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UC / Biology / BIOL 4011 / What are some aseptic techniques used to prevent contamination?

What are some aseptic techniques used to prevent contamination?

What are some aseptic techniques used to prevent contamination?


School: University of Cincinnati
Department: Biology
Course: Microbiology
Professor: Dennis grogan
Term: Fall 2019
Tags: Microbiology
Cost: 50
Name: Exam 2 Study Guide
Description: These are filled out outlines for the exam!
Uploaded: 10/10/2019
31 Pages 81 Views 11 Unlocks

Outline 5a concepts batch culture; four phases, exponential growth continuous  culture; measurement methods constraints on microbial growth (temperature, pH,  solute conc., oxygen)  

What are some aseptic techniques used to prevent contamination?

 Microbiologists grow huge populations of clonally pure microorganisms   Culture media  

o complex vs. defined  

 Culture media: nutrient solutions used to grow microbes in lab  Defined media: exact chemical composition known

 Complex media: composed of digests of microbial, animal, or  plant products  

 Enriched media: contain complex media plus highly nutritious  materials

 Used to culture fastidious microbes

o liquid vs. solid  

 Liquid has all nutrients  

 Solid- Agar as gell aid- no nutrients

 Solid media are prepared by addition of gelling agar to  

What happens during the stationary phase of cell growth?


 Solid help isolate cell masses

o selective vs. Diagnostic

 Selective media: contains compounds that selectively inhibit  growth of some microbes but not others  

 Differential media: contain an indicator, usually a dye, that  detects particular metabolic reactions during growth

o aseptic transfer: series of steps to prevent contamination during  manipulations of cultures and sterile culture media, both liquid and  solid We also discuss several other topics like How can the franchisee lose his right in operating a franchised business?

 Flaming in order to sterilize the loop and tube

o isolated colonies: visible, isolated masses of various shapes and  sizes depending on the organism, the culture conditions, the nutrient  supply, and other physiological parameters  

Why do bacteria die during the death phase?

 colony morphology depends on culture  

 Batch culture: a closed-system microbial culture of fixed volume o Typical growth curve for population of cells grown in a closed system is  characterized by 4 phases

o fixed volume of medium leads to four distinct culture phases   lag phase (cells re-adjusting metabolism) We also discuss several other topics like What is bride price payment?

 Interval between inoculation of a culture and beginning of  


 Time needed for biosynthesis of new enzymes and to  

produce required metabolites before growth can begin

 Exponential phase (accelerating) 

 Cells in this phase are typically in the healthiest state  

 Stationary phase (metabolic shut-down) 

 Growth rate of pop is 0

 Either an essential nutrient is used up or waste products  


 Metabolism continues at greatly reduced rate

 Some cells grow while others die, balancing  

 Death phase (cell damage) 

 If incubation continues after cells reach stationary phase,  cells will die

 Exponential rate

 Typically much slower than exponential growth

 Viable cells remain for months or years

o  Exponential growth: growth of a microbial population in which cell  numbers double within a specific time interval We also discuss several other topics like What was the main philosophy of the renaissance?

o basis = rate of population increase is proportional to population size  (all cells are reproducing)  

o Relationship exists between initial number of cells present in a culture  and the number present after a period of exponential growth

o Microbial cells undergo exponential growth, and a semilogarithmic plot  of cell numbers with time can reveal the doubling time of the  population.  If you want to learn more check out What is a person who hides their feelings called?

 Various growth expressions can be calculated from cell number  data obtained from an exponentially growing culture.

 Key expressions here are n, the number of generations; t, time;  g, generation time, and k, the instantaneous growth rate  


o derivation: dN/dt = µN N = Noe µt 

o alternate forms N = No2vt 

o N=N(o)2^n

 N is final cell number

 No is initial cell number

 n is the number of generations during the period of exponential  growth  

 dN/dt= kN, N=N(o)e^ut , N=No*10^kt  

o Generation time (g) of exponentially growing population g=t/n  T is the duration of exponential growth  

 N is the number of generations during the period of exponential  growth

o Consequence: population size increases by same factor over any time interval of a given length  We also discuss several other topics like After hitler came to power, which division of the military did he deem a threat?

 Ex. 1/v = g (doubling time); 1/k=increase 10-fold  

 Continuous culture: an open system microbial culture of fixed volume  o Chemostat: most common type of continuous culture device;  continuously adding medium and removing medium plus cells o Both growth rate and population density of culture can be controlled  independently and simultaneously If you want to learn more check out Who introduced the palladian style to england?

o Depends on

 Dilution rate: F/V (F is flow rate of adding fresh medium and  removing spend medium, V is culture volume)

 Concentration of a limited nutrient  

 Steady state: cell density and substrate concentration do not change over  time  

o represent exponential growth where µ = D (experimental control) o Exponential uses

o Maintain exponential phase for weeks/months

o Used to study physiology, microbial ecology and evolution, enrichment  and isolation of bacteria from nature

 Measuring microbial growth  

o primary goal: measure concentration or titer (cells/mL)  

o microscopic cell counts: 

 require special slides and phase-contrast/darkfield microscope   Observing and enumerating cells present  

 Counting chambers with squares etched on a slide for liquid  sample  

 Steps  

 Sample added

 Microscope observation; all cells counter as large squares

 Calculate # by multiplying cells and squares

 Plate count  

 **Limitation of microscopic cell counts  

 Cannot distinguish between live and dead cells without  

special stains  

 Precision is difficult to achieve  

 Small cells can be overlooked

 Phase-contrast microscope required if stain is not used

 Cell suspensions of low density hard to count  

 Motile cells need to immobilized  

 Debris in sample can be mistaken for cells

 ****direct microscopic count reveal more organisms than other  counting options because they show dead cells as well

o viable counts: measurement of living, reproducing population  spread plates vs. pour plates vs. membranes; quantitative  dilution  

