• Example of mutualistic association between free-living anaerobes.
• M. omelianskii was an interesting find several decades ago - seemed to be methanogen that converted ethanol to methane - unique. 
• In fact, was two organisms living together: 
• "S - organism": fermenter that converted ethanol to acetic acid + H2. 
• true methanogen: CO2 + H2 to methane

• Because ethanol à acetic acid + H2 equilibrium lies to the LEFT if hydrogen present. 
• This reaction will only go forward if the H2 is used as quickly as it's made. 
• "S - organism" cannot live by itself, in absence of organism that will use up H2 as it is produced. 
• Methanogen gets fed, S organism gets to make ATP if both live together.

o All the variety that exists with respect to energy-generating mechanisms exists in the microbial world.

o Multicellular organisms are metabolic cripples compared to prokaryotes.

o Dietary differences that exist among animals are superimposed on one basic energy-generating foundation.

o Dietary differences that exist among microorganisms are sometimes the reflection of profound differences in energy-generating pathways (eg. compare glycolysis and methanogenesis).

o Energy source has ecological implications.

o Differences in energy-generating mechanisms and waste products allow cross-feeding.

o Differences in energy-generating mechanisms are the foundations of biogeochemical cycles.

• How to add phosphate back onto ADP to make ATP? 
• Process depends on oxidation-reduction reactions that release more energy than making ATP consumes to drive forward the addition of P onto ADP to make ATP.

• Hydrolysis of ATP releases more energy than most synthetic reactions in cells consume 
• Organisms couple ATP hydrolysis, directly or indirectly, to synthetic reactions. 
• ATP: three phosphate groups held close together. 
• Electron clouds repel each other. 
• Like forcing together two magnets at the same pole and holding them there 

• NH4+ + O2 ---> NO2- (nitrite) + H2O 
• Ex.: Nitrosomonas

• The conversion of ammonia to nitrate (“nitrification”) is an important part of the nitrogen cycle.
• Two groups of eubacteria, those that oxidize ammonia to nitrite and those that oxidize nitrite to nitrate, have been known for over a century.
• Metagenomic studies and subsequent cultivation efforts have recently demonstrated that a large group of Archaea, representatives of the new phylum Thaumarchaeota, can also do this – which was not known before people began sequencing DNA isolated from environmental samples.  

• Astoundingly, given the fact that their existence was unknown until recently, members of this group of Archaea are present in nearly every environment on earth and typically outnumber the known bacterial ammonia oxidizers by several orders of magnitude where they occur. 
• Three species have been cultured (sort of), and studying the cultured forms has shown that they are chemoautotrophs that fix carbon via a novel pathway. 

• Anaerobic ammonium oxidizing bacteria – first species doing this identified in 1999.
• NH4+ + NO2− → N2 + 2H2O
• Although only a few species of bacteria that do this have been cultured, a large number of 16S rRNA sequences from this group have been found in the environment – it must be an important process 

• Amplification of 16S rRNA gene sequences from human skin has revealed the existence of hundreds (at least) of eubacterial species living there, rather than the dozens that had been catalogued during traditional culture-based studies.
• And up to 4.2% of the skin microbiome is composed of Archaea – mostly Thaumarchaeota.

Yes, although the energy yield is low. Oxidizing the inorganic compounds releases less energy than oxidizing organic molecules like glucose, and respiring with the non-O2 electron acceptors releases less energy than using O2, so combining the two can drop the energy yield to impractically low levels. 

• Note that proton gradient exists:
• More protons outside than inside
• pH lower outside than inside
• Outside more positively charged than inside
• The cell has built up a store of potential energy
• Electrochemical gradient now exists across membrane - can potentially do work if protons allowed to go down gradient. There is enzyme in cell membrane that can take advantage of this – ATPase (= ATP synthase). It allows protons through, combining P in the interior of the cell with ADP in the cell to make ATP.  

1. (Using the components in a typical electron transport chain) NADH + H+ passes Hs on to flavoprotein, reducing it, and creating NAD+ 
2. Flavoprotein reduces non-heme iron proteins in membrane. 
• Problem: they accept electrons, not whole H. 
• Solution: electrons passed to iron proteins, protons expelled across membrane (to the outside of mitochondrion or respiring bacterial cell)  

3. nonheme iron proteins reduce quinone. 

     -Problem: quinones are reduced by picking up two hydrogen atoms, not just electrons. 

     -Solution: quinones pick up two protons from inside the cell at the same moment that they accept electrons from the nonheme iron proteins 

4. quinones reduce cytochromes. 

     -Problem: cytochromes only accept electrons.

     -Solution: protons expelled from cell or mitochondrion as cytochromes accept electrons. 

