cycle of the squid At least 30 genera in 11 families of teleost shes have specialized light organs which contain the bacteria many other species generate their own light The bacteria is supplies with nutrients and protection the sh is supplied with light Light may be used to attract prey assist in escaping predation or communication Angler sh have a specialized lure containing luminescent bacteria and use it to attract smaller sh May use the light to communicate Can cause ashes by lowering a dark membrane over the pocket containing the bacterium Some sh have bacteria that make their ventral surface glow This may make them less susceptible to predation They are less apparent from below when there is downwelling light In the case of the pony sh a light organ is located deep in the body as a side pocket from the esophagus and the body of the sh is such that the light is dispersed downward through the sh to produce an even ventral glow G A unique species of Vibrio has been con rmed to be associated with several shes on the basis of 16s rRNA analysis This suggests that the endosymbiotic species are not always the same species within the genus and not necessarily the common ones found as planktonic bacteria Haggood and Distel 1993 Nature 363 1 541 56 VIII Symbiotic bacteria associated with a unque annelid that lives on the bones of dead whales Rouse et al 2004 Science 305668671 presentation IX Oxidation of NH4 and methane by intracellular bacteria found inside Siboglinum poseidom39 a small pogonophore found in mud A A small worm living in sediments have methanotrophic bacteria within some of their cells the bacteria can assimilate radiolabeled C02 or methane B C02 assimilation linked to oxidation of NHX Bacteria can also assimilate methane butane pentane and ethane The gasses are produced in the anaerobic muds so there is a continuous ux of the gasses The radioactivity is transferred form the bacteria to the worm X Sul de mining bivalves use foot to move bacteria to sul de Dufour and Felbeck Nature 42665 67 XI Bacteria isolated from the skin of sh may reduce drag When these bacteria were tested grown on surfaces in turbulent ow they reduced the drag Probably related to polymers that they excrete Bemadsky and Rosenberg 1992 Microb Ecol 246376 What type of interaction is this XII Bacteria attach to outside of some colonial ciliates Crustacea and nematodes that can oxidize sul de Polz et al 2000 ASM News 66531539 A The animals move into sul de rich anoxic zones and then back into oxic areas This allows the bacteria to oxidize sul de at rates much greater than would occur otherwise B The shrimp swim in and out of sul de rich plumes of waters from deep sea vents the nematodes move up and down into anoxic sediments C The animals eat the bacteria from their surfaces and gain nutrition XIII More weird stuff bees scavenge bacteria as they y through the air Lighthart et al 2000 Microbial Ecol 39314321 NH VV m4 L VV V 11 V me VV 3 V Lquot V 94 BIOL 687 Spring 2005 Interactions with animals A As they y through the air bacteria stick to bees by electrostatic charges B May move bacteria into hives and honey C May move genetically modi ed bacteria long distances 95 BIOL 687 Spring 2005 Unusual and Extreme Environments Microbial Ecology Topic 18 Unusual and Extreme Environments I Hotsprings A Physiological adaptations to life at high Nature 40910921101 1 Thermal energy causes large biological molecules to be less stable must maintain a certain degree of stability to function properly Lipids i As lipid become more liquid membranes start to loose their integrity ii Saturation of the lipids is high ie lots of single bonds few double bonds length of the chain increases and the number of methyl groups tends to increase the degree of branching tends to increase iii In archaebacteria there are actually covalent links between the lipids tetraethers so that the membrane is not actually a bilayer but actually a sheet iv The polar lipid head group often has carbohydrate sugars attached which may increase stability Proteins i As enzymes proteins transfer thermal energy to their active site to move molecules in speci c ways If they become to hot or to cold they are not able to maintain con rmation and deliver the right amount of energy to catalyze the reaction ii Increased levels of beta sheets and alpha heliX stabilize proteins More sul desul de bonds also increase stability iii These proteins have been used by biotechnologists to create PCR polymerase chain reaction and LCR ligase chain reaction a PCR takes a small amount of DNA and ampli es it by adding an enzymes speci c to a small primer region and nucleotides and going through repeated heating and cooling cycles The heat causes the DNA to split and a new complimentary chain to form against it b LCR uses speci c ligases and primers on both ends of the region of interest The 3 and 5 ends synthesize toward each other and when they reach the ligase interprets this as a nick in the DNA and binds them If there is no match on both sides of the target the entire piece of DNA is not synthesized The reaction is taken repeatedly from 65 to 95 C In both these cases the enzymes used must be thermostable to allow for the high temperatures of reactions These enzymes have been isolated cloned from thermophilic microorganisms to be used in these forms of biotechnology d PCR has been used to amplify DNA millions of years old from fossils to sense ma s active in cells at certain times and many other advances in molecular biology have come about with this technique Genetic apparatus i DNA and RNA both must be more stable at higher temperatures ii DNA may have more GC pairs why Actually this is true within groups but there are archaebacteria with low GC contents in the DNA so proteins may be more important in stabilization Kushner 1993 in Aquatic Microbiology iii mRNA has a tertiary structure that signals initiation of protein synthesis this must be more stable iv rRNA is the most studied De nitely has more stable structure There are speci c regions that must remain bound to keep structure These regions are more highly conserved in the hot springs organisms than in the mesophilic organisms v Has been hypothesized that rRNA structures are more likely to exhibit stability that DNA 1 eg 39 quotquotand 39 quot392001 2 V L V A n V 3 V 96 gt11 V BIOL 687 Spring 2005 Unusual and Extreme Environments taken from the entire genome because structure is so essential to function in this RNA Dixon et al 1992 J Mar Biol Assn UK 72519527 Algae are common in hot springs up to 75 C Cyanobacteria have highest temperature tolerance of the algae Some diatoms can survive up to 50 C Bacteria can survive up to boiling water handout p amp p Deep sea hydrothermal vents 1 L V 4 Volcanically in uenced water pours out below the sea at very high temperatures The extreme pressure does not allow for the water to boil There have been bacteria isolated up to about 150 C Claims have been made for life up to 250 C but these have not been substantiated The water is enriched in reduced compounds as it leaves the vents HZS may be the most important but reduced iron and manganese are also found i These reduced compounds can be combined with oxygen to yield energy Large populations of chemosynthetic organisms live this way ii Why is this mode of life not independent of photosynthesis iii Complex mechanisms to protect from high levels of sul de toxic to most organisms also important to organisms inhabiting anaerobic muds or at their interface see handout iv The waters are also emiched in metals These metals are coprecipitated with sul des in bacterially mediated interactions This has been shown to be a mechanism for concentration of sul des of silver arsenate and copper near the outfall of hydrothermal vents An entire community forms on the sea bottom i 10 foot long tube worms abound Riftia ii Have no mouth Entire gut is lined with large numbers of sulfur oxidizing bacteria see handout p iii Hemoglobin binds to oxygen different form to sul de brings both into the bacteria The bacteria oxidize the sul de and x C02 The worms subsist on the bacteria and their exudates Also may use nitrate to oxidize the sul de Hentschel amp Fulbeck 1993 Nature 366338340 iv Shown that thiosulfate will also bind to these hemoglobins but less so than sul de Childress et al 1991 Physiological Zoology 6414491470 Why is this adaptive v Clams or mussels with symbiotic microorganisms also do similar thing some also have methane oxidizers with both types of bacteria occurring in a single vacuole Fisher et al 1993 Mar Ecol 14277289 vi The tubeworrns are common to deep hotsprings at widely divergent areas of the ocean bottoms DNADNA hybridization of their bacterial endosymbionts not culturable suggests that they are the same also but different from the endosymbionts collected from clams Some but not all species of tube worms share the same species of endosymbiotic bacteria vii Crabs seastars and other scavengers congregate around these biological hotspots Have found an aracheal symbiont in deep sea vents i Associated with another Archael cell and can only grow with that organism ii Has very small cell size 04 pm and smallest known arehaeal genome 05 mgeabases iii Indicated by iv Pcr oerNA sequences Huber et al 2002 Nature 4176367 Hotsprings are attractive ecosystems to study because the high temperature makes the communities relatively simple Also the springs make for constant temperature and chemistry minimizing environmental variance G Studies of temperature maxima have shown that there are distinct maximum and peak 97 H E gt lt V 93 C BIOL 687 Spring 2005 Unusual and Extreme Environments temperatures of growth with even different strains of the same species partitioning the environment This leads to distinct gradations in the community as one goes down the temperature gradient See handout It is somewhat perplexing how hotsprings get colonized Most of the species do not have resting stages and there are few vectors Waterfowl and insects are often thought to transport freshwater species but these organisms cannot stand the extreme temperatures of hotsprings The springs are often upstream and separated by wide distances from each other Nonetheless some species of algae M asti gocladus are found throughout the world Many species yet to be cultivated Hot region for biotech companies Stetter 1995 ASM News 628 5290 Extreme cold Enzymes may be useful in biotechnology for food preparation and coldwater detergents Two types of cold microorganisms 1 Cold tolerant species are able to grow and tolerate freezing conditions 2 Psychrophylic species have optimal growth at about 10 but can grow lower Growth curves 3 At very low temperature 15 0 C growth requires relatively more substrate Weibe et al 1992 AEM 58359364 4 Some psychrophilic species can synthesize new more uid lipids upon exposure to cold White et al 2000 Antarctic Science 12386393 Marine Ice algae and associated community 1 Form in Arctic and Antarctic most are cold tolerant but a few species are psychrophilic 2 Limited by light and by temperature nutrients are often high in the ice 3 Some algae are just planktonic forms which are trapped in the ice others actively grow in the ice some species seem to thrive both in the plankton and in the ice in deep waters where there is little mixing from below it is a requirement to have some seed members of the planktonic communities held in the ice during the low light or dark winter Lizotte and Sullivan 1992 Polar Biol 12497502 Otherwise the algae will stop growing and sink If they are trapped in the ice the thawing in the summer reseeds the phytoplankton and allows relatively high rates of productivity This has been hypothesized to be one of the factors involved in establishing the high productivity of the polar seas even though there are extended periods of low light 4 Species mostly diatoms although greens and other species have been documented Bacteria also present some have has vacuoles highest concentrations associated with algal masses Gosink et al 1993 FEMS Microbiol Ecol 1028590 why 5 Some species of diatoms excrete carbohydrateprotein compexes that cause potting of ice and alter the surfaces Do not lower melting point however Raymond 2000 Polar Biol 23721 98 Q BIOL 687 Spring 2005 Unusual and Extreme Environments 729 6 19 of the total productivity in some polar marine areas may be a result of production by ice algae 7 Release large amount of bromoforrn CHBr3 may be important source of bromine to atmosphere where it is converted photochemically to bromine and reacts with ozone Sturges et al 1992 Nature 358660662 8 Often extremely adapted to low light high amounts of light collection pigments What photosynthetic parameter is high 9 The temperature they experience is often well below freezing as ice forms the salt concentrates creating brine channels which are several degrees C below 0 Consequently these ice algae also have to be tolerant of high salinity 10 Ice algae also found in high mountain lakes and in Antarctic lakes handout Recently bacteria found in ice above Lake Vostoc In 19741975 an airborne radioecho survey of Antarctic ice depths led to the discovery of a lake under the ice The ice sitting on the lake s surface is at relative to the surrounding ice sitting on land and remote satellite measurements of ice elevation have allowed determination of the size of the lake Kapitsa et al 1996 Estimates are that the lake covers about 15000 kmz is 125 m deep and rests below about 4 km ofice Preliminary calculations suggest that the residence time of the water in the lake is tens of thousands of years and that the lake basin is about 1 million years old However there is signi cant water exchange between the lake and the ice sheet Siegert et al 2000 Scientists have taken