 Spread-plate 

 Sample is pipetted onto surface of agar plate

 Sample is spread evenly over surface of agar using sterile  glass spreader  

 Incubation

 Colonies seen

 Pour-plate 

 Sample is pipetted into sterile plate

 Sterile medium is added and mixed with inoculum

 Surface and subsurface colonies  

 Count colonies on plates w 30-300 colonies  

 To obtain the appropriate colony number, sample counted  should always be diluted  

 Series of dilution  

 **Sources of error in plate counting  

 Depends on inoculum size, viability, culture medium,  incubation conditions

 Mixed cultures grow at different rates

 Plating inconsistencies

 Reporting in colony-forming units instead of viable cells  accounts for clumps

o Turbidity: measures of microbial cell numbers

 photometer measures apparent absorbance (light scattering)   Cell suspensions are turbid (cloudy) because calls scatter light  Most often turbidity is measured with a spectrophotometer and  

measurement is referred to as optical density at specific  wavelength  

 For unicellular organisms, OD is proportional to cell number  within limits  

 To relate a direct cell count to a turbidity value, a standard curve must be established  

 Advantages  

 Quick and easy to perform

 Typically do not require destruction or significant  

disturbance of sample  

 Same sample can be checked repeatedly  

 Disadvantage  

 Sometimes problematic

 Not always accurate

o each method has strengths & weaknesses; none of them suited for  natural microbial communities

 **The diversity of micro-organisms is seen in durability & adaptation to  environmental conditions

 Temperature  

o Temp is a major environmental factor controlling microbial growth o Cardinal temps: minimum, optimum, max temp at which an organism  grows  

o Range of <40 degrees C

o  plots of growth rate T look similar in shape for all microbes &  define 3 “cardinal” temps.

 can be considered a parallel to the corresponding behavior of  enzymes  

 below Topt, thermal energy contributes to enzymatic catalysis   above Topt, thermal energy destroys catalyst (enzyme  

thermostability of species tracks Topt)  

 limits to this analogy:  

 growth actually hits zero (unlike reaction rates)  

 cells can adapt physiologically (HSPs, etc.)  

 Microorganisms can be classified into groups by their growth temp optima o Psychrophile: low, found in cold environments; glaciers, polar oceans  Organisms with

 optimal growth temp <=15 degrees C

 Max <=20 degrees

 Min <=0 degrees  

 Inhabit constantly cold environments  

 (sensitive to ‘cool’ T)----- glaciers, polar oceans (regions of  constant cold)

o Psychrotolerant/psychotroph: organisms that can grow at 0  degrees but have optima of 20-40 degrees

 More widely distributed in nature than psychrophiles  

 Isolated from soils and water in temp climates and food at 4  degrees

 soils, lakes, refrigerated foods

o Mesophile: midrange, most commonly studied; many environments  o Thermophile: high, found in hot environments; hot springs, hot water  heaters  

 Organisms with growth temp optima between 45-80 degrees   Inhabit moderately hot and intermittently hot environments   hot springs, water heaters

o Hyperthermophile: very high, found in extremely hot habitats such  as hot springs and deep-sea hydrothermal vents; hot springs,  geothermal environments  

 Organisms with optima greater than 80 degrees  

 In hot springs  

 Chemoorganotrophic and chemolithotrophic species  


 Low generation time

 High prokaryotic diversity  

o Extremophiles: organisms that grow under very hot or very cold  conditions  

 hot springs, hydrothermal vents

 Enzymes and membranes of diff classification

o Psychrophiles  

 Enzymes: active in cold, flexible, denature  

 More polar and fewer hydrophobic AA

 Membrane: short-chain fatty acids, unsaturated  

o Hyperthermophile

 Enzymes: inactive in cold, rigid, stable  

 Stable at high temps  

 Hydrophobic interiors  

 Membranes: long chain, saturated

 pH  

o Like temperature  

 pH extremes can denature proteins

o Unlike temperature

 the CM can insulate cells from external pH  

o also, pH is a component of the PMF which is essential for all  prokaryotes pH + )R)  

o Types:

 Neutrophiles: organisms that grow optimally at pH 5.5-7.9  small ΔpH, Δ R inside-negative

 Inside cell is negative  

 Acidophiles: organisms that grow best at low pH (<5.5) acid   large Δ pH but Δ R inside-positive

 Δ pH large and inside can be positive

 Alkaliphiles: organisms that grow best at high pH (>=8) basic  Δ pH inverted, Δ R very large

 ΔpH is small and inside is negative (strongly –ve)  

 Have alternative ATPase  

o Use sodium motive force rather than proton motive  

force because more available

o Effect of pH

 Intracellular pH must stay relatively close to neutral even if the  external pH is highly acidic or basic  

 Buffers  

 Cells have a net negative charge

 Solute concentration (water activity)  

o Water activity (aw): water availability; the ratio of vapor pressure of  air in equilibrium with a substance or solution to the vapor pressure of  pure water  

 Varies from 0 (no free water) to one (pure water)

o Osmosis: water diffuses from high to low concentrations   Cytoplasm has a higher solute concentration than surrounding  environments---> water moves into cell (positive water  

balance)--> hypotonic 

 When a cell is in an environment w higher external solute  concentration, water will flow out unless the cell has a  

mechanism to prevent this --> hypertonic 

o Cell wall resists diffusion of water and this creates a pressure (2-10  atm)  