• electrons are passed through a series of cytochromes until finally being dumped on O2 inside the membrane. 
• The negatively charged O2 is split, and combines with protons, creating water. Water is the final resting place of the hydrogen atoms.  

Components play hot potato with the reducing equivalents, passing them along. Process is set up to create imbalance in protons on the two sides of the membrane, with more protons outside than inside

• Found in sulfide containing water in Switzerland by Winogradsky. 
• Bacteria contained sulfur granules
• Granules disappeared when bacteria removed from sulfide-containing water. 
• Back in sulfide, granules reappeared. 
• Winogradsky concluded H2S ---->  S 

• What was S converted to? Showed that it was SO42- 
• Whole path: H2S ---> S ---> SO42-. 
• Why? process releases energy 
• Winogradsky showed that they weren't oxidizing (CH2O)n for energy, suggested that H2S ---> SO42- was substituting for (CH2O) ---> CO2.  

• Hulcr J, Latimer AM, Henley JB, Rountree NR, Fierer N, et al. (2012) A jungle in there: Bacteria in belly buttons are highly diverse, but predictable. PLoS One 7(11): e47712
• On average,  they found 67 bacterial taxa per belly button. 
• However, the belly button communities were strongly dominated by a few taxa: 6 taxa occurred on >80% humans, and accounted for about 1/3 of all the sequence reads. 

o Metabolism from an element's point of view

o What compounds are involved

o What transformations are involved

o What are the sizes of the reservoirs of the various compounds

o How quickly is the material in a reservoir turned over

o Yes – aerobically:

o NH3 ---> NO2- ---> NO3-

o Transformation of NH3 to NO3- is called nitrification.

o No eubacterium oxidizes NH3 completely to NO3- . One set is capable of NH3 ---> NO2- and another set does NO2- ---> NO3-. Each of these oxidations yield energy.

• Incomplete oxidation 
• Internal electron acceptor 
• Substrate level phosphorylation 
• Comparatively low energy yield (glycolysis as we do it yields 2 ATP)  

• Potentially complete oxidation 
• External electron acceptor
• Electron transport chain 
• Phosphorylation mediated by ATPase 
• Higher energy yield
• (Does it require oxygen?)

o Many ciliates living in anaerobic habitats lack mitochondria  (useless without oxygen), but have hydrogenosomes instead 

o (Eg. the ciliates  in sea urchin intestines). Fermentation. 

o End products are hydrogen, CO2,  acetic acid (plus maybe some other low molecular weight C compounds,  depending on species). 

o Removing hydrogen as it's produced helps the fermentation reactions go forward, helps ciliate make ATP. 

o Ciliates contain methanogens in cytoplasm, often pressed up against hydrogenosomes. 

o Use H2, CO2, make methane.  

• Microbes in a mutualistic relationship are often physically connected - is to their advantage in staying together. 
• Examples: 
• Paramecium bursaria + Chlorella 
• Foraminifera + various algae
• Anaerobic ciliates + methanogens

Complex feeding associations based on the exchange of waste are especially characteristic of anaerobic habitats, because of the wide variety of metabolic waste products produced.

• Example - whey particle: 
• Desulfovibrio vulgaris oxidizes ethanol (from anonymous fermenter) to acetate while converting bicarbonate to formate. 
• Methanobacterium formicicum converts formate to bicarbonate while producing methane 
• Methanosarcina barkeri cleaves acetate into CO2 and methane (note that this is a fermentation in which acetate is split in two, with one product being more reduced and the other more oxidized than in the parent molecule)

• The relationship between Desulfovibrio and Methanobacterium is exactly analogous to the Spirillum-Chlorobium association, except that 1-carbon compounds rather than S are being recycled - one partner is oxidizing it, one partner reducing it. 
• Both can live independently. Synergism. 
• Methanosarcina barkeri is using acetate produced by Desulfovibrio. CO2 produced by Methanosarcina can be converted to H2CO3 (H2O plus CO2 --> H2CO3 à Na+HCO3-) which can be used by Desulfovibrio. 
• Both benefit. Synergistic. 
• It is a short step from this type of synergistic association to mutualism.

• Abiotic factors (temperature, salinity, pH, etc.) (autecology)
• Biotic factors (parasites, predators, competitors, etc.) (synecology)

• Needed for: 
• Uptake of nutrients 
• Movement 
• Making synthetic reactions go forward

Proteins containing a riboflavin derivative - reduced by picking up two Hs at a time 

• Encompasses two very separate things: 
• source of material for making protoplasm. 
• source of energy for making protoplasm. 
• We use the same class of compounds (reduced carbon compounds) for both components of cell matter and energy production, but many microbes don't. 