cores through the ice sheet to 3950 meters depth about 120 m above the lake The ice at 3310 m is about 420000 years old and was formed by refrozen lake water Jouzel et al 1999 Analyses of the refrozen lake water from the ice cores indicate the presence of a microbial community Priscu et al 1999 Karl et al 1999 and some of these bacteria may still be viable Karl et al 1999 Sampling the lake without contaminating it will be technically dif cult but is certain to yield interesting results Greenland glacier contains bacteria They can be grown and have been frozen for over 100000 years Sheridan et al 2003 AEM 6921532160 Unique organisms have been found in other Antarctic lakes that are not as deep under the ice Karr et al 2003 AEM 6949104914 presentation Snow algae Common in mountainous permanent snow elds throughout the world Aristotle noted pink snow elds the algal which causes it was rst recorded in 1835 and was also noted by Charles Darwin in snow elds of South America Most common species Chlamydomonas m39valz39s It has a red pigment astaxanthin which gives rise to the watermelon snow often seen in Rockies Cells tend to be concentrated on the surface but cells have been found up to 30 cm into the surface These algal cells at the surface may experience the highest light experienced by any alga The pigment probably protects from photoinhibition Motile stages germinate at the soil and swim up through the snow to the surface when light gets extremely high they swim back down to a depth at which they are not photoinhibited Nutrients provided by wind blown particles generally nutrients are low Chlamydomonas nivalis appears to be a psychrophile with reproduction only occurring below 4 C Algae x carbon leak some out so there is also a bacterial community There are mites which live only on the bacteria and algae Bacteria isolate that are active at subzero temperatures in small pockets of liquid water in Antarctic ice pack Carpenter et al 2000 Appl Environ Micro 6645144517 V N V L V 3 V V 2 O U J 1 VV V V V so V O V 99 III CB 92 3E 92 2 O V BIOL 687 Spring 2005 Unusual and Extreme Environments 10 Deep under arctic glaciers there is liquid water at very high pressures Bacteria active there including denitri ers and methanogens Skidmore et al 2000 Applied and Environ Microbiol 6632143320 Endolithic communities Desert crusts a number of organisms live within rocks Occurs throughout the world even in building stones It may provide protection from extreme conditions or predation In the Antarctica there are very low numbers of any type or organisms In some locations the main primary producers are those found growing inside of rocks usually right under the surface Mainly in rocks which can transmit some light Sometimes called cryptoendolithic Generally dominated by cyanobacteria Prokaryotes seem to be able to handle extremes Because there are only a few months of each year where these rocks heat up enough for photosynthesis to occur growth and production is slow 014 ug C m392 y39l about 100 times less productive than open oceans one of the least productive habitats on earth oceans that is Turnover times of carbon are about 17000 years forests have the next slowest reported turnover times with about 27 years Some species are psychrophilic others just survive and do well embedded in rocks in the desert as well Cyanobacteria abundant in quartz stones where light can be transmitted Smith et al 2000 Antarctic Science 12177184 Such microbial communities also occur in th Mojave Desert Schlesinger et al 2003 Ecology 8432223231 Microorganisms associate with many types of rocks Lichens and other microorganisms can vastly accelerate the weathering of these rocks Lichens can also protect from weathering Ari o et al 1995 The Science ofthe Environment 167353363 Bacteria and fungi have been isolated from sandstone on a weathering building Levels low relative to soil but signi cant Metabolism lowers pH but this does not seem to be the primary route of increased degradation Excretion of weak organic acids which actually attack the stone is more important Bacteria also form nitric acid by nitri cation 39 39 and have been isolated from building stones and produce signi cant amounts of nitrate from ammonium Signi cant algal communities have been reported several cm into fairly translucent sandstones found on the bottom of streams Crusts are important components of desert soils These crusts can withstand intense heat desiccation and light Often have algae in them May be important in soil formation Nostoc commonly present The cyanobacteria can respond within hours to rewetting Nostoc has survived over 100 years in an herbarium dry and then been rehydrated successfully Oscillatoria can move up toward the surface of soils if they become wet and then move down as they dry Pringault and GarciaPichel 2004 Microbial Ecol 47366373 presentation Antarcticadry valleys The dry valleys of Antarctica are the driest habitats known Precipitation is sparse and relative humidity about 10 Soils support about 100 times less bacterial cells per unit volume than do arid desert soils from temperate zones Initially thought that these areas were sterile but simply did not have the appropriate culture techniques Needed low temperatures extremely low nutrients and long incubation times 2 3 O V 7 V 1 1 L 2 3 4 V 100 V m V 5333 E 3 U V m V gt11 V BIOL 687 Spring 2005 Unusual and Extreme Environments months Many forms of bacteria found are peculiar to the Antarctic Spore forming bacteria are rare as there is rarely time for the bacteria to grow enough to form spores Recently human invasion has increased the numbers of nonnative fungal and bacterial species isolated the bacteria associated with soils around human settlements are distinctly different than that which is found in more pristine areas This may be the closest terrestrial analog to the martian habitat Signi cant algal population also can be found 05 cm from soil surface Daves and Clarke 1991 Antarctic Science 3257263 these species are cold and drought tolerant and are adapted to low light Research on Siberian soils shows thin layers of unfrozen water exist in permafrost soils and metabolism continues with doubling times of 1 day at 5 C 20 days at 710 C 160 days at 720 C Rivkina et al 2000 Applied Envron Micrbiol 6632303233 Deep marine Characterized for the most part by extreme pressure combined with low temperature Primary variable in pressure gradient with depth Morita 1986 pp 171184 in Microbes in the Extreme Environments Low energy common except carcasses of large vertebrates that sink Extreme pressure effects membrane uidity High pressure increases melting point of lipids Kimimura et al 1992 J Oceanogr 4893104 Kimimura et al 1993 AEM 59924926 This is separate from temperature resistance pressure C17 lipids temp C12 and c18 are less uid Increasing pressure can reverse anesthetic effects These anesthetics seem to work by increasing membrane uidity When mice are anesthetized to the point where they are falling down and the atmospheric pressure is returned to atmospheric they recover Model is a follows WNH VVV 3 V This is why it is difficult for microorganisms to be very active in the deep ocean temp is about 4 C When deep submersible was being worked upon in deep water in Atlantic a wave entered the hatch and it sunk Workers had left a lunch on it Three months later it was retrieved The sandwich left in the lunch although compressed was still edible What would happen to a sandwich left for three months in a refrigerator Very difficult to do work in deep ocean conditions the materials in seawater corrode steel under high pressure Barophilic and bar tolerant bacteria both present at deepsea sites Barophilic bacteria also tend to be psychrophilic and require both low temperature and high pressure Barotolerant like high temperatures Kato et al 1995 Biodiver Conserv 419 Deepsea sediments may have signi cant numbers of bacteria These bacteria may include Archaea that thrive in deep cracks with nutrients from volatile compounds from magma Little is known about these organisms but they are candidates for analogs to life on other planets Delanye et al 1998 Science 281 222230 Deep or shallow bubbling methane seeps can be the site of communities that are based on methylotrophic bacteria Jensen et al 1992 Mar Ecol Prog Ser 83103112 Vestimeniferan tubeworrns similar to those in hydrothermal systems occur Scott amp Fisher 1995 Amer Zool 35102111 Deepwater bacterial communities are probably diverse When rRNA sequences were isolated from deep marine systems of 61 total clones 11 were closely related to marine cyanobacteria all Lquot V 6 V 101 BIOL 687 Spring 2005 Unusual and Extreme Environments of the rest were more than 10 different from any previously sequenced organisms Never more than 2 cooccurred in the same sample Fuhrman et al 1993 AEM 5912941302 G Deepsea bacteria are generally starved very small cells low available organic C and they are not tolerant of high temperatures These cells are more tolerant of temperatures 17 0C when starved than when cultured in nutrient rich medium Preger and Oliver 1993 AEM 592653 2656 hand out p have unusual survival strategies Simu and Hagstrom 2004 AEM 702445 2451 presentation H Deep water foraminifera single celled eukaryotic organisms are diverse and many require high pressure for survival Turley et al 1993 DeepSea Research 40643652 1 Protozoa show different levels of pressure sensitivity 1 Paraphysomonas butcheri isolate from ocean can reproduce up to 100 atm Bodo curvifilus could reproduce up to 300 atm 200 atm 2000 m depth Cercomonas species could only reproduce at greater than 300 atm 2 Feeding strategies have to change with depth Particles become more nutrient poor and processed with depth So not only do the protozoa need to tolerate high pressures but the food limitation becomes more sever with depth I Nodules of manganese 1 Bacteria inhabit mineral nodules in which there is signi cant precipitation of heavy metals caused by the bacteria 2 The metals come up from the sediments in reduced form and are oxidized by chemoautotrophic microorganisms 3 There has been a push to mine these ore rich nodules 4 Some negative reaction to this given the highly unusual deep sea communities and the unknown impact of this mining on deep sea communities At present the technology is not there to mine these with enough ef ciency K Active bacteria have been found in sediments as much as 500 m below the sea oor This microbial action may be important for geochemistry of ocean Parkes et al 1994 Nature 371410413 L Diamond anvil cells used to demonstrate bacterial metabolism up to 16 Gpa 16000 atmospheres Sharma et al 2002 Science 29515141516 VI Hyper saline environments A Hypersaline environments most often form in closed basins with high rates of evaporation relative to the water input chemistry can vary widely depending upon the initial ion concentration of the incoming water B Saline environments are extreme because ions such as magnesium become highly hydrated When they are concentrated they compete for water In a sense water becomes a limiting resource to algae in hypersaline environments This can also preserve organisms One group of researchers claim that bacteria have been isolated and grown from inside a 250 million year old salt crystal Vreeland et al 2000 Nature 407897900 C Protein and lipid stability decreases This is because these molecules retain conformation from having ionic bonding ie phospholipids on the surface of membranes hydrophilic portions of protein The increased levels of positive ions tend to shield out these interactions 1 Organisms can require high levels of salts just as they require high temperature once they have evolved to tolerate it 2 This tolerance requires stabilizing proteins by hydrophobic interactions and more sul de bonds and lipids by making them less uid similar to thermal environments D Solubility of oxygen drops as salinity increases Anaerobic microorganisms are found at the highest salinity hand out p E Methane oxidation is inhibited by high salinity but methanogenesis is not Thus these environments may form an important source of atmospheric methane Conrad et al 1995 FEMS 102 gt11 V 53 gt lt V lt3 BIOL 687 Spring 2005 Unusual and Extreme Environments Micro Ecol 16297306 Cyanobacteria are most halo tolerant of algae many can tolerate anaerobic conditions some other algae such as Duneliela have moderately halotolerant 1 Seawater 35 salinity is not considered hypersaline 2 Algae can tolerate up to 35 salinity 3 Photosynthetic bacteria can tolerate up to 45 salinity Algae in such environments are often highly productive when the salinity tolerant grazers such as brine shrimp are unable to survive above about 14 then the biomass can become tremendous Halotolerant Oscillatoria limnetica is used in solar ponds in Israel to regulate the temperature of the lower layer Using saline waters in aquaculture has promise because desert regions often have an abundance of saline waters Stromatolites large structure formed by cyanobacteria can form in hypersaline lagoons because the grazers that would normally eat the cyanobacteria that form them are killed by the high salinity Shark Bay in Australia is an example of this Halophilic bacteria do well up to even higher salinities As usual the archaebacteria are right in the thick of things but some species of bacteria are also important Anton et al 2000 Applied Environ Microbiol 6630523057 1 39 39 39 the red 39 l 39 39 Bacteria quot quot 39L Oren 2002 FEMS Microbiol Ecol 3917 Deep hypersaline basins 1 Water is trapped deep in the ocean because the salinity makes it much denser than the sea water surrounding it 2 Generally formed by input from hotsprings 3 Orca Basin in northern Gulf of Mexico 1800 2400 m deep covers