 Cell maintains turgor pressure and works to do it- solute stress   Purpose to maintain concentration  

 from low to high stress: they will  

o increase salts

o Increase intake of K+  

o compatible solutes: used by cell to maintain  

positive water balance; compatible with cell  

structure and content to maintain turgor pressure  

o Pumping solutes from environment to cell

o Synthesize cytoplasmic solutes  

o Highly-water soluble

o most prokaryotes are adapted to hypotonic conditions and they require turgor for growth  

o respond to external solute by increasing internal solute; these may  include specialized compounds  

 some archaea & bacteria have ‘innate’ adaptation to low water activity

o Halophiles: organisms that grow best at aw= 0.98 (seawater); have a  specific requirement for NaCl

o Halotolerant: organisms that can tolerate some additional dissolved  solutes but generally grow best in the absence of the added solute  o Extreme halophiles: organisms that require very high levels (15- 30%) of salt; often unable to grow at lower concentrations  


o Osmophiles: organisms that live in environments high in sugar as  solutes  

o Xerophiles: organisms able to grow in very dry environments

 Oxygen  

o not essential, not available everywhere, potentially reactive; cells need defense systems

o oxygen defines different classes of micro-organisms  

o Aerobes: requires oxygen (respiration) and grow at full oxygen tension o Anaerobes: cannot respire oxygen

o Types of microorganisms  

 obligate aerobe: require oxygen to survive

 require oxygen

 lack fermentation pathways  

 Aerobic respiration

 facultative aerobe “anaerobe”: can live with or without  oxygen  

 grow better with O2  

 Aerobic and anaerobic respiration and ferment pathways  

 aerotolerant: tolerate oxygen and grow in its presence even  though they cannot respite

 O2 -indifferent

 oxygen has no effect

 lack aerobic respiration

 Has fermentation  

 Microaerophile: can use oxygen only when it is present at  levels reduced from that in air due to limited respiration or  oxygen sensitivity

 require low conc O2, lower than atmsopheric  

 have respiration, lack fermentation

 lack defenses  

 obligate (strict) anaerobe: inhibited or killed by oxygen   killed by oxygen, only grow anaerobically

 lack oxidation defenses and respiration  

 Fermentation and anaerobic respiration  

o simple qualitative test: Thioglycollate tube  

o strict anaerobes and micro-aerophiles are challenging to culture;  various techniques  

 Aerobes need extensive aeration  

 Anaerobes need O2 excluded  

 Reducing agents: chemicals added to reduce oxygen  o Complex medium separates microbes based on O2  


o O2 can penetrate only the top of the tube

o Microbes grow at diff heights based on O2 exposure

 A- obligate aerobes  

 B- anaerobes

 C- faculative aerobes  

 D- microaerophile  

 E- aerotolerant


Outline 5b concepts preventing contamination or infection diverse methods to kill  microbial cells  

 Decontamination: treatment of an object to make it safe to handle  Disinfection: directly targets the removal of all pathogens, not necessarily  all microorganisms

 Sterilization: defined as the elimination (killing) of all micro-organisms [incl.  viruses]  

o Heat sterilization is the most widely used method to control microbial  growth

o Lethal conditions usually cause an exponential decay  

 as with growth, the instantaneous rate constant can be  

expressed with different bases

 slope of semi-log plot yields rate of death; can be expressed as  “decimal reduction time” (could also use t1/2)  

o Decimal reduction time (D): amount of time required to reduce  viability tenfolds (steam is best)

 Cells: 65*C in 0.5 min

 Endospores: 121*C in 5 min

o practical aspects -  

 real microbial populations not homogenous  

 the more-durable organisms extend the “tail”  

 extreme case represented by endospores (very common in the  environment, very durable)  

o most effective: high T combined with moisture (synergism)  

 get both with steam under pressure (1 atm = 121 C)  

 Autoclave: sealed device that uses steam under pressure  

o Allow temp of water to get above 100*C  

o Kills endospores  

o Not the pressure but the high temp that kills microbes

o two modes (fast vs. slow exhaust)

o hazards associated with large volumes of liquids  

 heat-killing of endospores also enhanced by low pH [practical application to  home canning]  

 Heat sterilization conditions are harsh, damage many substances (foods, for  example)  

o Less drastic approach first developed by Pasteur for wine (prevented  spoilage)  

 Pasteurization: process of using precisely controlled heat to reduce the  microbial load of heat-sensitive liquids  

o Does not kill all organisms, diff from sterilization

o Brief heating (71*C for 15 sec)

o usually applied to raw milk / function = destroy Mycobacterium, other  pathogens  

o typical: 71 C for 15 sec; most of the bacteria survive (milk must be  refrigerated)  

 more extreme heating will essentially sterilize the milk (store at  room temp)

 Sterilizing by radiation  

o microwaves -  

 sterilize by heating  

o UV - sufficient energy to cause modifications and breaks in DNA (260  nm)

 Useful for decontaminating surfaces  

 Cannot penetrate solid, opaque, or light-absorbing surfaces  sterilizes by damaging DNA (pyrimidine dimers block DNA  replication) used on surfaces (has little penetrating power)

 short-wave (UV-C, 260nm) most effective  

o Ionizing radiation: electromagnetic radiation that produces ions and  other reactive molecules upon collision

 Some microorganisms more resistant to radiation than others   Used for diverse items  

 (gamma, X-rays) used commercially to sterilize wrapped  


 highly penetrating

 fragments DNA  

 sensitivity generally inverse to genome size  

 Filtration 

o simple principle - remove all microbial cells from fluids  

o Physical control methods  

o Avoids use of heat on sensitive liquids and gases  

o Pores of filter are too small for living organisms to pass through but do  not trap most viruses  

o Pores allow liquid or gas to pass through  

o Membrane filters function more like a sieve

o need sterile filter with very small pores  

 air (laminar flow hoods, etc.)  

 liquids (culture media, medical solutions)  

 Antimicrobial chemicals  

 -cidal --> kills microogranisms (-lytic)