• Many interactions between microorganisms revolve around cross-feeding.
• Imagine something rotting in anaerobic sediment:
• Respirers:
• nitrate --> ammonia
• sulfate --> sulfide
• (methanogens) CO2 + H2 --> methane
• Fermenters:
• (CH2O)n--> ethanol, acetate, lactate, etc.

• All of this is diffusing upwards, and it is all food for aerobic organisms:
• ammonia --. nitrate (chemolithotrophs)
• sulfide --> S, sulfate (chemolithotrophs)
• methane --> CO2 (methylotrophs)
• ethanol, acetate, lactate etc. --> CO2 (aerobic chemoorganotrophs)
• These two sets of organisms are consuming each others wastes as sources of energy - feeding each other.

• All of this is diffusing upwards, and it is all food for aerobic organisms: 
• ammonia --. nitrate (chemolithotrophs) 
• sulfide --> S, sulfate (chemolithotrophs) 
• methane --> CO2 (methylotrophs) 
• ethanol, acetate, lactate etc. --> CO2 (aerobic chemoorganotrophs) 
• These two sets of organisms are consuming each others wastes as sources of energy - feeding each other.

• Many interactions between microorganisms revolve around cross-feeding.
• Imagine something rotting in anaerobic sediment:
• Respirers:
• nitrate --> ammonia • sulfate --> sulfide
• (methanogens) CO2 + H2 --> methane
• Fermenters:
• (CH2O)n--> ethanol, acetate, lactate, etc.

o Foraminiferans are  large marine amoebae that live in a shell (test) from which they extend  pseudopods. 

o They contain many different types of algae,  have the same  relationship to symbionts as P. bursaria.

o Giant Great Barrier Reef foram:  extends pseudopods with symbionts out during the day, back in test at  night.

• H2 + CO2 ---> CH3COOH 
• Electrons are passed onto CO2.
• Picking up protons convert it to acetic acid. 
• Ex.: Sporomusa spp. in the intestine of termites use H2 and CO2 from the fermentations of other organisms. 

• H2 + O2 ---> H2O 
• In some aerobic habitats, free H2 exists for organisms to use. 
• Alcaligenes and some Pseudomonas species do this 

• There are organisms which use compounds other than organic molecules as electron source and respire with O2. 
• There are many bacteria which take electrons from INORGANIC molecules and transfer them to O2. Collectively, they are called chemolithotrophs ("litho" = "rock"; "lithotroph" = "rock-eater")

• H2 + CO2 ---> CH4 
• Ex. Methanobacterium spp.
• Methanogens are very sensitive to the presence of oxygen, and for a long time could not be grown in pure culture for this reason.
• They can live in any highly anaerobic habitat; the rumen of cattle is a good place to find them. 

• Some microbes can respire and regenerate NAD+ like we do
• Some microbes can only dump H back onto the carbon skeleton emerging from glycolysis – these are the fermenters

• Numerous low-molecular weight carbon compounds are produced by fermenters (ethanol, acetate, butyrate, lactate, propionate, etc.) as a means to regenerate NAD+
• Converting NADH to NAD+ this way DOES NOT PRODUCE ATP
• Lactobacilli - electrons dumped on pyruvate to make lactate; lactate excreted as waste 
• Yeast (when growing without O2) - CO2 removed from pyruvate to make acetaldehyde, electrons dumped on acetaldehyde to make ethanol; ethanol excreted as waste

o At any given moment, methanogens are making methane, sulfate-reducing bacteria are making sulfide, autotrophs are fixing carbon dioxide, etc.

o Although the amount of material altered by an individual microbe is small, microbes are extraordinarily abundant.

o Studying these massive transformations of matter is its own scientific discipline.

Oddly, there are no known bacteria that can oxidize ammonia all the way to nitrate. They only go as far as nitrite, and then another set of oxidizers takes over. It is possible to tell from the prefix in the generic name which set of reactions these bacteria carry out; Nitrososomething oxidizes ammonia to nitrite, a Nitrosomething oxidizes nitrite to nitrate. 

o Nitrogen is present in all organisms, but not in quantities as large as carbon.

o Main nitrogen-containing cell macromolecules:

• amino acids

• nucleotides

• At the beginning of the 1990s, the Archaea were thought to predominate in only a few unusual and extreme habitats - hypersaline, extremely hot, or strictly anoxic places. 
• To everyone’s surprise, DeLong and his coworkers showed that pelagic Archaea constituted up to 34% of the prokaryotic biomass in coastal Antarctic surface waters. 
• They then went on to show that one archaeal group (the crenarchaeotes) occurred abundantly in the Pacific Ocean below the euphotic zone, increasing in numbers with depth, reaching 39% of the total picoplankton detected. The pelagic Crenarchaeota represent one of the ocean's single most abundant cell types, and yet, a year earlier, nobody had known that they were there at all!