and area of 20x25 km it is 180 m deep 4 High levels of NaCl warm water dated at about 8000 years old 5 The brine is high in nutrients bacteria and organic material Anaerobic high levels of methane suggest active methanogenesis Brines also common in slattems salt production facilities and enclosed marine bays 1 These habitats have extremely dense bacterial populations The salt companies are worried about what types of bacteria are there and how many because they can start precipitation of salts or may inhibit it Tend to increase the organic content of water Want to manipulate them for positive uses 2 These mats have seem much study because they are accessible very simple with no eukaryotes contain exotic microorganisms and have been seen as an analog habitat for primitive life Enzymes have been isolated from alkaline hypersaline environments with commercial use Cellulase used in laundry detergent ignores crystal cellulose in cotton but opens up other regions where dirt gets trapped And amylase that breaks up starch causes formation of glucose doughnuts these are being used to microencapsulate drugs for time release Groundwater as a habitat In past were thought to be mostly sterile and not a signi cant habitat Work with oil exploration and drinking water contamination has shown otherwise See handouts for the different types of groundwater and zones What organisms live there In limestone aquifers and cobble with large spaces invertebrates can often be found Viruses can survive for long periods of time and the bacterial activity in the groundwater can alter their survival time Gordon and Toze 2003 J Appl Microbiol 95536544 Protozoa are often present if there is a signi cant amount of space between particles Sinclair amp Ghiorse 1987 AEM 5311571163 Their importance increases with greater C contamination Sinclair 1991 pp 339351 in Proc First Int Symposium on Microbiol 1 V 2 3 103 m V gt11 V 53 lt3 BIOL 687 Spring 2005 Unusual and Extreme Environments Deep Subsurface Bacteria are almost always present unless it is solid rock or very ne particles Levels lower than at soil surface but do not continue to drop with depth after this rather they become a function of the suitability of the habitat Even in very deep sediments under deserts there are signi cant numbers of bacteria Kieffer et al 1993 Microb Ecol 265978 Smallscale variation in chemistry can lead to distinct microbial populations just as on the surface Franklin et al 2000 Microb Ecol 38377386 Clogging of pores by microorganisms may be an important in many applications from construction of reservoirs to secondary oil recovery Baveye et al 1998 Critical Reviews in Environ Sci Technol 28123191 Understanding ecology including predation and competition may be important for these applications Systems are usually oligotrophic and often contain oxygen unless there is a signi cant subterranean c source or it is not rapidly mineralized on the surface Diversity is generally similar to that seen in soils most major groups of bacteria have been isolated from groundwaters Balkwill 1989 Geomicrob Journal 73352 Madsen and Ghiorse 1993 in Aquatic Microbiology However completely unique organisms have been isolated from deep habitats according to rRNA sequence analyses Chandler et al 1998 Microb Ecol 363750 Bacterial transport through sediments varies depending upon species and nutritional state For example some species form aggregates with mucopolysaccharides These move slowly through aquifers Heise and Gust 1999 Marine Ecology Prog Series 190141153 Nutrient limitations are not well described few studies We know that under prairie N is often limiting but under ag elds c limits bacterial production How did the very deep microorganisms get there It is known that the water in those systems turns over very slowly 100 s of years by isotope analysis Murphy et al 1992 Water Resources Research 28723740 Some suggest that the bacteria were locked in there upon sedimentation and have evolved separately since that time Sewage contamination a common problem in groundwater see handout on viruses in groundwater Viruses can survive for at least days in groundwater McKey et al 1993 Environ Sci Technol 2710751079 PCR can be used to amplify DNA from enteric viruses to detect them at low numbers in groundwater Abbaszader et al 1993 AEM 5913181324 May have implications for bottled water but techniques have become so sensitive this is controversial Gassilloud et al 2003 AEM 6939653969 presentation Deep wells 2001300 In show lithoautotrophic communities can occur in Columbia River Basalt Group Stevens amp McKinley 1995 Science 270450454 Showed that H2 generated from rock was used by methanogens to form a viable sub surface community without organic C inputs from above Since them suggest that chemistry may not be favorable enough to allow for rapid H2 production required Jury still out on this one Another group has shown that hydrothermal waters may be rich enough in hydrogen to support a bacterial community without external organic carbon or oxygen Chapelle et al 2002 Nature 415312315 Novel communities in Movile Cave Romania Sul de rich water contacts Oz containing caves Microbes oxidize sul de and produce thick layers of bacteria Isotope analyses carbon suggest that community is isolated from surface communities more depth in 13C than surface Supports a diverse animal community including isopods and amphipods Sarbu et al 1994 Geomicrobiol J 12175182 Deep vadose sediments in central Washington state 2490 In One pristine 2 contaminated with wastewater Few culturable bacteria in pristine sites many more in contaminated sites Water associated with contamination probably most important aspect Fredrickson et al 1993 Geomicrobiol J 1195107 3 V Lquot V O V V 2 3 104 BIOL 687 Spring 2005 Unusual and Extreme Environments M An anoxic aquifer in Texas VIII 1 30 m deep boreholes 2 Sulfate reducing bacteria common 3 System may be based on these sulfate reducing bacteria found in narrow bands next to organic C rich sediments lignitite Ulrich et al 1998 Microb Ecol 36141151 Basalt aquifer studied in Idaho from 7500 m depth Zheng amp Kellog 1994 Can J Microbiol 40944954 Dominant bacteria were gram negative mesophilic heterotrophs 149 cultures obtained include Psuedomonas Bacillus A 39 L 4 t Ly t 7 quot and Clavibacter Deep aquifers in ocean produce uids that can support microbial life giving support to the idea that deep groundwaters under the ocean can harbor bacterial biomass Cowen et al 2003 Science 299120123 presentation Oligotrophic habitats Has been suggested that oligotrophy is common Morita Many bacteria under nutrient stress most of the time It is possible that H2 interaction with iron allows for longterm survival of inactive cells This low rate of energy supply is important to counteract the effects of racemization and depurination Morita 2000 Microbial Ecology 38307320 Bacteria under nutrient stress are small and often inactive These bacteria may simply be waiting for better conditions Can often make these bacteria active again with correct techniques Schut et al 1997 Aquat Microb Ecol 12177202 Methods developed to culture oligotrophic bacteria NH VV 1 Standard methods too nutrient rich Native bacteria are either outcompeted or actually experience toxicity 2 High ow through miccrotiter plates and sensitive detection methods were used to culture marine bacteria at insitu nutrient concentrations Could culture 14 to 14000 times higher numbers than with standard laboratory media Connon and Giovannoni 2002 AEM 683878 38 85 Acid mine drainage Common in metal and coal mines Will talk about pH in acidi cation but it is an extreme environment Archaea have been isolated that can grow at pH 0 Edwards et al 2000 Science 2871796 1799 Oxidize pyrites releasing heavy metals into drainage increasing contamination 105 BIOL 687 Spring 2005 Bioremediation Microbial Ecology Topic 19 Bioremediation I H A B V 3 V Basic problems associated with bioremediation biological detoxi cation of hazardous wastes Can detoxify at source most efficient or use microorganisms to clean up spills or contaminated environment Much of the data is proprietary because industries make their pro t by these methods and new research is not rapid Cleaning up spills if you spill enough it will get into the groundwater Spills contaminate soils vadose zone and groundwaters draw diagrams from p40 of ground water and soil contamination remediation Steps in bioremediation of spills Remove contaminated soils Pump out water and treat may reinject Alternatively treat insitu either with the ambient bacteria or with injected bacteria injected bacteria generally don t do any better than ambient Fuller et al 1995 Microb Ecol 29311 325 i Problems with in situ treatment 21 Oz needed for the most efficient utilization of organic mater oxygen often directly b 0 9A we C9 injected Alternatively nitrate can be injected why would this work H2 Oz can be added but bubble formation may be a problem Adding SO4339 may cause problems because it will cause buildup of His that is toxic Edwards et al 1992 AEM 58794 800 why is sul de formed Nutrients need to be added to force carbon limitation often nitrogen and phosphorus Excess bacterial growth may plug the aquifer These bacteria typically form bio lms and excrete polymers that are sticky and clog things up Ross et al 2001 Water Res 3520292037 and stop further ow of additions to entire spill Grazing regimes of bacteria are not well understood Motile bacteria may move contaminant through an aquifer faster than would be expected otherwise Jenkins and Lion 1993 AEM 5933063313 but movement of bacteria can be a complex function of porosity and interaction of charges on cells with changes on particles in aquifer Lindquist amp Bertsson 1995 Soil Biol Biochem 27941948 Citrate may complex with heavy metals and make them unavailable for concentration or decontamination by microbes Francis et al 1992 Nature 356140142 ii Problems with pump and treat A 3 b V Very energy intensive Often takes a very large amount of time to remove the contaminated water and lots of wells If large amounts of the contaminant are adsorbed onto particles they may leach into the water very slowly so even of the water is cleaned it becomes recontaminated 106 III 93 E E2 92 m V E BIOL 687 Spring 2005 Bioremediation down in the aquifer d Have to keep pumping for a long time never will be able to completely clean up a spill Nitrate contamination of groundwaters 90 of rural drinking waters come from wells 33 of Kansas wells are contaminated by nitrate Excessive nitrate will be converted by stomach acids to nitrite and this binds irreversibly to hemoglobin Can cause asphyxiation Causes blue baby syndrome when contaminated water used to make formula The baby turns blue because the hemoglobin does not get oxygenated Also stomach acids convert nitrate and nitrite to nitrosamine carcinogenic and acutely toxic If much of the nitrogen comes from ammonium additions to elds some importance or in the form of ammonium and urea from eld lots probably most important why is it in the form of nitrate Why does some of the nitrate hang around even in anaerobic waters Can remove some of the nitrate by injecting organic carbon sources May get problems of aquifer plugging Protozoa bacteriverous soil agellates may reduce this clogging Mattison et al 2002 AEM 6845394545 Time series of nitrogen and 02 under a feedlot Degrading petroleum pollutants see Atlas amp Cemiglia 1995 BioScience 45332338 Problem widespread tanker spills dramatic but more than 250000 tanks are currently leaking in service stations Basic research in 60 s amp 70 s on marine oil spills Different paths of breakdown occur with different types of organisms see handout Most prevalent in 39 order P A F lavobacterium Nocardia Arthrobaacter Vibrio Bacillus M icrococcus Acinetobacter Very complex mixtures of compounds the degradation pathways of the fungi are very poorly understood Most breakdown aerobic but anaerobic oxidation by sulfate reducing bacteria has recently been documented Rueter et al 1994 Nature 372455458 This also leads to higher sul de coals and oils Fungi are more important than bacteria in soils or still surface slicks including Cardida Rhodotorula Aureobasidium amp Sporobolomyces yeast like amp Penicillium Aspergillus and Cladosporium Gasoline spills important Gasohol may be problematic because ethanol increases the solubility of petroleum products Microbes that may bioremediate gasoline Powers et al 2001 Crit Rev Environ Sci Technol 3179123 also prefer it A case study of diesel removal in Arctic soil tundra In the early 1980 s a diesel fuel pipeline at an air force station in northern Alaska ruptured and caused massive contamination of the hillside down slope from the pipeline Two bioremediation approaches were tested 1 In situ bioremediation 2 Above ground treatment of contaminated soil For above ground 1 Soil was removed and clean ll was placed above permafrost to keep it from thawing 2 Spread 18 inches thick over a paved area lined with plastic sheeting 107 9 VI VII BIOL 687 Spring 2005 Bioremediation 3 Surfactant added to soil to loosen the nonwater soluble oil from the soil surfaces and make it more available to soil microbes 4 Organic carbon fertilizer was added to stimulate natural microbial community 5 Soil mixed once per week to oxygenate and fertilized every 2 weeks 6 By 6 weeks diesel concentrations had been reduced by 42 after 6 weeks the soil was covered for the winter 9 months later the cover was reduced and the levels were down by 60 from start another 20 after another 2 months treatment the levels were down to 25 of initial contamination Not perfect but much better Native tundra was also treated in place 1 Dilute surfactant and fertilizer were sprayed directly over the soil 2 By the