 -static --> inhibits growth  

 Antibacterial agents classified as bacteriostatic, bacteriocidal  and bacteriolytic  

 Bacteriostatic agents: inhibit biochemical processes  

such as protein synthesis and bind weakly

 Bactericidal agents: bind tightly and kill the cell

 Bacteriolytic agents: kill by lysis  

o measuring effectiveness:

 MIC (quantitative, but depends on many variables)

 Minimum inhibitory concentration (MIC): smallest  

amount of an agent needed to inhibit growth of a  


 Kirby-Bauer (fast, easy, but gives only relative effectiveness)   Disc diffusion assay 

 uses solid media

 Antimicrobial agent added to filter paper disc, diffuses  

into agar  

 MIC reached at some distant  

 Zone of inhibition: area of no growth around disc  

 Steps:

o Inoculate plate w liquid culture of test organism

 Nutrient agar plate  

o Discs containing antimicrobial agents are placed on


o Incubation

o Test organisms shows susceptibility to some  

agents, indicated by inhibition of bacterial growth  

around discs

 Zones of inhibition

 Sterilant, disinfectants, sanitizers, and antiseptics all used to prevent growth  on inanimate surfaces and external body surfaces

o Sterilant/sterilizer/sporcide: destroys all microorganisms including  endospores

 needed for objects that must be sterilized but cannot be  

autoclaved (examples: ethylene oxide, formaldehyde, H2O2,  peroxyacetic acid, bleach)

o Disinfectants: used on surfaces to kill microorganisms but not  necessarily endospores  

 milder, may not kill endospores, intended primarily for surfaces,  objects

o Sanitizers: reduce microbial numbers but do not sterilize   milder, may not kill endospores, intended primarily for surfaces,  objects

o Antiseptics (germicides): kill or inhibit microbial growth but are  nontoxic enough to be applied to living tissue

 Used on skin

 Determined by FDA

Outline 14a concepts  

importance of harvesting energy & electrons from environment multiple strategies  of phototrophy in prokaryotes -------  

 Many bacteria & archaea are physiologically durable -- could any of them  colonize another planet?  

o What are the critical environmental resources that a single cell needs  in order to grow and reproduce?

 Sources of Energy

 Carbon, P, H, N, S... other elements found in cell material  

 Reducing equivalents (e-)

 Water

 -> a cell that harvests energy can use it to drive many essential reactions  o i) take electrons from an environmental source, even if it is not very  ‘reducing’  

o ii) transfer them to CO2 to make cellular material  

o iii) transfer them to N2 , SO4 , etc.  

 This explains the importance of photosynthesis, which combines two  distinct processes:  

o energy harvest = ‘phototroph’ vs. CO2 fixation = ‘autotrophy’  o conversion of light energy to chemical energy  

o Take electrons from Enironmental sources and transfer to CO2  o Phototrophs carry out photosynthesis  

 Also autotrophs that use CO2 as sole carbon source

 Use light to reduce CO2 to organic compounds  

 Photoheterotrophs: phototrophs that use organic carbon as  carbon source

o Light reaction: produce ATP

o Dark Reaction: reduce CO2 to cell material for growth

 Requires ATP and electrons (NADH/NADPH)

 NADH/NADPH requires electron donor from environment

 Green plants are photoautotrophs (phototrophy is a good way to support  autotrophy)  

o but many prokaryotes exhibit other metabolic strategies (e.g.  photoheterotrophs, chemoautotrophs)  

 How do cells capture light energy?  

o major players in the process:  

 Chlorophylls and bacteriochlorophylls 

 bacteriochlorophylls absorb in infra-red

 Use diff pigments that allow diff phototrophs to absorb diff


o critical features of these pigments:  

 absorb energy of a photon and retain it  

 transfer the energy without loss (“exiton”)  

 use the energy to oxidize one molecule and reduce another  

o the reduced molecule is high on the redox tower, it will be able to  initiate a series of ‘downhill’ e- transfers  

 photosynthetic membranes

o converting light into ATP requires a membrane  

o helps organize the components; enables formation of a PMF o Chlorophyll/bacteriochlorophyll are not free in the cell and is found in  photocomplexes containing proteins housed within membranes  reaction centers vs. antenna pigments  

o Reaction centers: contain some pigments and participate directly in  energy conservation

o Antenna pigments: surround and funnel light energy to reaction  centers

o most of the (bacterio)chlorophyll serves to capture photon’s energy  and transmit it to RC  

o RC contains specialized cluster of (bacterio)chlorophylls, site of ‘charge separation’  

 accessory pigments  

o Phycobilins: antenna pigments in cyanobacteria

 main light-harvesting systems

 Assemble aggregates called phycobilisomes that attach to  thylakoid  

 Pigments integrated into cytoplasmic membrane

o Carotenoids: protect other pigments

 Function primarily as photoprotective agents, quenching toxic  oxygen species and prevent dangerous photooxidation

 Hydrophobic accessory pigment that is embedded in membrane  Basic photosynthesis: the Purple Bacteria  

o Purple bacteria: phototrophic bacteria that make their own food and  pigmented due to diff pigments including various carotenoids that give  them this color; simple photosynthesis  

o membranes contain the reaction centers surrounded by antenna  pigments (all bacteriochlorophylls)  

o exitons migrate to special pair, makes it strongly reducing; an electron  is transferred to Bchla, then passed to quinones and down a series of  carriers; this forms a PMF  

 Exciton carry energy and roam around to give energy (electrons  in this case)