• In 1987, when sequencing studies were first starting to be applied to microbial ecology, there were about 12 known eubacterial phyla and two archaeal phyla.
• There are now about 57 known eubacterial phyla, 29 of which contain one or more species that people have succeeded in culturing.
• There are now about 5 known archaeal phyla, three of which contain one or more culturable species.

• Rinke et al. reminder:
• Partially (10-90%) amplified the genomes of 201 individual cells!
• Many were from eubacterial and archaeal groups that had never been cultured
• Found many of the uncultured species metabolized hydrogen or sulfur (by comparison with known genes)
• Found about 20,000 new protein families!

• Many microbes respire with nitrate as electron acceptor:
• (CH2O)n + NO3- ---> CO2 + NH3 (or NH4+ - same thing; NH3 picks up proton) or CO2 + C
• Electrons are passed down an electron transport chain onto nitrate.
• Organisms are respiring with nitrate – anaerobic respiration. 

• MANY bacteria can do this if O2 is absent. Pseudomonas species commonly do this (and E. coli carries out the first step: NO3- ---> NO2- )
• One eukaryote is known to do this: The freshwater ciliate Loxodes.

• NO2- + O2 ---> NO3- (nitrate) + H2O 
• Ex.: Nitrobacter. 

Proteins with sulfur and iron reaction centers, but not heme - reduced by picking up one electron at a time 

• Oxidation-reduction reactions do not necessarily involve transfer of O2. 
• By definition they involve the transfer of ELECTRONS. Protons may or may not accompany the electrons.
• If a compound accepts electrons or H atoms, it is being reduced. 
• If it loses electrons or H atoms, it is being oxidized.
• Neither oxidation nor reduction can take place by itself in cells; if a cell oxidizes something, it reduces something else (electrons have to be put somewhere). 

o P. bursaria is a phagotrophic ciliate capable of feeding on bacteria in its habitat. 

o Also contains green algae belonging to the genus Chlorella. 

o P. bursaria provides algae with ammonia, CO2 and motility.  

o Chlorella provides host with O2, reduced C (mostly maltose).

o Ignoring the donor and acceptor of electrons, ATP is made in the same way in both phototrophy and respiration!

o Some non-sulfur purple bacteria can either respire or photosynthesize using their electron transport chain according to whether light is present or absent.

o Cyanobacteria and their descendents – chloroplasts in eukaryotes - employ noncyclic photphosphorylation:

• Electron flow from chlorophyll is a one-way street

• An external electron source is required

• ATP and reducing power are both generated

• Two photosystems are used

o Many protozoans and invertebrates contain symbiotic phototrophs.

o Three names left over from past, before identities were known:

   • symbiotic cyanobacteria - cyanelle

   • symbiotic green algae - zoochlorellae

   • symbiotic dinoflagellates – zooxanthellae

o organic N (R-NH2) -3 (oxidation state)

o ammonia (NH3/NH4+) -3

o nitrogen gas (N2) O

o nitrous oxide (N2O) +1 (average per N)

o nitrogen oxide (NO) +2

o nitrite (NO2-) +3

o nitrogen dioxide (NO2) +4

o nitrate (NO3-) +5

• Consider our oxidation of glucose. Three parts: 
• Glycolysis  - a fermentative process 
• Krebs cycle - a means of stripping H from the glycolytic leftovers 
• Respiration - Electron transport chain in mitochondria 
• When our muscles are working hard and cannot be supplied with oxygen rapidly enough, glycolysis is operating as a pure fermentation and lactate is being produced. 

• Dominant protozoan in gut
• 105 bacteria (single type) in each protozoan
• Account for 70% of the bacteria in the gut of C. formosanus

Small hydrophobic molecules (not proteins) - reduced by picking up two hydrogen atoms at a time. 

• Our muscles during strenuous exercise (O2-deprived) - electrons dumped on pyruvate to make lactate – fermentation (can later be completely oxidized) 
• If our muscles are not deprived of oxygen, H on NADH has different fate: is carried off on electron transport chain in mitochondrial membrane, and pyruvate is not converted to lactate - respiration

o It was thought for decades that bacteriorhodopsin was restricted to halobacteria.

o Venter et al. 2004. Environmental Genome Shotgun Sequencing of the Sargasso Sea. Science 304: 66-74.

o BAC clones turned up that seemed to contain bacteriorhodopsin genes.