next summer contamination was reduced by 50 and some plants had regenerated Soil was retreated and another 5 improvement was seen over the summer Marine oil spill biodegradation Atlas amp Cerniglia 1995 BioScience 45332338 1978 Amaco Cadiz Brittany shoreline Before this it was thought that weathering was required for biodeterioration and a few weeks lag needed to occur However degradation of small molecular weight components happened quickly more quickly than volatilization Probably because of microbial population that had already been exposed to oil in ballast water In this case wave action keeps aeration and coastal eutrophication fertilizes bacteria IXTOC well blow out in Gulf of Mexico 1979 Thought degradation would occur rapidly because of high temperatures and adapted bacteria However oil formed an emulsion mousse and bacteria could only attack surface of droplets Exxon Valdez 1989 Bragg et al 1994 Nature 368413418 Used branched markers to assess biodegradation but bacteria in this region got to the branched compounds quickly They were like pine turpines that were naturally occurring in environment Nutrient additions mainly N were necessary to stimulate biodegradation Used slow release fertilizers urea laureth phosphate and oleic acid Yielded dramatic results 35 times faster degradation with treatments Using a bioluminescent indicator organism to indicate naphthalene exposure and biodegradation The genes from Vibrio sheri for bacterial luciferase lux were inserted into a naphthalene catabolic plasmid in Pseudomonas uorescens Inserted using a suicide plasmid This gene then can serve as a marker of catabolic activity ie atp availability drives light production The plasmid codes for enzymes that convert naphthalene to salicylate and salicylate to acetaldehyde and pyruvate The gene was inserted in a place that it will only produce luciferase when the naphthalene degrading genes are induced induced by salicylate This causes the bacterium to uoresce when exposed to naphthalene vapors or salicylate The higher the rate of naphthalene degradation the more light was emitted in cultures In soil slurries contaminated with naphthalene and with the modi ed P uorescens added there was induction of luminescence over 1 hr in contaminated but not uncontaminated soils May be used in unconfined aquifers to sense naphthalene May be used in bioreactors especially with mixed cultures to monitor activity Other chemical degradation systems may be modi ed in a similar fashion in the future Above ground treatment facilities bioreactors Need to optimize surface area for bio lm to develop on and for contaminated water to contact Need to remove microorganisms that slough off of the surface before using the water Organisms in this case can simply take up the contaminant but if they do not process it it must be treated as hazardous waste and still disposed of Cause for heavy metal contamination problems Concentrate and dispose of safely Bender et al 1995 J Indust Micro 14 1131 1 8 108 1X X 3 O V 9 m V BIOL 687 Spring 2005 Bioremediation Theoretical considerations using bio lms in bioremediation and other bio lms as well When a substrate is toxic it can be processed as low levels but as it increases in level it starts to inhibit growth If we have a substrate S then we can write S H H max S2 S K S K i 1 Where u growth rate umax maximum with no inhibition Ks the half saturation constant with no inhibition and K the inhibition constant As K goes to in nity we approach monad growth kinetics graphs are a follows take from P 570 international conference on physiochemical and biological detoxi cation of hazardous waste vol 2 Burgay 1994 J Biotech 3497100 Furthermore we can think of a bio lm known as periphyton in streams and realize that there will be a decrease in substrate as we move into the bio lm that is determined as a complex function of the thickness of the diffusion boundary the rate of consumption the thickness of the diffusion boundary and the amount of inhibition Considerations like these can be used to model the performance of bioreactors Complex need to consider equations like those above and small scale spatial gradients such as this Scanning laser confocal microscopy has been used to show that bio lms have organization Wolfaardt et al 1994 AEM 60434446 The compounds that are being degraded can in uence this architecture May be mutually bene cial bacteria growing closer to each other Bacteria may have plasmids that allow conjugation and induce bio lm development Ghigo 2001 Nature 412442445 Bacterial motility may play a role in movement of toxins away from a site but also movement of bacteria toward a remediation site Pandey and Jain 2002 AEM 6857895795 presentation The roles of eukaryotes in bioremediation Eukaryotes are important predators of bacteria so they may inhibit or help bioremediation efforts 109 BIOL 687 Spring 2005 Bioremediation 1 Lab experiments show that an exponential decrease in introduced bioremediating bacteria can be attributed to bactivory by bactiverous protists Snyder et al 2000 Microb Ecol 40189 199 Protists may actually stimulate bacteria that use more labile organic C Kinner et al 2002 Env Sci Technol 3643124218 presentation B At the edges of a organic contamination of groundwater there is increased bacterial activity and an increase in protozoan bactivory and an associated increase in the numbers of protozoa Madsen et al Nature article 1 In this case the organic carbon stimulates part of the groundwater bacterial community and they grow faster as the rates of growth increase the food base for the protozoa increases and they are able to grow more rapidly 2 The increase in predation may keep the aquifer from plugging even with the fertilization 3 The continuous cropping of bacteria may keep bacteria in log growth and make them process the material at maximum ef ciency 4 The true structure of groundwater communities is poorly understood Very difficult to study C Eukaryotes can also contain enzyme systems that can break down xenobiotic compounds 1 Organophosphate pesticides are a common contaminant 2 mussels protozoa squid and other organisms have enzymes that can break down these compounds organophosphate acid anhydrases The utility of such organisms has been poorly evaluated as insitu degraders of organic compounds XI An aerobicanaerobic bioreactor to degrade polychlorinated hydrocarbons Fathepure and Vogel AEM 5734183422 A In anaerobic bioreactors initial rates of degradation are rapid when chlorine sidegroups are numerous as degradation progresses there is a signi cant decrease in efficiency To completely degrade all of the organics to C02 may be difficult if impossible see review by Wiegel and Wu 2000 FEMS microbiology ecology 32115 except now there is a bacterium that can do it It is an obligate anaerobe Adrian et al 2000 Nature 408580583 Aerobic organisms are able to ef ciently utilize organic molecules with few substituted side groups C A two stage bioreactor system was tested with rst an anaerobic stage followed by an aerobic state When acetate was used to feed the anaerobic stage hexachlorobenzene tetrachloroethylene and chloroform were degraded to tri and di chlorinated products 99 80 and 35 respectively The effluent was then passed through the aerobic columns The nal ef ciency of removal 14CO production from 14C labeled precursors was 99 80 and 83 respectively Reactors made using columns packed with glass beads as a surface for the microbes to grow on Aerobic sludge from a sewage plant was used to seed the aerobic column aerobic activated sludge for the aerobic bio lm Oxygen was added to keep the aerobic portion going Water pumped through at about 1 replacement per hour 5 Did not scale up to industrial scale setup D Another approach has been taken Put genes from aerobic and anaerobic pathways into one bacterium Psuedomonas Wackett et al 1994 Nature 368627 XII To remove PCBs from soils a bioreactor was used Inoculated bio lms with indigenous bacteria Repeatedly washed soil and ran the wash water through the bioreactor After 18 days of recycling the wash water through the bioreactor more than 99 of the PCB s were removed 25000 gallons of water were treated soil processed at 500 pounds per hour N V 3 V 03 V WM VV V 3 V 9393 110 XIV 93 BIOL 687 Spring 2005 Bioremediation This pilot study illustrates the feasibility of using bioreactors to clean soil as well as waters Study from river showed that indigenous organisms will also remove PCBs with little change in rates with addition of bacteria known to remove PCBs Harkness et al 1993 Science 259503 507 Sewage treatment is the oldest form of bioremediation Modern sewage treatments plants use natural processes to oxidize organic carbon and remove pathogens Some new techniques include using wetlands to remove nutrients It has been suggested that plant root zones can serve to treat domestic sewage Rivera et al 1995 Wat Sci Technol 32 21 1218 Bacteria may also be useful in remediation of mine wastes Increase solubility of metals actually may be used in mining Can also cause a decrease in solubility of metals making them precipitate 1 Some bacteria were stimulated by addition of acetate These bacteria stimulated transformation of soluble U VI to insoluble U IV 2 This led to immobilization of a toxic contaminant Holmes et al 2002 AEM 6823002306 lll BIOL 687 Spring 2005 Acid Precipitation Microbial Ecology Topic 20 Acid Rain and Microbial Processes I A B C 11 O V 111 m V Formation of acid rain Acid rain has been recorded with pH less than 20 2 aci s l Sulfuric main one HZSO4 gt 2H SO42 2 Some nitric acid HN03 gt PF N03 Sources Coal and oil with high sulfur content i Europe and NE US are mostly dependant on coal Smokestacks emit S02 which is oxidized to sulfuric acid i Smokestacks used to be short and they adversely effected local populations ii Smokestacks are higher and transport pollution further from source This makes it harder to justify cutting back emissions Cars emit nitrogen oxidizes photochemically oxidized to nitric acid Microorganisms emit some sul de Lightening forms some nitric acid 5 of sulfuric acid and 510 of nitric acid now from natural sources Physical effects of acid rain Some lakes which are not well buffered change pH easily 1 Buffering is related to the bicarbonate concentration 2 Bicarbonate and acid form C02 and water like vinegar and baking soda 3 Lakes in regions with limestone have lots of carbonate and are not effected lakes here 4 Lakes with granite basins are low in carbonate and not able to take much acid precipitation without changing pH 5 Soils are generally more buffered and the microbial populations are less strongly effected pH of lakes 1 Ultraoligotrophic 2 Mesotrophic 7 3 Eutrophic 79 lots of buffer 4 Saline soda lake 10 soda lake tons of buffer zzes with acid addition Water with low pH can dissolve some metals 1 Aluminum concentrations increase in lakes with acid input even though pH of water does not change 2 Copper and other metals are effected the same way handout p 28 geochemistry effects on t V N V 3 4 5 6 6065 little buffer 5 1 The extent of the problem There are 100000 Scandinavian lakes Over the last 30 years 35000 have become shless Pollution in Industrial Europe was killing vegetation around the plants so they increased smokestack height and prevailing winds took acidity to Scandinavia In Norway half of all of the lakes are currently endangered in 1989 the number of shless lakes in southernmost Norway has doubled in the last 10 years A 75 reduction in acid loading would only allow for recovery of half the effected lakes Presently 70 of the lakes in southern Norway have lost all buffering capacity Many lakes in United States have been effected at least 1180 lakes and 4670 streams have been shown to be acidi ed according to 1989 EPA survey 1 Fish are dead in poorly buffered lakes in US and Canada There is strong evidence for acid precipitation throughout Canada Analysis of sediment cores has shown shifts in diatom communities which are related to acid precipitation ll2 1V V BIOL 687 Spring 2005 Acid Precipitation i Some species are more sensitive to acidi cation than others ii The silicon frustule of diatoms preserves in the sediments hence there is a historical record of acidi cation see handouts p Calculate curve iii The decease in acidi cation on the handout is related to International Nickel Company 150 km distant building a taller smokestack about 8 years ago iv Acidi cation coincides with the in ux of industry in a speci c region 4 Historical data from the streams in the Catskill mountains indicates that acidic deposition has signi cantly in uenced water quality and that the major changed occurred before 1945 as an increase in the Ca and Mg concentrations Large scale Biological effects A Fish die between pH 4555 1 Stop being able to reproduce much before that 2 Since they reproduce in streams areas which have high snow melt can cause reproductive failure i Snow makes streams low pH ii Fish spawn at time snow melts in streams 3 Ph and aluminum interact to eat away sh gills 4 Makes it hard for sh to get calcium malnourished the change in sh population may cascade down to the microorganisms B Even in high alkalinity streams there can be an acid surge with high precipitation events 1 With high precipitation events or snow melt the underlying geology has little effect and the alkalinity may drop suddenly 2 The pH can drop rapidly for the duration of the event 3 pH has been shown to drop from 7 to 4 over a few days in a Swedish stream killing sh and bottom fauna C All snails and other mollusks need calcium carbonate for their shells low pH eats away their shells and they die below pH 60 p handout microbes pH Microbial effects much data from acid mine drainages a common problem in southeast Kansas