 Light hits, makes strong donor, electrons get cycled around and  makes PMF

 2 H for every e- cycled

 Light energy transferred to and excites special pair--> strong  electron donor  

 Electron flow thru a membrane from low E to high E and  

generate PMF

 ATP synthesis= photophosphorylation

o Cyclic photophosphorylation: electrons move  

within closed loop; no net input or consumption

o process is like respiratory electron transport, except electrons come  back to RC (not transferred to oxygen)  

o this “cyclic photophosphorylation” represents almost unlimited supply  of ATP provided by light  

 however, CO2 fixation (and N2 fixation) requires “low-potential” electrons  (strongly reducing: NADH or Fd)  

o Reducing power is necessary to reduce CO2 to cell material  Comes from many sources, NADH, H2S, etc

 External sources  

 For biosynthesis, need electron source from the environment,  such as organic sources

o Requires reverse ETC (against electrochemical gradient) for NADH  production in purple phototrophs

 photosynthetic bacteria use different strategies but all use their  photosystems to ‘energize’ these electrons  

o Purple bacteria

o Green sulfur bacteria  

 Use FeS reaction centers

 Reverse electron flow is unnecessary  

 **Ferredoxin critical for electron transfer

 Unclear whether electron transfer in green sulfur or  

Heliobacteria is cyclic or noncyclic

o Heliobacteria  

 Same as green sulfur bacteria  

 the electrons taken from the PS must therefore be replenished from external  donors (organic compounds, H2S, etc.)  

 summary of energy capture:  

o - antenna pigments absorb photons, transmit energy to RC o - RC converts exiton to reduced Bchla  

o - electron transport generates ATP, or it reduces CO2 (and e- must be  replaced)

 Electrons shuffled thru and 2H+ per e- but e- is recycled and not released  

 Oxygenic photosynthesis (Cyanobacteria)

o oxygenic photosynthesis--> H2O donates electrons to drive CO2  fixation, and O2 is a by-product

 Two separate but interconnected photosystems in oxygenic  phototrophs, PSI and PSII, whereas anoxygenic phototrophs  

contain a single photosystem

 Noncyclic  

 Uses both FeS and quinone  

 Uses water to break and make energy; O2 byproduct

o Electron flow and ATP synthesis in O2 photosynthesis

 PS2 splits water into O2 and electrons as water oxidizing  complex

 PMF generated by electron transport thru quinones and  


 PS2 transfers energy to PS1, terminating reduction of NADP+ to  NADPH  

 12H+ translocated per O2 produced  

 Noncyclic photophosphorylation: electrons do not cycle back and reduce NADP+ to NADPH

 Cyclic photophosphorylation will occur ONLY if cell  

requires less NADPH to produce more ATP

o  Therefore- 3 diff photosynthesis

o Purple bacteria/anoxygenic  

o Green sulfur bacteria/heliobacteria  o Cyanobacteria/oxygenic photosynthesis 

Outline 14b concepts: oxygenic photosynthesis, multiple pathways of C fixation, N  fixation chemolithoautotrophy  


 ‘purple bacteria’ illustrate simple approach to photosynthesis: one  photosystem (RC + ETC) to harvest energy

o the photosystem can also “energize” electrons for biosynthesis, but an  environmental donor is needed

 other bacteria (‘cyanobacteria’) have a more complex system: two  photosystems operate in tandem  

o this enables electrons to be taken from water  

 basic features of the Z-scheme 

o i) has two distinct reaction centers involving chlorophyll a

o ii) PSII operates at high E0 , takes e- from water to form oxygen o iii) short ETC leads to PSI, generates PMF

o iv) PSI reduces FeS protein, then ferredoxin, then NAD(P)  

o - plant chloroplasts show extensive relatedness to cyanobacteria  (endosymbiont hypothesis)  

o - PSII resembles RC of Purple bacteria; PSI resembles RC of Green  Sulfur bacteria  

 Autotrophy (the ‘synthesis’ in photosynthesis)  

o with energy and reducing power, possible to make cell carbon from  CO2 (very abundant)

o Autotrophy: process by which CO2 is reduced and assimilated into  cells

o Cells require carbon and nitrogen to form biomass  

o Atmospheric sources (CO2 and N2) must be chemically reduced for  assimilation (fixation)

o Requires ATP and reducing power

o In phototrophs, called dark reactions

o multiple strategies  

o Calvin cycle (CO2 + pentose diP => 2x glaP)  

 key (unique) enzymes:  

 Phosphoribulokinase

 ribulose bis-phosphate carboxylase (RubisCO)  

 the cycle has to turn six times to make one hexose-P (requires  18 ATP and 12 NADPH)  

 “product” of the cycle = glyceraldehyde 3P; converted by  

reversal of glycolysis to Fructose 6P  

 Requires CO2, a CO2 acceptor, NADPH, ATP, RubisCO, and  phosphoribulokinase

 RubisCO/Ribulose bisP carboxylase oxygenase: 

carboxylation of C5 compound to yield two C3  


o Oxygenation is an alternative activity

 Phosphoribulokinase: regeneration of CO2  

acceptor, adds P group onto ribulose-5-phosphate  

using ATP

 First step catalyzed by RubisCO from ribulose bisphosphate and CO2  

 PGA then phosphorylated and reduced to G3P

 Glucose formed by reversal of glycolysis  

 Easiest to consider cycle as 6 CO2 required to make one  molecule of glucose; 12 NADPH and 18 ATP required

o many autotrophic bacteria package their RubisCO in “carboxysomes”  (protein shell, RubisCO)  

 Carboxysomes: inclusions containing and improving of  RubisCO in many autotrophs  

 Inorganic carbon first incorporated as bicarbonate  

and is converted to CO2

 CO2 cannot escape carboxysome

 Carboxysome also protects RubisCO from O2, which  

competes w CO2

 two apparent functions:  

 concentrate CO2  

 decrease O2 (and the competing oxygenase reaction)  

 two distinct CO2 -fixation strategies are found among “green sulfur” bacteria  o Chlorobium: Reverse Citric Acid cycle  

 made possible by reduced ferredoxin

 most of the same enzymes as CAC, except  

 - citrate lyase instead of citrate synthase

 carboxylations require Fd as reductant

 other key enzymes:  