• A more indirect way of making ATP
• Requires a series of components that are alternately oxidized and reduced
•  electron transport chain components of mitochondria and O2-using bacteria: 
• flavoproteins, cytochromes, nonheme iron-sulfur proteins, quinones
• (The exact components of the respiratory chain vary from organism to organism)

• Anaerobic respirations release less energy than respiration with O2.
• Some organisms, nitrate respirers especially, can respire either with O2 or something else (eg. nitrate), and such organisms usually respire aerobically when they can.

• For every molecule of glucose that enters the pathway:
• four ATP molecules are made from 4 ADP molecules and the 4 Ps on the carbon skeletons. There is a net creation of 2 ATPs. 
• 2 NAD+ molecules are reduced.

o Cloned one of these genes in E. coli.

o Added retinal.

o pH changed in light but only of wavelengths that might be used by this rhodopsin-retinal complex.

o found E. coli made ATP using this rhodopsin as a proton pump – a novel way of using light energy.

o So these clones DID contain bacteriorhodopsin genes – but not from archaebacteria!

o Eventually showed that this type of phototrophy widespread in ocean.

o As of 2010, Venter's group had gotten 1 billion base pairs, 1.2 million new genes, including 782 new EUBACTERIAL rhodopsin genes!

o They are found in proteobacteria and other groups of bacteria never suspected of using light.

o Wavelength of light used varies with depth.

o The concentrations of compounds used by organisms are not changing much in the short term - NOT because nothing is being done to them, but because opposing processes produce a steady-state concentration of these materials.

o All elements used by organisms are cycled.

• Preparatory reactions: 
• 2 phosphates from ATP added on to glucose, which is split into 2 3-carbon pieces
• inorganic phosphate is added onto the 3-C compounds 
• (these reactions CONSUME energy)
• ATP generating reactions:
• Hydrogen atom equivalents are removed and added onto NAD+, converting it to NADH + H+. 
• This change in the carbon skeleton increases the energy of the phosphate bonds; their hydrolysis now releases more energy than adding phosphate onto ADP consumes, so an enzyme can catalyze the transfer of these Ps onto ADP and does so.

• Many microbes respire with sulfur compounds as electron acceptors:
• (CH2O)n + SO42- (or S) ---> CO2 + H2S 
• Electrons are passed down an electron transport chain onto sulfur compounds. 
• Organisms that do this are, in essence, breathing with sulfur compounds rather than oxygen – anaerobic respiration.

• Ex.: Desulfovibrio spp., which use sulfate. 
• Sulfate reducers are more prominent in marine environments (more sulfate there). 
• Prefer smaller organic compounds like lactate, acetate, pyruvate etc. - compounds likely to be produced by fermenters in the same environment. 

• H2S + O2 --->  (S), SO42- + H2O
• Very common process
• Ex.: Beggiatoa – first chemolithotroph found

• Chlorobium spp.+ Spirillum spp.: 
• Chlorobium (green sulfur bacterium, anoxygenic phototroph): anaerobe which splits H2S, S is waste. Fixes CO2 
•  Spirillum (anaerobic or microaerophilic): respires with S, transferring H from small organic molecules like formate to generate H2S 
• Sulfur cycles between them, changing oxidation state at each exchange.

• Chlorobium spp.+ Spirillum spp.: 
• Neither runs out of the form of sulfur that they require, even if concentration is low, since it is constantly being provided by the other. 
• Very dense cultures produced when the two are grown together. 
• Isn't commensalism, since both benefit. Mutualism or synergism? 
• Is synergism since both can live independently.

• Macromolecules are improbable structures for C, O, H, N atoms to be in. 
• Will not assemble spontaneously into macromolecules in any reasonable time frame. 
• Organisms must create the necessary bonds themselves if protoplasm is to be built and cells grow and multiply. 
• Cells synthesize material by coupling a synthetic, energy-consuming reaction to a reaction which releases an even greater amount of energy (=coupling endergonic and exergonic reactions). 
• This will drive the energy-consuming reaction forward. 

• For organisms that can respire, NADH is not an annoying product to be gotten rid of anymore - its electrons can be used to make ATP. 
• We, and many other respirers, use the Krebs cycle (aka citric acid cycle, tricarboxylic acid cycle, TCA cycle) to MAXIMIZE NADH production - pyruvate is completely oxidized to CO2, most electrons used to make NADH 

• Note that in glycolysis H is transferred to NAD+ to make NADH + H+. 
• NAD+ is expensive to make. 
• If NAD+ were not regenerated, glycolysis would not work, since it would take many more than the two ATP made during glycolysis to make the NAD+ needed in the process 
• NAD+ has to be reused in order for the process to work. 
• Electrons have to be dumped somewhere to regenerate NAD+.  Something must be reduced.