A Bacterial activity has been shown to change 1 Leaf decomposition lowered in aerobic situations Sulfate reducers do better because of the sulfate input 2 Anaerobic bacteria may do better with more acidic conditions ie sulfate reducers 3 Organisms that live in sediments are less likely to be effected because the sediments tend to buffer pH changes 4 Bacterial activity on leaves and rocks in Smokey Mountain Streams are lower with acidi cation 5 It has been suggested that total microbial number and activity is not strongly in uenced by acidi cation particularly in sediment Tranvik et al 1993 Can J Fish Aquat Sci 51 2529 2536 B Microbial processes which change pH 1 Increases i Photosynthesis with concurrent uptake of PO4339 or N0339 requires assimilation of protons and tends to increases pH Some have suggested eutrophication by addition of these nutrients to counter acidi cation ii When nitrate is respired with NH4 as an end product protons are used up alkalinity increases iii Sulfate reduction also causes an increase in alkalinity and a subsequent buffering of pH but this only works if the H2 S escapes the system as a has or is precipitated with iron as pyrite Other wise it is oxidized within the system and there is no net effect 2 Decreases 113 VI BIOL 687 Spring 2005 Acid Precipitation i Heterotrophic processes tend to decrease pH especially fermentation which results in the production of organic acids Fig 4 on p C The Okefenokee swamp as a model for longterm acidi cation of a system V N V L V The swamp is 1000 s of years old it gradually became acidi ed over 8000 years as microbial acids have accumulated Overall rates of activity are similar to nonacid swamps because microorganisms can use the easily degradable organic carbon as ef ciently However the rates of lignin decomposition are lower leading to black waters and peat deposition The microbial communities are distinctly different ie less efficient at degrading complex carbon compounds and have different capacities in the swamp even thought there has been 8000 years for the communities to evolve toward or acquire more acid tolerant species Effects on phytoplankton l 2 Jgt b V V Lquot V 6 In unaffected oligotrophic lakes planktonic algae are chrysophytes green algae and diatoms When acidi ed dino agellates and different species of chrysophytes take over Some people report that cyanobacteria do not do well under acidic conditions but more recent data contradict this idea Largest changes appear to occur between pH 56 and 47 where the water looses its buffering capacity As acidity increases the levels of dissolved metals increase Many species of algae are sensitive to dissolved metals Grazers are also sensitive to pH changes and alteration in grazing pressure can change dominant algae Often productivity decreases over all leading to quite clear lakes Effects on benthic algae periphyton V 2 3 V Green algae do well total biomass increases diversity decreases This is because more light reaches the sediment as the water clears and grazing snails are no longer able to survive It has been suggested that increased lamentous mats of algae are an early sign of acidi cation Solutions Technology is available to t smokestacks with scrubbers Low sulfur fuel is available the clean air act now requires use of low sulfur fuel SUV s continue to burn gas at high rates Lime can be directly added to lakes and streams to neutralize the acid This is very cost intensive and V 2 3 4 5 must be repeated as long as input continues every year In addition this can cause unstable response of the organisms The liming can cause death of the lamentous algae on the bottom of the system releasing nutrients The nutrients cause algal blooms which subsequently crash and deplete oxygen The sediment release phosphate when the lake goes anaerobic causing further oscillations It is currently not known how to avoid such extreme oscillations Can fertilize lakes Phytoplankton photosynthesis will make lake more basic and tend to neutralize the acids Davison 1995 Nature 377504507 The sources and the solutions have been known for at least 25 years 114 BIOL 687 Spring 2005 GEMS Microbial Ecology Topic 21 Transfer of Genetic Material in Natural Populations Release of Genetically Modi ed Microorganisms I Importance A Transfer of genetic material important to microorganisms in selective sense Transfer in range probably about the same order of frequency as mutation Cohan 1994 TREE 9175180 DNA may also rearrange in resting bacteria Amber et al 1994 FEMS Microbiol Ecol 15514 B Importance in terms of systematics if there is a large amount of bacterial phylogeny based upon speci c catabolic functions if later transfer between widely divergent groups is common then the characteristic less useful Thus taxonomy of speci c genes particularly those on plasmids does not necessarily match taxonomy of the bacteria they are found in Heinemann and Billington 2004 ASM news 70464471 C In sexual organisms genetic exchange stabilizes genetic diversity by preventing divergence Unlike in sexual organisms lateral exchange may not lead to same consequences Cohan 1996 ASM News 62631636 D Useful information if genes are inserted via genetic engineering that would be harmful if they ended up spreading through the natural population E Antibiotic resistance seems to have spread to a large number of unrelated strains of bacteria Found in the open ocean Found in pristine ponds and rivers in Australia Boon 1992 Aust J Fresh Mar Res 43847859 Antibiotic resistance found in all habitats near Savannah River in South Carolina but frequency varied between sites and between days at same sites Leff et al 1993 FEMS Microbiol Ecol 13135144 see handout F Antibiotic resistance spreads more rapidly in areas with heavy metal pollution McArthur and Tuck eld 2000 Applied Environ Microbiol 6637223726 This is because of increased selection for transmission of plasmids in general G Spread of antibiotic resistance may be encouraged by genes that can be turned on by stresses Beaber et al 2003 Nature 42772 presentation H Does the material spread or does it evolve independently I Spread of genetic material caused by human activities may be considered to be genetic pollution As with species introductions the pollution self propagates and is difficult if not impossible to remove from the environment once it has become established I Antibiotic resistance may be considered a form of pollution in itself When the resistance spreads the probability that disease organisms will occur increases In Denmark banning the use of antibiotics for livestock growth promotion did not lead to decreases in production but did lead to decreases in the total number of antibiotic resistant bacteria Wenener 2003 ASM News 69443448 Transfer often mediated by plasmids this section and the next in Paul et al 1991 AEM 571509 1515 Transduction virus transports transformation from environment conjugation A Plasmids are common in 23 to 46 of bacteria isolated from natural environment B Occur in marine bacteria that are antibiotic resistant These are bacteria that have been isolated from an environment where antibiotics have never been important Thought to have spread on a plasmid or plasmids C Not all plasmids can transfer genetic information most are less than 20 kb kilobases and do not have the space to code for the genes of self transmission D PCR can be used to detect mobile genetic elements in natural samples even thought they are low density can be ampli ed Smalla and Sobecky 2002 FEMS Microb Ecol 42165175 E Plasmid transfer is probably most often associated with conjugation but can also be associated with transformation or transduction Marine isolates that are transformable by plasmids that have a broad range of acceptable hosts have recently been described 115 III IV V BIOL 687 Spring 2005 GEMS F Transfer of plasmid DNA to indigenous marine bacterial populations has been directly documented Fischer et al 1994 FEMS Microbiol Ecol 15127136 10 ofbacterial isolates were transferred by plasmids 14 by chromosomal DNA In this case numbers associated with corals sponges and sea cucumbers were the most transformable Plasmid transfer in bio lms may be limited by the channels through the bio lm that control the ability of donor bacteria to enter the bio lm Wuertz et al 2004 Water Science and Technology 49327336 A study of gene transfer in the natural water column and a sediment were conducted to determine what the rates of transmission actually were A Used 25 ml incubations for 12 h generally Have isolated high frequency of transmission strains of a marine Vibrio species that are naturally transformable with a variety of broadhostrange plasmids and possess a unique colony morphology that makes it easy to assay for the species against the background ora Transformants are veri ed by DNA probes for the transformed DNA added 5 ug of transforming DNA and 107 to 108 recipients per ml ie 10 to 100 times more than natural ambient recipients and 10 to 20 times the total ambient levels of dissolved DNA in natural waters Needed to use this much to even detect the transformations note that ocean has 1021 L and their rates observed were 103911 per 25 ml 12 h so it could be important in evolutionary sense B In water columns transformation efficiencies ranged from 17 X 10396 to 27 X 103910 transformants per recipient Higher rates of transformation occurred with addition of low amounts of nutrients Rates were about 10 times lower with an ambient marine microbial community than with sterile seawater Nutrient requirement has been further con rmed for lakes in neuston Jones et al 1991 Microb Ecol 221525 C In sterile sediments nutrient addition had no effect on transformation in sediment with the ambient community there was no detectable transformation D This suggests that in natural communities transformation is more likely to occur in the water colunm than in the sediments This may be because sediments bind the DNA and make it more available However clays also make DNA resistant to degradation so it may make it hang around longer in natural environs thus increasing the chance of transformation Miller 1993 in Aquatic Microbiology E Hermansson amp Lindberg 1994 FEMS Microbial Ecol 154754 suggest that there is a higher movement of genetic materials in surface associated communities than bulk water in their review Transfer of a plasmids to degrade 3chlorobenzoate in a freshwater ecosystem Fulthorpe and Campbell AEM 5715461553 A Bacteria are isolated which are Alcaligenes sp that have a strain with a plasmid containing a transposable element coding for the 3 Chlorobenzoate gene BR60 B These bacteria were inoculated into 50 L followthrough systems containing various levels of 3 chlorobenzoate The initial innoculum died out rather rabidly before the 3 chlorobenzoate was all degraded C A number of bacteria were isolated from the systems that had the BR60 1 Most of these were motile yellow pigmented gram negative rods related to the group III pseudomonades They were capable of complete 3 chlorobenzoate degradation at both millimolar and micromolar concentrations Two more distantly related Pseudomonas species were isolated that could not completely degrade 3 chlorobenzoate and could only survive at millimolar levels of the compound Case study of plasmid transfer in natural soil Klingmuller FEMS microbial Ecology 85107116 A Strains of the nitrogen xing Enterobacter agglomerans isolated from the rhizoshpere of cereals ave the genes for nitrogen xation encoded on a large plasmid This plasmid is self 2 3 116 BIOL 687 Spring 2005 GEMS transmissible between closely related strains B The plasmid was marked with labeled transposons and labeled bacteria were released into non sterile and sterile soil under laboratory conditions Cells that were labeled donor and unlabeled recipient were added to the soil C Rates of plasmid transfer were low in nonsterile soil 106 per donor and no transfer was seen in sterile soil Adding nutrients to nonsterile soil increased transfer rates 100 fold addition of wheat seedlings decreased transfer rates why we don t know VI Multiple antibiotic resistant Ecoli added to ryegrass miniplots sampled over a 13 year period Sjogren 1995 Water Air Soil Poll 81315335 A Started 107108 cfumL fell rapidly in rst 41 d to 12 cfug B By 2 years the tracer had moved down into groundwater 16 m C 62 of Ecoli with the antibiotic resistance were the original serotype after 8 years by 13 years 43 were of original serotype D Downslope dispersion was 10 m d39l soil to groundwater 002 m d391 hardpan clay VII Movement and survival may be dependent on other organisms A Added a modi ed Psedomonas uorescens to ash leaf litter with and without isopods B The bacteria survived passage through the isopod gut C Present in feces for at least 6 days after eXposure to the organism even though only 5 h transit time for food in gut D May lead to movement of GEM s not predicted otherwise Clegy et al 1994 FEMS Microbiol Ecol 15161168 VIII Bacillus thuringiensis is a natural bacterium that kills lepidoptera and colepoptera A Used in biological control of caterpillars B Showed that plasmids transferred well in active infections when bacterial population high and growing rapidly Thomas et al 2000 Appl Environ Microbiol 66118124 C Note toxin is now directly incorporated into corn IX Genetically modi ed microorganisms in the natural environment from eld testing genetically modi ed organisms framework for decisions 1989 A Potential bene ts are enormous 1 Degrade pollutants 2 Virus to control insects 3 Plants suited to special environments B Questions about dangers and success rely upon knowledge of microbial ecology 1 Are we familiar with the properties of the microorganisms What will survival be i Protozoan grazing may be important to