 2-ketoglutarate synthase

 pyruvate synthase  

 CO2 reduced by reversal of steps of CAC

 More efficient, requiring 4 NADH, 2 reduced ferredoxins, 10 ATP

 Requires unique NEW enzymes not found in CAC  

 Alpha-ketoglutarate synthase

 pyruvate synthase

 citrate lyase- splitting citrate  

 fumarate reductase

 Net reaction: 3 CO2 + 12 H + 5 ATP ---> C3H6O3PO3 + 3 H2O o Chloroflexus: Hydroxypropionate pathway 

 may be most primitive form of CO2 fixation

 key enzymes:  

 acetyl-CoA carboxylase

 propionyl-CoA carboxylase  

 all the intermediates are CoA thioesters

 two of them (acetyl CoA & propionly CoA) are  


 end product is glyoxylate (2-C acid)  

 Needs a coenzymeA intermediate to invest energy

 hydroxypropionate, a three-carbon compound, is a key  

intermediate and it couples two cycles, one that fixes two  

molecules of bicarbonate into one molecule of glyoxylate and a  second that adds a third molecule of bicarbonate to ultimately  yield pyruvate  

 Cyclic phosphorylation  

 Less efficient than reverse CAC cuz needs 16 ATP

 Summary:

 CoA thioesters

 2 carboxylations  

 Glyoxylate end product

 some bacteria use yet another strategy that requires H2 : Acetyl CoA  pathway/acetogenesis 

o key enzyme=  

 CO dehydrogenase  

 one CO2 reduced to methyl

 2nd CO2 added as carbon monoxide  

o Other factors  

 Special cofactors (THF, CoFeSP)

 From environment: H2 and CO2

o final step of the pathway, the methyl group is combined with CO by the activity of both CO dehydrogenase and acetyl-CoA synthase to form  acetyl-CoA

o Conversion of acetyl-CoA to acetate is the last step in the pathway,  generating one ATP by substrate-level phosphorylation

o Net reaction: 4H2 + H+ + HCO3- --> acetate + 4H2O + .3 ATP o Reductive acetyl-CoA pathway of CO2 fixation is not a cycle o Acetogens conserve energy by the generation of an ion motive force

 microbial cells face other challenges: NH3 or NO3 not always available  o cells that can convert N2 to NH3 have an advantage, but process  requires strong reductant + energy  

o AKA nitrogen fixation 

o Nitrogen needed for proteins, nucleic acid, other organics

 Nitrogenase: enzyme for nitrogen fixation

o large enzyme complex

o two main components  

 Dinitrogenase

 dinitrogenase reductase

o special cofactor (FeMoCo) contains iron and molybdenum   Some may contain V and iron instead of MO or iron only

o primary reductant (dinitrogenase reductase) reduced by Fd &  “activated” by ATP hydrolysis  

o Inhibited by oxygen  

 very strong Eo makes the dinitrogenase reductase is very  sensitive to oxygen  

o Nitrogenase protective strategies:  

 genetic regulation

 cell differentiation (heterocysts)

 Anoxic heterocyst formation

 Inside the heterocyst, conditions are anoxic, while in  

neighboring vegetative cells, conditions are just the  

opposite because oxygenic photosynthesis is occurring.  

 Oxygen production is shut down in the heterocyst, thus  

protecting it as a dedicated site for N2 fixation

 encapsulation (slime layer)

 Production of Oxygen-retarding slime layers

 high respiration (high rate of consumption)

 Removal of O2 by respiration

 “conformational” protection of enzyme itself  

o Electron flow in N2 fixation  

 Triple bond stability makes activation and reduction very energy  demanding  

 6 electrons needed; 8 actually consumed because H2 must be  produced  

 Electron donor--> dinitrogenase reductase --> dinitrogenase -->  N2  

 ATP required to lower reduction potential (total 16)

 nitrogenase activity is commonly measured by the acetylene reduction  assay  

o Assaying nitrogenase: acetylene reduction  

o Nitrogenases reduce other triple bonded compounds, including  acetylene to form ethylene

o Proof requires N2 marker to form NH3

 some cyanobacteria can fix N2 , making them very independent nutritionally  o used as natural fertilizers in some parts of the world  

o N2 fixation very important for global ecology (required for CO2  fixation)  

(return to theme of energy harvesting)  

 Summary of our comparisons:

o Heterotrophy vs autotrophy (carbon

o Phototrophy vs chemotrophy (energy)

o Lithotrophy vs organotrophy (electron)

 Branched off of chemotrophy  

 Chemolithotrophy: get energy and electrons from inorganic sources =>  involves respiration

o Examples of chemolithotrophy:  

 H2 oxidation: seems a simple way to harvest energy (all from  gas phase)  

 Oxidize hydrogen gas (H2) with the use of hydrogenase 


o Electrons from H2 are transferred to quinone, thru  

cytochromes, making PMF and then reduce O2 to  


 Reducing O2 to water  

 respire H2 aerobically, forming water and ATP as the final  


 Synthesis of ATP during H2 oxidation by O2 is the result of

electron transport reactions that generate a proton  

motive force

 overall reaction: H2 + ½ O2 --> H2O

 Exergonic and coupled w synthesis of ATP

 Sulfur oxidation: lower energy yield than H2 oxidation; produces  acid  

 Capable of oxidizing wide variety of reduced sulfur  


o hydrogen sulfide (H2S), elemental sulfur (S0), and  

thiosulfate (S2O3 2-); sulfite (SO3 2-) can also be  


 final oxidation product is sulfate (SO4 2-)

 occurs in stages, with the first oxidation step yielding  

elemental sulfur, S

 Enzyme: sulfite oxidase

o [in both cases, cells need certain key enzymes (hydrogenase, sulfur  oxidase)]  

o both processes are oxidations: the inorganic chemical is the cell’s  electron donor  

 for other organisms, a 2nd inorganic chemical may be the  electron acceptor  

o in the latter cases, important to distinguish  

 Dissimilative vs. Assimilative redox processes  

 dissimilative reactions provide energy for chemotrophs;  

plants & many bacteria use similar processes to  

assimilate these elements as nutrients, which consumes  

energy (C, N fixation, e.g.)  