Oxidizes H2S with nitrate as electron acceptor. Gigantic vacuole filled with nitrate, replenished when bacteria temporarily suspended in water column. 

• Difference in trophic connections in macro- and microorganisms:
• Since microorganisms are physiologically so diverse waste of one can be food of another.
• Animals require (CH2O)n and produce CO2, water - can NOT live on the metabolic waste products of other animals.
• (Are eg. flies in cow dung an exception? NO. Flies are living on (CH2O)n that the cow didn't use, NOT on CO2).

• Difference in trophic connections in macro- and microorganisms:
• Since microorganisms are physiologically so diverse waste of one can be food of another. 
• Animals require (CH2O)n and produce CO2, water - can NOT live on the metabolic waste products of other animals. 
• (Are eg. flies in cow dung an exception? NO. Flies are living on (CH2O)n that the cow didn't use, NOT on CO2).

• Physiological uniformity of animals and physiological diversity of microbes determines the trophic connections that exist. 
• Animals consume other organisms - good source of (CH2O)n. 
• Microbes consume other microbes’ metabolic waste. 
• Is one important connection in our macro world analogous to microbial cross-feeding: 
• plants produce O2 as waste, which we use. 
• We produce CO2 as waste, which plants use.

• chemotrophs ("chemical eaters") use the energy in chemical bonds 
• phototrophs ("light eaters") use the energy in light 

• ATP is used by releasing phosphate groups to generate ADP (or AMP)
• ADP must be converted back to ATP
• An individual molecule endlessly changes back and forth between ATP and ADP
• Food used as a source of energy is used to convert ADP back to ATP (AMP is converted back to ADP by transfer of P from ATP)
• Intricate pathways exist for getting ADP back to ATP

• The only absolute requirement for an oxidation-reduction reaction for making ATP is that it release more energy than making ATP consumes. 
• It is not necessary for respirers to start with organic carbon, and it is not necessary to transfer the electrons onto O2. There is more variety in the microbial world. 

• The standard (from a human point of view):
• (CH2O)n + O2 ---> CO2 + H2O 
• (CH2O)n simply means a reduced organic compound of some sort, like glucose. 
• Electrons are removed from a compound and eventually passed down an electron transport chain onto O2, which picks up protons and becomes water (aerobic respiration).

–Determine a gene sequence underlying the process of interest and compare it to homologous sequences of known microorganisms.

•Procedure only works if a large number of the gene sequences from various organisms are available

–Determine the rRNA sequence of the organisms possessing the metabolic gene of interest

•Procedure only works if the two genes are physically connected in cloned material

• Many of the transformations are mediated by living organisms

• Prokaryotes carry out most of the transformations (considering that chloroplasts and mitochondria are eubacterial, do eukaryotes do much of anything?)

• Prokaryotes were the first to carry out these transformations

• The transformations affect earth's geology and atmosphere - eg.

• Phototrophs oxygenated the earth’s atmosphere

• limestone deposits are from millions of years of CaCO3 deposition

• petroleum and coal deposits are the remains of organisms exposed to heat and pressure

• Order of energy release of different terminal electron acceptors in respiration:
• O2 >NO3- >SO42- >CO2 
• Shows up in physical zonation of organisms, eg. vertical core through beach sand. Organisms that obtain more energy from given unit of food outcompete other organisms. 

• ATP is being made without

• an external electron donor equivalent to (CH2O)n in us

• an external electron acceptor equivalent to O2 in us

• Nitrate isn't toxic, BUT it can be reduced by microorganisms in the intestinal tract to nitrite, with all the consequent problems.

• More of a problem in ruminants and human babies (One "blue baby syndrome") than adult humans (differences in pH make this less likely to occur in human adults).

• Necessary to monitor nitrate levels in water supplies. Is occasional problem in eg midwest cornbelt and California's San Joaquin valley

• nitrite combines with amino compounds to produce nitrosamines - carcinogenic

• nitrite binds to hemoglobin, interferes with oxygen transport to tissues

• ATP is being made with

• an external electron donor equivalent to (CH2O)n in us

o (what is it?)