survival of GEMS in soil Recorbet et al 1992 FEMS Microbiol Ecol 101 251260 ii Particles in lakes may increase the probability of survival of GEMS E call but how important is not known Brettan and Ho e 1992 AEM 58 22012210 iii Generalist strains or species may survive better than specialist McArthur et al 1998 PNAS 8596219624 iv Even EColi used in Lab K12 may be able to survive in natural habitats Matsui et al 2001 Environ Contam and Toxicol 66139145 Can the organism be controlled or contained What are the effects on the environment Introduced species that are pests are the worst type of pollution known They never go away once they establish in the environment We know this from macro organism invasions Zebra Mussels Starlings rabbits in Australia Risk analysis will be necessary to determine when organisms should be released Giampietro 2002 Ambio 31 466471 bWN VVV Lquot V 117 C Q m V gt11 V 53 BIOL 687 Spring 2005 GEMS 6 You are probably starting to get an idea of how little we actually know about microbial ecology If we don t know what all of the organisms are in any 1 environment how can be predict the population dynamics of an introduced organism Two approaches deleting genes or adding genes problems with both 1 Deleting genes i Genes can be deactivated with transposons the transposons are labeled with antibiotics to screen the organisms that the transposons have been successfully inserted in The transposon can jump back out and the gene can be reactivated and worse if the organism is released to the natural environment it may spread antibiotic resistance to other species Special transposons have been designed that will insert but can not excise again There is still a remote possibility that antibiotic resistance can be transferred from these organisms With detailed knowledge of the gene sequence to be deleted 1 amino acid can be deleted or changed to inactivate the gene This is reversible because a back mutation can change it Alternatively the gene can be removed cloned part of it excised and the defective gene reinserted There is no chance of reversal in this case 2 Addition of genes i Can add plasmids but plasmids can be transmitted not good if the gene should not be transmitted to other organisms ii can add bits to chromosomes using defective transposons or gene replacement need to nd a unimportant part of the DNA so vital genes are not broken up with this process Utilizing Bacillus thuringiensis as an insecticide 1 This bacterium has a protein that kills larvae of caterpillars used to control gypsy moths ten caterpillars etc 2 Does not multiply on the surface of plants so repeated applications are necessary 3 No known negative environmental effects have been recorded even though there has been extensive use 4 Toxin genes are being added to Pseudomonas uorescens a bacterium that can survive on leaves so repeated applications are not necessary 5 Larvae have evolved recently that are not affected the toxin just like synthetic chemicals Rhizobia has been added to many environments with no apparent negative effects has been added for over 100 years 1 Currently there are efforts to increase rates of nitrogen xation by Rhizobia by altering genes in the bacterium or by inserting the genes from the bacterium into plants this probably won t work 2 In Louisiana there was a eld trial of soybeans inoculated with a genetically engineered Rhizobia with several marker genes The Rhizobia had superior nitrogen xing capacity After the experiment the plants were removed and burned and the elds plowed The EPA signed off on the experiment The site was revisited and it was determined that the GEM was out competing the native Rhizobia Nobody knows what will happen to it in the future Cairns and Orous 1992 Rev Environ Contam Toxic 1241939 On the other hand large additions of E coli and Psuedomonas putida had little effect on bacterial community structure in a lake but additions of nutrients did Ho e 1992 AEM 5833873394 Pseudomonas cepia Burkholderia cepia is being considered as abiocontrol agent and a plant stimulator Holmes 1998 Emerging Infectious Diseases 4221227 Speert et al 1994 Journal of Infectious Diseases 17015241531 Isolated from plant roots Can suppress plant fungal infections May replace important fungicides Many strains antbiotic resitant G n H 118 BIOL 687 Spring 2005 GEMS 5 Infects lungs of cystic brosis patients 6 Should we use it H A grab acterium radiobacter produces an antibiotic agrocin 84 that kills the crown gall bacterium Ryder 1994 FEMS Microbiol Ecol 15139146 1 The gene that codes for the antibiotic is on a plasmid along with the gene for immunity to the antibiotic 2 If the bacterium is used to try to control the crown gall bacterium the plasmid may get into the crown gall bacterium and give it immunity Used for 15 years and hadn t happened yet but it could so new product was tested 3 The portion of the plasmid that codes for transmissibility was identi ed and removed 4 Field tests were conducted in Australia Open pot eld tests showed it did not spread Currently being sold in Australia Genes for bioremediation of organic contaminants 1 Trichloro ethylene TCE is the most frequently reported contaminant at hazardous waste sites i It is degraded aerobically anaerobically it may be converted to even more toxic compounds such as vinyl chloride ii Methylotrophic bacteria have been isolated that degrade TCe completely in the presence of phenol genes have been isolated from these organisms iii Genes have been inserted in E coli and the altered bacterium can completely degrade TCE gt lt V iv Methanotrophs can naturually use TCE One treatment approach is to pulse methane and oxygen to encourage buildup of methanotrophic bacteria Baker et al 2001 Environ lIicrobiol3187l93 X Use of bioluminescence to detect genetically engineered organisms in the environment Shaw et 992 AEM 58267273 Labeled X anthomonas campestris with the luX gene The bacterium is a plant pathogen Released it into the eld cultures were taken and luminescence light production used to indicate bacteria Luminescence and plate counts agreed well luminesce less work Detected the added bacteria up to 6 weeks after addition on leaf surfaces and in rhizosphere but not in the area immediately outside of the treated area Suggest such techniques could be used to track genetically engineered bacteria Was detected up to 6 weeks after initial inoculation XI Although rates of transfer are low large scale may be important and long time frames Such transfer may be important in an evolutionary sense even if it has little overall importance in the human time scales XII Summary A Worst form of pollution introduction of organisms B Don t know enough about microbial communities to predict the ultimate consequence of adding microorganisms to the environment C Immense bene ts to using GEMS so not likely to stop it Only control to some degree D Have been several successful introductions of GEMs with minor if any problems 3333 99 3933 H 119 Methods Microbial Ecology Lecture 22 Methods biomass production and in situ identi cation 1 Summary of Biomass methods cannot good with organisms may include resting stages or spores counts not ATP per constant etrazolium CTC all cells low redox doesn t work with anaerobic environments amount acid C02 may vary H Determination of bacterial biomass Serial dilution 1 Use nonselective media 2 Dilute initial inoculum in series of steps 3 Incubate until tubes or plates show growth of bacteria 4 Figure the lowest dilution neXt to the one that allowed no growth started with 1 cell 5 Back calculate to the number of cells in the original sample 6 Problem that there is no such thing as a nonselective medium some if not most of the cells present will not grow in culture and this proportion is not known so the method gives minimum numbers 7 Will work to make estimates of a single type of bacteria if you have highly selective media B Direct counts 1 A nonspeci c stain can be used but then it is not known if you are only counting living cells How long do dead bacterial cells hang around In addition it is dif cult to tell between a living bacterium and a particle of detritus at the limit of resolution with a light microscope 2 Vital stains useful only stain the living bacteria i acridine orange stains DNA and lots of other organic molecules It is used to stain bacteria and the sample is ltered onto a black 02 pm pore size lter and counted with epi uorescence uoresces orange or green 3 120 gt11 V 53 Methods ii Dapi and Hoechst are other uorescent dies that are much more speci c to DNA Fluoresce blue are used more frequently at present Direct counts often give estimates of bacterial numbers that are 100 times higher than the culture methods Scanning confocal laser microscopes can be used to image more deeply into bio lms directly One study used multiple methods to count bacteria from marine sediments 7074 of the cells were dead Only 4 were actively growing counting nucleoids and using antibiotic inhibition Of these 6 to 11 could be activated by adding nutrients carbon Suggests many dormant cells at any one time Luna et al 2002 AEM 6835093513 Sublimation used to concentrate adenine and biomass determined from adenine Galvin et al 2004 AEM 7059235928 presentation ATP estimation as an estimate of biomass 1 All cells use ATP in theory if you can estimate the amount of ATP then you know how many living bacteria there are Luciferase from re ies generally is used to detect ATP one molecule of ATP creates l photon with the enzyme resulting in 1 what Photons are counted in a sample from which the ATP has been extracted and an estimate of total ATP present is made Problem more active cells may have more ATP per cell nonbacterial cells may have ATP Not necessarily a perfect measure of number lipid analysis for diversity amp biomass Bene t it is much easier and less time consuming than counting cells Maybe slightly more expensive Tetrazolium salts reduction CTC uorescence 1 The inside of all active cells nonphotosynthetic is reduced 2 Tetrazolium salts go into the cells and are converted to formazan The formazan can be extracted and measured spectrophotometrically or cells with formazan crystals inside of them can be counted 3 Problem What environmental condition would interfere CTC expensive chemicals and equip 4 These counts show that 520 of bacteria are active that otherwise stain with acridine orange Measurement of DNA 1 Can isolate and purify DNA 2 React with uorescent dyes and can detect small amounts 3 Eukaryotic DNA and contaminants in isolation steps can interfere Other useful compounds for biomass L V Mb VV 6 V N V L V 4 5 1 Cell walls of most bacteria have muramic acid 2 Gramnegative bacteria contain lipopolysaccharide as part of the cell wall If this is extract it can be analyzed 3 Fungal cell walls have signi cant amounts of chitin can be analyzed 4 I 39 39 pigments 39 39 quot 39 39 etc can be measured Chloroform fumigation 1 Kill all of the microorganisms in soil with chloroform 2 Add new microorganism to killed soil and control unkilled soil 121 Methods 3 Measure amount of C02 evolved It should be greater in the killed sample because of the released organic C from the microorganisms there fore the C02 release rate is proportional to biomass some systems Menten hard to know speci c not easy to use dif cult to know speci c activity Need to use conversion factors as evolution radioactive or can estimate rates of use of effectiveness problems IV Productivity activity A Uptake of substrates 1 Generate an uptake curve for a radiolabeled substrate similar to a Michaelis Menten curve VmX is then the maXimum activity and this is dependent upon the total biomass Assumes VmX is the same for different species Can nd uptake of individual species Used 14C labeled phenol then isolated RNA from speci c species to show it was taken up into a growing cell Mane eld et al 2002 AEM 6853675373 B Synthesis of DNA 1 thymidine is added labeled with tritium and incubated Fraction containing DNA is extracted after a set amount of time Radiolabel is estimated good estimate of activity because it gives a rough estimate of growth DNA synthesis Some methodological problems including knowing what the actual thymidine concentrations are in the cell so knowing what your speci c activity is knowing how fast your pool is diluted 5 Is growth DNA increase total increase in mass total numbers of cells increasing C Synthesis of protein 1 Radiolabeled leucine is added 2 Protein extracted and counted on a scintillation counter after incubation 3 Correlates well with the thymidine method D Evolution of C02 1 Radiolabeled 14C organic material is added 2 CO2 is collected and counted in a scintillation counter to estimate the rate of respiration of the added substrate 3 Useful to determine the use of speci c substrates less useful determinant of the total community activity E Proportion of dividing cells has been counted to estimate growth rates F Preliminary indications that the amount of cellular RNA may be an indicator of growth probably works best with single species when growth is relatively slow Kemp et al 1993 AEM 592594 2601 Alternatively uorescence probes for rRNA have been used to estimate the number of ribosomes and thus growth Poulsen et al 1993 AEM 5913541360 both new techniques that V bWN VVV Jgt WM V VV V 122 Methods have not bee evaluated well yet Techniques to identify speci c species and numbers of bacteria in the environment A Marchin will talk about isolating and characterizing species present in a particular environment Still the problem of how many are there and how much Systems similar to the biolog have been developed that are more speci c for enteric bacteria Kuhn et al 1991 AEM 5731713177 B Flow cytometry 1 Move particles rapidly through a bacteria sized optical