 Assimilative  

o Purpose of reducing an inorganic form to an organic


o Making organic compounds

 Dissimilative  

o Purpose of reducing during energy conservation,  

not for purpose of making organic compounds  

o Use of it as an electron acceptor for the purpose of  

energy conservation

 nitrate reduction  

o NO3 used as terminal e- acceptor; requires a corresponding terminal  oxidase: nitrate reductase  

o some bacteria stop after producing nitrite others continue, reducing  nitrite to NO, etc.  

o these additional reductions all require corresponding terminal oxidase  o


 All subsequent enzymes of the pathway are coordinately  

regulated and thus also repressed by O2

 In addition to anoxic conditions, nitrate must also be present  before these enzymes are fully expressed

 >note that all the products of the additional reductions are gases  o this process called denitrification; represents “loss” of “fixed” nitrogen   Denitrification: reduction of NO3- to gaseous nitrogen  

compounds, such as N2

 NO3- is alternate final electron acceptor to O2  

 Dissimilatory nitrate reduction  

o from fertilizer -> wasteful  

o from sewage -> beneficial (prevents unwanted algal growth  downstream)  

o nitrate reduction often used in identifying bacteria (simple color test  for nitrite in culture medium)

Ch 14c concepts respiration & chemolithotrophy, assimilative vs. dissimilative  reductions environmental electron donors & acceptors, diverse strategies of  fermentation -------------  

 CO2 fixation and N2 fixation remind us that . . .  

o - many prokaryotes can build cell material from simple chemicals if  they have a supply of energy  

o - all other forms of life depend on these processes (important link in  global cycles)  

 now we focus on the fact that some prokaryotes can use simple chemicals to  generate energy (ATP)  

 Chemolithotrophy (“growth on minerals”- contrast with both phototrophy & heterotrophy)  

o get energy from inorganic chemicals => involves respiration   Review defining features of respiration-->  

o controlled electron transport leading to PMF & ATP electron acceptor  provided by the environment

 Examples of Chemolithotrophy  

o H2 oxidation 

 seems a simple way to harvest energy (all from gas phase)  

 Hydrogen (H2) is a common product of microbial metabolism  key enzyme needed: hydrogenase (electrons from H2 pass  down a short ETC)  

 electrons travel through a series of cytochromes to  

generate a proton motive force and eventually reduce O2  

to water

 Synthesis of ATP during H2 oxidation by O2 is the result of

electron transport reactions that generate a proton  

motive force. The overall reaction

o H2 + ½ O2 --> H2O

o Sulfur oxidation 

 lower energy yield than H2 oxidation; produces acid  

 cell needs a sulfur oxidase to begin the process  

 reduced sulfur compounds can be electron donor

 final oxidation product is sulfate (SO4 2-)

o note that these are both oxidations (use inorganic chemical as electron donor)  

o other chemolithotrophs use inorganic electron acceptors (alternative to oxygen)  

 before continuing, make an important distinction: Dissimilative vs.  Assimilative reductions  

o processes resembling chemotrophic reactions are used by plants &  many bacteria to incorporate N, S for biosynthesis; these processes  assimilate the element & consume energy  

 nitrate reduction  

o NO3 is terminal e- acceptor (almost as good as O2 in terms of energy  yield)

o requires cell to have a corresponding terminal oxidoreductase: nitrate  reductase 

o multiple possibilities for the extent of this process

 However, while some bacteria stop after producing nitrite,  others continue reducing nitrite to NO, etc.  

 these additional reductions all require corresponding terminal  oxidases

o >also note that all the products of the additional reductions are gases  o this process called denitrification / has agricultural, ecological  significance  

 NO, N2O, and N2 are all gases, they can be lost from the  

environment, and their biological production is called  


o represents “loss” of “fixed” nitrogen (could have been assimilated by  plants, algae)  

 from fertilizer -> wasteful  

 from sewage -> beneficial (prevents unwanted algal growth)   the ability to reduce nitrate is a characteristic often used in  identifying bacteria  

 Other types of anaerobic respiration--> that support chemolithotrophs do not  use the ‘complete’ electron transport chain present in mitochondria and  many bacteria  

o sulfate reduction 

 Sulfate (SO4 2-), the most oxidized form of sulfur, is reduced by  the sulfate-reducing bacteria, a highly diverse group of  

obligately anaerobic bacteria widely distributed in nature

 electrons can come from various organic compounds, sulfate is  the electron acceptor  

 ecological significance: requires anaerobic conditions and  produces H2S

 sulfate reduction is a common source of unwanted H2S (oil  wells, water systems)  

 other strictly anaerobic processes reduce CO2 and produce  energy without using ‘normal’ electron transport  

o acetogenesis 

 (H2 + CO2 --> CH3COOH)  

 uses the reductive acetyl CoA pathway of CO2 fixation, plus  some unusual features

 very short ETC (Fd to NAD)