• an external electron acceptor equivalent to O2 in us

o (what is it?)

o chemolithotrophs. Chemolithotrophs oxidize INORGANIC compounds for energy, and most satisfy C needs by fixing CO2.

o Ammonia to nitrite conversion is carried out by genera with the prefix Nitroso- in their names. (eg. Nitrosomonas)

o Nitrite to nitrate conversion is carried out by genera with the prefix Nitro- in their names. (eg. Nitrobacter)

o Both kinds of genera usually occur together, so usually there is no buildup of nitrite. This is a hard way to make a living, as the energy yield from these oxidations is small:

• ammonia to nitrite: delta G is about -66kcal/mol

• nitrite to nitrate: delta G is about -17kcal/mol

o Consequently, the growth of these species is slow

o ANaerobic AMMonium Oxidation

• NH4+ + NO2− → N2 + 2H2O

o The bacteria mediating this process were identified in 1999, and at the time were a great surprise for microbiologists

• Planctomycetes

o Exact pathway still unknown, but involves hydrazine

o Only prokaryotes do this

o Many different kinds of prokaryotes fix nitrogen, especially cyanobacteria and rhizobia

o It is energetically enormously expensive

o it is essentially an anaerobic process; nitrogenase is very oxygen-sensitive

o Aerobic prokaryotes that fix N have figured out how to remove O2 from the site of N-fixation – eg. the heterocysts of cyanobacteria

o What is ammonia added onto?

o alpha-ketoglutaric acid + ammonia ---> glutamate

o glutamate + ammonia ---> glutamine

o Organisms may have either or both pathways. The second reaction requires ATP; the first does not. Once whichever amino acid is made, transaminases can transfer the amino group to other carbon skeletons, creating other amino acids.

o In our world, phototrophs are also autotrophs – but this is not necessarily true among phototrophic microbes

o Possibilities:

• Live without autotrophy

• Use H2S as a source of H

• Use H2O as a source of H

o Conversion of NO3- to NH3: ASSIMILATIVE nitrate reduction.

o NO3- is converted to NH3 immediately prior to incorporation into organic N.

o Pathway repressed by ammonia (no need to run it if ammonia is present).

o Prokaryotes, algae, fungi, higher plants do this, but we can't.

o conversion of NO3- to NH3 or N2:

• DISSIMILATIVE nitrate reduction.

• NO3- is converted to NH3 or N2 through respiration

• Pathway repressed by oxygen, if the organisms are capable of respiring with either oxygen or nitrate (respiring with oxygen yields much more energy, so nitrate not used this way if O2 present), but not by ammonia

o Note that generating N2 undoes what N fixers are busy doing

o light excites chlorophyll e-

o excited e- passes down electron transport chain

o proton gradient established across membrane

o ATP made by ATPase which lets protons move across membrane down gradient

o chlorophyll is both donor and acceptor of electrons; electrons run around in a loop.

o Purple and green sulfur and nonsulfur bacteria use cyclic photophosphorylation.

o Cyanobacteria use noncyclic photophosphorylation (usually) – it is what their photosystems are designed for.

o Removing the amino group, releasing free ammonia, is called ammonification.

• Why would any organism do this?

• In order to get energy by oxdizing the remaining carbon skeleton - the amino group has to be removed before the C can be oxidized.

o Charge changes in process from positive to negative.

• Ammonia binds to clay particles, which are positively charged.

• Nitrate cannot bind.

• Nitrate is therefore much more readily leached from soil. Fertilizer lost.

• Solution: add ammonia fertilizer together with compounds that inhibit nitrification (eg nitrapyrin inhibits NH2 ---> NO2-)

o Eubacteria (major groups):

• (sulfur and nonsulfur) purple bacteria

• (sulfur and nonsulfur) green bacteria

• cyanobacteria (bluegreen algae)

• (proteobacteria (and a few others))

o Archaebacteria (halobacteria)

o Eukaryotic algae

o Purple and green sulfur bacteria ARE photoautrophs

o Hs for the conversion of CO2 to (CH2O)n come from H2S.

o These bacteria convert H2S to S or SO42-using the Hs for conversion of CO2 to sugars.

o Purple and green sulfur bacteria use different mechanisms for this

• Many purple and green non-sulfur bacteria absorb organic material to make protoplasm with, but do not use it as an energy source (what are they called?) 
• Photoheterotrophs

o Anoxygenic photrophy

• The type of phototrophy employed by the purple and green sulfur and non sulfur bacteria – it does not release oxygen

• Many of these organisms use H2S (produced anaerobically by what?) and are oxygen-sensitive

• An unusual habitat for green sulfur bacteria

o Oxygenic phototrophy

• The type of phototrophy employed by cyanobacteria and their relatives – it does release oxygen

• They are not oxygen-sensitive

o NO3- ---> NH3

o NO3- ---> NO ---> N2O ---> N2 This is called denitrification.

• Lots of bacteria do this facultatively. Pseudomonas - common denitrifiers.

• N2 and nitrous oxide are both released. Proportionally more N2O is released at lower pH.

o Where is dentrification important?