chamber 2 Can use lasers to look at light absorption 3 Speci c labels can be used to look at uorescence of sub populations 4 Can count many individual cells rapidly but may not work well when lots of other particles are around ie sediments Works best in open ocean sediments C Ribosomal rRNA analysis from natural populations 1 If rRNA can sequences can be taken from the natural environment they can be compared to known sequences Several techniques have been used 1 Since there are known sequences in all rRNA s DNA has been isolated from natural populations the polymerase chain reaction has been used to amplify the genes and the resulting ampli ed DNA cloned and sequenced ii Alternatively the 16S RNA can be isolated and complementary DNA synthesized and that DNA cloned Two initial studies of natural microbial populations using these techniques have been published 1 Isolates from Octopus Spring in Yellowstone national park a hot spring have been sequenced rRNA was cloned Of 8 sequences retrieved only 1 had been cultured before 4 belonged to cyanobacteria like organism large just not cultured yet and 1 was a group not yet reported Weller et al 1991 AEM 5711461151 ii rRNA from Sargasso sea ultra oligotrophic center of ocean was isolated and cloned Britschgi and Giovanonni 1991 AEM 5717071713 In this study 51 16 S rRNA distinct genes were isolated and partial sequences found these sequences fell into 7 distinct groups 2 of these 7 groups failed to correspond to any know cultivated and sequenced microorganisms The sequences related to the bacteria sized phyto picoplankton that had previously been cultivated from the Sargasso sea and sequenced were not the same These results suggest that unknown bacterial species are present in large numbers in this habitat 4 rRNA probes are being developed now for common groups of freshwater bacteria Zwart et al 2003 AEM 6958755883 presentation 5 Can be used in concert with other techniques to elucidate spatial patterns and community composition Kindaichi et al 2004 AEM 7016411650 presentation D Using antibodies to detect speci c organisms Antibodies can be produced to speci c species of bacteria Monoclonal antibodies are highly speci c Fluorescent molecules can be attached to the antibodies the antibodies reacted with the natural 1 l 39 39 and 39 1 used to identify labeled bacteria some probes use phycobiling conjugated to the antibodies Very uorescent Vibrio cholerae studies by Rita Colwell or the natural occurrence of the bacterium 1 Causes dysentery if not treated early with antibiotics can cause death by dehydration Currently an epidemic in south America ii At the time of her studies there was an epidemic in Bangladesh in a estuary area large portion of country on a estuary river delta iii Tried to isolate the bacterium on agar known to support growth could not do it with N V L V N V 123 E Methods natural environmental samples mud iv Fluorescent antibodies showed that the pathogen was present in the waters v Found if the mud was injected into living rabbits and then serum from the rabbits was cultured that bacterium could be isolated vi NeXt used the antibody on the Mississippi river delta Found it there health of cials accused research laboratories of releasing it into the environment Subsequently tested a large number of estuaries found out that Vibrio cholerae is present in many estuaries throughout the world vii Shows how isolation techniques do not always give the true picture 3 Monoclonal antibodies have been used to detect the pathogens Giardia and Cryptosporidium in natural waters LeChevalier et al 1991 AEM 5726102616 including Marchin and Upton here i Tested the source waters of 66 water treatment plants from across the eastern and north central US ii Found 81 had Giardia and 87 had Cryptosporidium 97 of the samples had one or another in Had recovery rates using known additions of the assay of 48 and 42 respectively Suggest that treatment standards set by EPA are not stringent enough 4 Immunogold antibodies have been used to identify Legionella pneumonphila in bio lms of compleX communities Rogers and Keevil 1992 AEM 5823262330 Campbell et al 1994 Plankt Res 163551 lt 5 pm cells from eukaryotic microplankton of antibodies to culture off of what were thought to be most common 4 species only hit about 10 of total number Using gene probes to detect individual organisms in the natural environment 1 If a DNA sequence is known for a speci c organism desired to detect and that sequence is speci c for that type Then a complementary DNA can be labeled with radioactivity or with a uorescent probe and the probe can be used to detect the gene in the environment 2 The genes for nitrogen xation have been sequenced Can be used to detect the location of nitrogen Xing bacteria in an environment Kirshtein et al 1993 Mar Ecol Prog Ser 95 305309 i Different types of organisms have slightly different sequences of nif genes probes can be designed to be speci c to certain groups of nitrogen Xing organisms ii Has been applied to mats of 39 39 39 showing bacterial populations within the community that are able to X nitrogen Micro autoradiography with added 14C combined with uorescent probes for nitrogenase showed that photosynthesis occurs in the center of the laments and nitrogenase at the terminal ends 3 Now taken a step further in using microarrays gene reactions spotted onto glass slides or chips Has been used for genes for N cycling Wu et al 2001 AEM 6757805790 4 Has also been successfully used to nd bacteriophages in the natural environment Ogunseitas et al 1992 AEM 5820462052 5 polymerase chain reaction can be used to amplify the signal and detect low numbers of microorganisms in the environment Bej et al 1991 AEM 5735293534 i Filter the cells on Fluorpore lters others inhibit the reaction ii Amplify the DNA by PCR on the lter in Then probe for the desired gene iv Can detect as few as 1 cell per 100 ml with this technique 6 High throughput DNA sequencing can be used to identify individual genes in the environment regardless of what genes they are associated with 12 million new genes from 1500 liters of sea water Falkowski and de Vargas 2004 Science 3045860 Ventner et al 2004 Science 3046674 presentation 391 124 7 so V O V Methods Nitrate reductase gene was isolated and characterized to probe for denitrifying bacteria Smith and Tiedje 1992 AEM 58376384 i Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from 5 separate genera but not from nondenitrifying bacterial genera 6 tested ii Detected bacteria in an aquifer microcosm a bioreactor and denitrifying toluene enriched soils denitrifying bacteria could not necessarily be cultured from all of these habitats An alternative technique is to make a lter with areas of known DNA sequences on it sort of like biolog system extract DNA from the natural population label it and see which spots on the lter retain the label Voordouw et al 1991 AEM 5730703078 i This technique was used to identify sulfate reducing bacteria in oil eld samples ii Showed that at least 20 different genotypes were present in these samples iii Has since been used to ngerprint and compare microbial communities from different oil elds Voordouw et al 1993 AEM 594114114 iv If sequences that are highly replicated in the microorganisms are chosen the probability ofa positive is much higher Waterhouse and Glover 1993 AEM 5913911397 rRNA sequences for identifying organisms i Sequences known for a wide variety of organisms ii Some regions are hypervariable and can be used to distinguish between species others are general to all species iii A three kingdom probe protocol has been set up where the three probes exclusively label eubacteria archaebacteria or eukaryotic ribosomal DNA has been used in natural communities Hanks et al 1992 AEM 5821582163 iv Individual subspecies can be detected as well such as Lactococcus lactus subsp cremoris Salama etal19915713131318 21 Isolated sequence from the subspecies that makes the best cheddar cheese b Made a probe that did not cross react with other less desirable subspecies c Makes it possible to rapidly identify the species using culture techniques is much more cumbersome v Works in drinking water also Manz et al 1993 AEM 5922932298 and for sul de reducing bacteria in bio lms Amann et al 1992 AEM 58614623 vi Found novel Archaea 16S sequences in Yellowstone Made 16S probe conjugated to a uorescent molecule Took sample stained it found single stained cell on microscope Used optical tweezers to isolate it and added it to culture medium Huber et al 1995 Nature 3765758 vii Application to deep sea water suggests several uncharacterized types of bacteria Suggests that uncultivated archaebacteria dominate the deep sea and may be the most abundant group of organisms on earth Fuhrman et al 1994 Microb Ecol 28133145 viii 16S rRNA used to identify a bacterium isolated from Baltic Amber as Bacillus subtilis Amber was dated as 2030 million years old Hamamoto amp Hoikoshi 1994 Biodiv Conserv 3567572 ix It is important to freeze samples for analysis immediately because they can change composition after several hours Rochelle et al 1994 FEMS Microbiol Ecol 15215 226 x Used 16S rRNA probes for Fe amp Mn reducing bacteria Shewanella putre faciens Showed that in situ probing was as sensitive as enrichment cultures using media where FeH precips with sul de DiChristina amp DeLong 1993 AEM 5941524160 xi The amount of rRNA varies twofold through cell cycle so intensity of labeling may not give much information Ruimy et al 1994 FEMS Microbial Ecol 15207214 Also can use rRNA for diversity estimates Use PCR to amplify 1 RNA sequences and y to 125 Methods compare sequences Have shown that bacterial community in streams can be altered by human disturbance Cody et al 2000 Hydrobiology 432207215 F Very small scale sampling for bacterial diversity 1 Now can take 1 pl samples and identify bacteria in samples Difference in species composition at less than 1mm in seawater was found Long and Azam 2001 Aquat Microbial Ecol 26103113 Samples taken lysed ampli ed by PCR and 16S rRNA sequence diversity determined by denaturing gradient gel electrophoresis 126 BIOL 687 Spring 2005 Microscale techniques Microbial Ecology Topic 23 Microscale Techniques 1 Many new microscale identi cation and habitat characterization techniques are being developed Molecular and microsensor techniques are at the forefront of microbial ecology and their use will only increase Amann and Kuhl 1998 Current Opinion in Microbiology 1352358 H Oxygen microelectrodes A Measure oxygen by measuring the ow of electrons amperage between a cathode and a reference anode 1 Use a gold surface and a membrane permeable to oxygen 2 Size of probe dictates its response because of the scale of diffusion At very small size the rate of diffusion is sufficiently large to negate any requirement for stirring as there is in larger scale oxygen electrodes The smaller the tip the more rapid the response Generally constructed for environmental research with tips lt lOum Fick s law of diffusion dCdT D dCdX Where D diffusion coef cient c conc t time x distance B Two types Clark type and cathode type 1 Cathode type simple 2 Clark type has an internal reference has added advantage of insensitivity to sul de will kill a cathode type electrode 3 Extra consuming probe can be placed inside to remove back ow of oxygen l27 BIOL 687 Spring 2005 Microscale techniques C Measuring photosynthesis with an Oz microelectrode 1 When oxygen is at steady state Photosynthesis respiration 2 When lights go off photosynthesis stops and rate of respiration the former rate of photosynthesis D One important use in microbial ecology has been to measure the oxygen gradients at the sea bottom 1 Need special probes which can stand great pressure 2 Send down on sea bottom landers When the lander hits the bottom the probes are gradually lowered into the sediments remotely and data is collected continuously Usually several probes are attached because they break readily and this is a way to ensure data and measure heterogeneity This is important because the oxygen pro le in sediments determines what the rates of organic mater decomposition will be and the rates of inorganic chemical deposition Deposition is the main loss of inorganic chemicals from the ocean Eventually all dissolved inorganics are remixed but if they sediment they are lost to the system until much longer terrn processes geological bring them back to the surface if ever Ocean biogeochemistry and productivity thus can be controlled by what happens on the sea oor E New method planar optrodes A lm that is sensitive to oxygen clear on back Chemicals uoresce depending upon Oz concentration Can use microscope to get detailed crosssectional information May require time for community to grow naturally against the optrode L V 111 Microscale measurement of lightfor photosynthetic measurements and ne scale pigment determination A Small optical bers have high resolution with a diameter of about 100 pm Can collect light over very small size scales B The end of these bers can be made pointed to an even smaller sensing tip C After the light is collected it needs to be sensed by a very sensitive detection system single ber does not carry much light 1 a sensitive photodiode can be used but this may be sensitive to a wide range of wavelengths this can be altered by addition of lters to make the photodiode sensitive to speci c wavelengths 2 Spectral radiometers can be attached with a monochrometer to render the photodiode response to only a few nanometers D Spectral distribution of light in microbial mats has been used to determine the position of pigmented microorganisms within the mat This is useful because the position can change with time if they are motile New species have been cultured following identi