 Na+ ions substitute for H+ ; Use sodium for PMF

 Energy conserving pathway

 Making acetate from H and CO2

 Not a lot of energy but enough  

 All bacteria

o methanogenesis 

 (H2 + CO2 --> CH4 )

 similar to acetogenesis in many ways (also strictly anaerobic,  also has unusual features)  

 unique cofactors (carry C1 intermediates)  

 uses both Na+ and H+ to drive ADP phosphorylation   biological production of methane  

 reduction of CO2 by H2 to form methane (CH4) is a major  pathway of methanogenesis and is a form of anaerobic  


 Conservation involves one last step of cleaving off a methyl  group  

 Similar to acetogenesis  

 All archaea

o **note > all methanogens are archaea; all acetogens are bacteria   some bacteria can use methane as a carbon/energy source  (similar to H2 oxidation)  

o Methanotrophy/ methane oxidation 

 CH4 + O2 –> CO2  

 oxidation of methane (and all hydrocarbons) begins with key  enzyme mono-oxygenase (inserts O atom)  

 steps in CH4 oxidation to CO2 can be summarized as   CH4 –> CH3OH –> CH2 O –> COOH –> CO2  

 like H2 oxidation and other respirations, energy conservation  relies on respiration, ETC, PMF  

 CH3OH is oxidized by an alcohol dehydrogenase, yielding  formaldehyde (CH2O) and NADH, and the CH2O is either  oxidized to CO2 or used to make new cell material

 unlike H2 , however, CH4 also has the potential to provide  cellular carbon

 The strategy:  

 convert formaldehyde to intermediary metabolites  

 two approaches  

o serine pathway 

 acetyl-CoA is synthesized from one molecule  

of CH2O and one molecule of CO2

 requires reducing power and energy in the  

form of two molecules each of NADH and  

ATP, respectively, for each acetyl-CoA  


 employs a number of enzymes of the citric  

acid cycle and one enzyme, serine  

transhydroxymethylase, unique to the  


o ribulose mono-phosphate pathway 

 more energy efficient than the serine  

pathway because all of the carbon for cell  

material is derived from CH2O

 Because CH2O is at the same oxidation level  

as cell material, no reducing power is needed

for its incorporation

 all of the NADH from the oxidation of  

methane can be oxidized in the electron  

transport chain

 finally, think about living on organic compounds without respiration . .   Fermentation strategies  

o Many different types, all represent cells harvesting energy from a  carbon compound (examples: Table 14.8)  

o all these variations must still address two basic features of  fermentation:  

 i) substrate-level phosphorylation (= energy harvesting) >  phosphorylate and rearrange to a molecule that can be  

“converted” to ATP (Table 14.7)  

 ii) redox balance (= sustainability) all electron carriers must be  regenerated (no external acceptor)  

 for (ii) we have seen (with EMP pathway) the approach of  reducing a “leftover” of the carbon compound  

o another approach is  

 to reduce protons, thus producing H2 (requires only one  


o lactic acid: homo- vs. heterofermentative  

 already discussed homofermentive production of lactic acid by  EMP route  

 some bacteria use other forms of glycolysis that oxidize and  decarboxylate the sugar;  

 these pathways produce one lactic acid and one ethanol+CO2  o other bacteria have more complicated pathways that produce multiple  end-products  

 mixed-acid:  

 pyruvate + NADH2 –> lactate / formate / succinate /  

ethanol / H2 / CO2

 the relative amounts (ratios) of these products can vary  

with culture conditions  

 a variation of this pathway produces a non-acidic compound:  butanediol  

 key intermediate (acetoin) can be detected by  

chromogenic assay  

 Would expect obligate anaerobes to have diverse fermentation pathways  (good examples = clostridia)  

o fermentation of sugars by saccharolytic clostridia produces several  acids

 as growth proceeds, low pH triggers shift to solvent production  o solvent production sacrifices some energy but results in less acid stress on cells  

 historically important source of solvents (acetone, butanol)

o some clostridia can ferment amino acids  

 complex rearrangements involved; common feature = fatty acyl  CoA leads to ATP  

o a specialized process (?fermentation or anaerobic respiration?) =  “Stickland reaction”  

 pair of amino acids; one serves as e- donor, the other as e acceptor  

o Some clostridia can ferment other fermentation products, but best  example comes from another genus, Propionibacterium (ripens  Emmentaler cheeses /can ferment lactate)  

 complex series of transfers generates ATP from a ‘waste  

product’ of other anaerobes waste products (propionic acid,  

acetic acid, CO2 )  

 alternatives discussed above occur during or after the glycolytic phase and  affect the end-products but  

o bacteria also vary with regard to which sugars can be fermented   other hexoses (must be converted to glucose or fructose)   disaccharides, polysaccharides (must be hydrolyzed to  


o reflects specialized isomerases and hydrolases that act before the  glycolytic phase  

 example: lactose (metabolism requires beta-galactosidase,  galactose epimerase)

o in contrast, polysaccharides must be hydrolyzed extracellularly (cannot be brought in)  

o many of these properties are used in diagnostic tests to help identify  bacteria  

o production of acidic or gaseous products can be revealed by a pH  indicator + inverted tube  

 [photograph posted: sugar fermentation test]  

 Metabolic differences are commonly applied in identifying bacteria  o sugar metabolism  

 Which sugars can be fermented?  

 (detected as growth on that sugar in the absence of  


 Is acid produced? Is gas produced?  

 (results indicate heterofermentative or mixed-acid  


 Is acetoin produced?  

 (indicates butandiol pathway)  

o extracellular enzymes  

 Is starch hydrolyzed?  

 (detected by staining starch plate with iodine solution;  

indicates secretion of amylases)  

 Is gelatin liquified?  

 (indicates secretion of proteases)  

 anaerobic respiration

 Is nitrite produced from nitrate in the growth medium?  o (indicates nitrate reduction)  

 Is nitrate consumed without producing nitrite?  o (may indicate denitrification)

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