• waterlogged soils

• standing water rather than running water

• hypolimnion of stratified lakes

• Oxic environments: 
• CO2 ---> (CH2O)n 
• oxygenic phototrophs: cyanobacteria, algae, terrestrial plants 
• chemolithotrophs: Thiobacillus, Nitrosomonas, etc. 
• (CH2O)n ---> CO2 
• heterotrophs that respire: us, other animals, nearly all the bacteria you saw in introductory microbiology, fungi 
• Do the same cycles operate anaerobically? Yes, with different organisms 

• CO2 ---> “(CH2O)n “ (reduced carbon)
• anoxygenic bacteria: purple sulfur bacteria (eg. Chromatium), green sulfur bacteria (eg. Chlorobium) 
• methanogens (although some cannot generate all the C compounds they need from CO2, and require - usually - a single amino acid) 
• some other anaerobic archaebacteria, eg. Archaeoglobus 
• acetogenic bacteria 
• (CH2O)n --> CO2 
• Numerous fermenters 
• Anaerobic respirers

So the (CH2O)n <---> CO2 cycle can operate aerobically or anaerobically - and material produced in one of these environments can be transferred to the other for further processing. 

• A carbon cycle usually requiring the presence of both oxic and anoxic environments: 
• CH4 <---> CO2 
• CO2 ---> CH4 - methanogens carry this reaction out only under strictly anaerobic conditions 
• CH4 ---> CO2 - methylotrophs (=methanotrophs) carry this out under (usually) aerobic conditions 

Consider intestinal fermentation in animals: plant material produced in an oxic environment is fermented in an anoxic environment, and fermentation products are then completely oxidized to CO2 in an oxic environment. This transfer is absolutely required for the symbiosis to work. 

• Part of the carbon cycle representing an interruption of (CH2O)n ---> CO2: 
• (CH2O)n ---> fossil fuel 
• If dead organic material is buried by sediment before decomposition is complete, it can be transformed by heat and pressure over geological time into fossil fuel; both petroleum and coal are the remains of organisms that lived 100s of millions or billions of years ago. 
• Petroleum can seep to the surface and be oxidized by microorganisms that are specialized to use this energy source. So the carbon in fossil fuels can very slowly be brought back into circulation again. 

• A cycle involving metabolism in one direction, abiotic changes in the reverse direction: 
• CO2 <---> CaCO3 
• Many organisms secrete calcium carbonate tests, can accumulate as limestone. Tests dissolve again under pressure as eg foraminiferan tests sink to bottom of ocean. Exposed limestone strata eg along roads can be slowly dissolved by rain. C winds up dissolved in lakes, rivers and oceans again. Slow process. CaCO3 is slowly cycled C reservoir. 

• (CH2O)n ---> CO2 part of the carbon cycle. 
• Animals can eat plants (grazers) or eat animals that eat plants (predation) or eat animals that eat animals that eat plants etc. 
• Each of these steps is called a trophic level. 
• At each level, part of the carbon is absorbed into an animal's own tissue without a change in oxidation state, part is oxidized to CO2, and part is excreted without being used. 

The part that is excreted without being used, and indeed, the animals themselves after they die, can be oxidized by microorganisms that are part of the decomposer food web.

• Food webs with many trophic levels are more a feature of aerobic than anaerobic environments. 
• Why? Aerobic oxidation of glucose to CO2 yields about 686kcal/mol, fermentation of glucose yields about 50kcal/mol. 
• It isn't possible to put so many organisms in an anaerobic habitat that a long food chain of many steps can be supported. 
• Food chains do exist in anaerobic habitats, but they are short and mostly microbial (eg ciliates eat bacteria, and that's the end). 

• Instead, anaerobic communities are built up by microbes using other microbes' waste products. Eg. glucose ---> ethanol ---> acetate ---> CO2 ---> CH4 with each species carrying out a different step. 
• Anaerobes partially oxidize what they get. 
• Aerobes completely oxidize part of what they get. 

• Relative importance of grazing/predation to decomposition varies between habitats. 
• • Forest - decomposition is dominant. 
  • Many aquatic systems - grazing/predation is more important. 
• In aquatic habitats, primary producers are microbial (algae). 
• In terrestrial habitats, primary producers are macroorganisms (grass, trees) 
• The decomposer food web is microbial. 
• Food webs based on predation are metazoan. (except protozoa can be in both) 

• Humans are interfering with the carbon cycle. By oxidizing fossil fuel at an enormously faster rate than it has ever been oxidized before, CO2 is being added to the CO2 pool faster than it is being removed by other processes. From 1860 to 2014, atmospheric CO2 has risen from 270 ppm to nearly 400 ppm. Possible consequences: 
• “greenhouse effect” (majority view)
• cloud cover idea
• CO2 consumption idea