cation of a unique pigment signal E Smallscale photosynthesisirradiance pro les can be measured with this technique Addition of a spherical sensing tip can make the probe able to sense irradiance from all directions without this light is only sensed in a narrow cone 128 VI 3 3 0 V 3 03 V BIOL 687 Spring 2005 Microscale techniques pH microelectrodes Same as larger scale pH electrodes glass which allows only hydrogen ions to go across is put in solution with a voltage potential between the probe and a reference The H ow into the probe causes an electrical current to ow The techniques for this in microbial ecology have been taken from microphysiology where pH probes are constructed to measure pH in single cells Useful when measuring sul de with microelectrodes because sul de can be a gas or an ion given different pH and sul de probes are only sensitive to ions Sul de microelectrodes Similar to oxygen probe except that they are coated with silver instead of gold and no membrane is necessary Sensitive to SZ39 and HS39 but not HZS so need pH measurements to calculate the total sul de present Useful in highly anaerobic mats where sul de is produced by sulfate reduction in anaerobic portions and consumed in aerobic portions by biological and abiological sul de oxidation Data from microbial mats shows diurnal variation in sul de pH and Oz Nitrous oxide microelectrodes Similar to oxygen microelectrodes except a silver cathode is used which is sensitive to N20 The silver is also sensitive to 02 so an oxygen microelectrode is put in a case right in front of the and behind the N20 sensor to remove oxygen before it hits the silver sensor tip N20 is in the pathway from nitrate in denitri cation 1 If acetylene is added the conversion from nitrate to N2 is stopped or blocked at N20 2 The probe can be used to sense denitri cation with high sensitivity 129 VII 3 03 V VIII 1X 93 59 gt11 V X 3 BIOL 687 Spring 2005 Microscale techniques Ion speci c electrodes Speci c polymer membranes have specialized permeability to speci c ions Many different macroelectrodes present for ammonium nitrate uoride chloride bromide etc Generally require fairly high concentrations to get good measurements Microelectrodes have been made for nitrate and ammonium After the speci c membrane is added the measurement process is similar to pH measurement where the ow of ions causes a current which can be measured and is proportional to ion concentration Small scale microscopy Laser scanning has become an important way to optically section bio lms Lawrence et al 2003 AEM 6955435554 presentation Biosensors A new eld not yet applied to microbial ecology but it will In this technique bacteria with speci c properties are immobilized upon the surface of a microprobe When they come into contact with a speci c substrate they metabolize more rapidly and create a measurable electrical current For example a bacterium could be selected for which is highly selective for glucose this bacteria could be used to sense smallscale variations in the levels of glucose Alternatively enzymes can be attached to these directly and the byproducts then can be sensed Another twist is to add bioluminescent bacteria and attach to a sensitive optical ber when their rate of metabolism increases the light generated will increase Bioluminescent LUX gene Pseudomonas has been used as an organism to screen toxicity of xenobiotic compounds Boyd et al 1997 Chemosphere 919671985 The more toxic the less they glow because the lower their metabolism An interesting biosensor to detect the interaction of bacteria with solid surfaces Bacteria are attached to a forcesensor used in a force microscope to sense interaction strengths Lower et al 2001 Geomicrobiology 186376 Silicon technology pH electrodes have been constructed upon silicon wafers can make 100 s of pH probes only several um apart with this technology May make very precisely controlled microelectrodes by this technique some day Will probably be combined with biosensors Microscale sectioning Cytological techniques can be used to nely section portions of mats can make sections as thin as 1 pm Can be used to sense ne scale rates of nitrogen xation add 14N2 gas incubate section and analyze for 15N in the organic material Can be used in conjunction with electron microscopy to determine morphologically distinct species this doesn t work often because of the lack of morphological differences between species of bacteria Confocal microscopy Microscopes can be used to make optical sections through transparent 130 BIOL 687 Spring 2005 Microscale techniques material by focusing on different planes can construct three dimensional images XII Microscale measurement of ow A Can use very small beads with small diameter Take photograph with the shutter open for a known amount of time and the length of the streak is related to its current velocity B Pitot tubes Pressure at face is transmitted to height at the top of the tube Can not work very small because viscosity gets to high C Thermistor probe Can get 1 mm resolution Use 2 thermistor Each thermistor has a resistance which is proportional to temperature The reference probe has a low resistance so additional current does not change the temperature of the probe itself The sensing probe has a high resistance so it heats itself as the current passes through it The probe measures the amount of electrical current it takes to heat the probe to 10 C higher than the reference It takes more and more current as the water velocity increases because the rate of heat transfer away from the probe increases with the rate of heat transfer from the surface of the probe XIII Optical trapping Use laser beam under a microscope to trap cells can be removed from complex community and cultured in unicellular culture Michell et al 1993 Microb Ecol 25113119 131 BIOL 687 Spring 2005 Human microbial ecology Microbial Ecology Topic 24 Human Microbial Ecology I 11 III In general there are bacteria that do not obviously harm the person The general idea is that these bacteria outcompete harmful types and are not pathogenic to humans unless the person is debilitated ie stressed autoimmune de cient etc E B E E 03 V For example normally 107 Salmonella necessary to initiate a gastric infection in individuals treated previously with antibiotics 10 cells can initiate an infection Some investigators are researching the effects of supplementing gut bacteria to protect against diseases or eating foods that stimulate bene cial bacteria Gibson and Rastall 2004 ASM news 70 22423 1 Understanding microbial ecology may be important in understanding human infections For example virulence factors that allow Candida albicans to infect other fungi may allow this fungi to infect humans Hogal and Kolter 2002 Science 2962229 Other research has 39 39 the 39 l of 39 l T l 39 39 for iron in the pathogen Pseudomonas aeruginasa G1if n et al 2004 Nature 10241027 presentation Oral micro ora Habitats 1 Anaerobic to aerobic 2 Top of tongue has many ssures inhabited by microorganisms 3 Soft mucosal membrane characterized by rapid cell growth and continuous sloughing 4 Teeth hard surface to attach to lots of abrasion 5 Pouch between teeth and gums has large populations 6 All habitats have lots of washing by saliva and associated enzymes At least 200 species have been cultured with more being cultured all of the time 1 Some are found in most everybody eg Streptococcus sp that can be found in most humans and many animals 2 Others are fairly speci c for example the yeast Candida albicans is found in only 3050 of humans tested Normal ora can cause disease under speci c conditions 1 Sugar increases acidity by causing more anaerobic zones 2 Lactic acid is formed in these zones 3 Attaches the surface of teeth 4 Pits form with a more permanent anaerobic zone 5 pH continues to drop cavities form 6 Also infections can occur associated with denture use The normal sloughing of skin is retarded and the microbes are not removed fast enough Intestinal tract Esophagus 1 High rates of sloughing hot drinks washed with 15 L of saliva per day 2 Tends to have low numbers of microbes Stomach Initially thought that stomach contents were sterile This is true in western cultures 12 hours after fasting needed people to fast so they don t vomit much when they stick in the sampling tube pH low 1825 with optimal acid excretion Most intestinal microorganisms are not tolerant of pH levels below 5 how does intestine become colonized Infants have little stomach acid not fully developed until 1520 days after irt Later it was shown that there is a diurnal uctuation in the bacterial numbers see handout 1 V N V L V 132 BIOL 687 Spring 2005 Human microbial ecology 4 Helicobacterpyloris has been found in the stomachs of people with ulcers Olson and Maier 2002 Science 2981788 presentation Stimulated by hydrogen stimulate own hydrogen by hydrogenase i Found in the mucosal layer which is separate from the low pH stomach ii Also can be cultured from people with no ulcers may be an opportunistic infection 5 In areas with high ber diets low nutritional value there are often signi cantly more bacteria associated with the stomach contents Small intestine 1 Still a hostile environment Bile salts and proteolytic enzymes in high levels in upper parts 2 In Western populations 104ml bacteria in South American and Indian populations the levels are generally higher than this 3 Where higher levels of bacteria are found mostly gram cocci the intestinal villi are leaf like in western populations they are nger like What is cause and what is effect 4 Still more bacteria than in stomach less than in large intestine D Large intestines 1 Bacterial numbers high many are fastidious anaerobes Facultative anaerobes scavenge any oxygen very effectively Gut populations form quickly after birth Breastfed babies primarily have Bifidobacterium and formula feed babies have a more complex microbial community by 2 years of age a more adult composition occurs with more that 500 bacterial species Gibson and Rastall 2004 ASM news 70224231 Distinct successional patterns evident from birth to adult with diet determining the nal climaX state Uncooked vegetarian diet shifts bacterial ora as shown by GLC analysis ofbacterial fatty acids Petonen et al 1992 AEM 5836603666 Obligate anaerobes numerically dominant with at least 3040 species consisting of 99 of the total bacterial mass Most common genera Bacteroides Bi dobacterium and Eubacterium Bacteroides is number 1 Initially thought that Escherichia coli was the dominant but this is a facultative anaerobe Could be isolated in aerobic conditions and was not harmed by them When anaerobic techniques were developed it became clear that they are only a minor component Still useful to indicate fecal contamination because they can survive in the outside environment and can be counted there 5 May assist in digestion of food about 10 of daily energy content is from organic acids from microbial fermentations Gibson and Rastall 2004 ASM news 70224231 May ultimately be used to deliver drugs to humans Hooper and Gordon 2001 Science 29211151118 Also may be important in transfer of antibiotic resistance to pathogenic bacteria the antibiotic resistance can develop in nonpathogens but be transferred in the gut where populations are high and many bacteria are in close contact with eachother Andremont 2003 ASM news 69601607 Giardia and Cryptsporidium are common water contaminants that cause gastric upset in adults i Can be detected in 97 one or the other of surface water samples taken in the United States and Canada ii In virtually all surface waters in Kansas iii Even in all high mountain surface waters don t drink the water without treatment when backpacking Urogenital bacteria A Often thought to be mostly sterile B Have found very small bacteria 0205 pm in diameter in the blood and many organs C May be implicated in formation of kidney stones form calcium shells that may trigger larger 0 V 2 V L V 3 V O V l V so V 133 VI 93 599 VII A V B C D VIII BIOL 687 Spring 2005 Human microbial ecology deposits Vogel 1998 Science 281153 Female Genital tract Upper mostly sterile lower has signi cant populations Lactobacilli probably most common between 4595 of cells 1 Thought that they ferment organics resulting in lactic acid lowering pH and restricting the species present to those which can tolerate lowered pH levels 2 At least 60 other species of bacteria have been isolated Cervix may exhibit a unique ora there may be other subhabitats in the vagina Menstrual cycle pregnancy menopause can all alter the populations found Lactobacilli have been shown to inhibit a large number of species of disease causing bacteria Skin Major components from three groups LI w coryneforrns and gram negative principally Acinetobacter Humidity a major factor The high salt content makes the skin even drier for microorganisms Increasing moisture at the surface of the skin immediately increases bacterial counts Several distinct habitats see handout 1029 Immunoglobins occur directly on the skin and may retard microbial growth Lysozyme is also oun n 1 1 Mycobacterium tuberculosis Hallovan 1994 TREE 97275 Most common cause of death in world Some populations have up to 33 rates of infection Enters lungs goes to lymph nodes immune system response encapsulates bacteria usually without much spread In adults it infects lungs in children it affects the bones 60 die of disease in developing countries the number is lower as health increases For example during WWI people had poor nutrition and disease rate went up In the US levels dropped until 1985 since then 39000 more cases have occurred than would be expected given the decreasing trend There are 2 drugs 30 of isolates can resist one 19 both 134
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