Microbial metabolism

Microbial metabolism is the means by which a microbe obtains the energy and nutrients (e.g. carbon) it needs to live and propagate. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe’s environmental niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles.

Types of Microbial Metabolism

Main article: Primary nutritional groups

All microbial metabolism can be arranged according to three principles:

1. how the organism obtains carbon for synthesising cell mass:

 * autotrophic – carbon is obtained from carbon dioxide (CO2) 
 * heterotrophic – carbon is obtained from organic compounds
 * mixotrophic – carbon is obtained from both organic compounds and by fixing carbon dioxide 

2. how the organism obtains reducing equivalents used either in energy conservation or in biosynthetic reactions

 * lithotrophic – reducing equivalents are obtained from inorganic compounds
 * organotrophic – reducing equivalents are obtained from organic compounds

3. how the organism obtains energy for living and growing

 * chemotrophic – energy is obtained from external chemical compounds
 * phototrophic – energy is obtained from light

In practice, these terms are almost freely combined. Typical examples are as follows:

1. chemolithoautotrophs obtain energy from the oxidation of inorganic compounds and carbon from the fixation of carbon dioxide. Examples: Nitrifying bacteria, Sulfur-oxidising bacteria, Iron-oxidising bacteria, Knallgas-bacteria

2. photolithoautotrophs obtain energy from light and carbon from the fixation of carbon dioxide, using reducing equivalents from inorganic compounds. Examples: Cyanobacteria (water as reducing equivalent donor), Chlorobiaceae, Chromaticaceae (hydrogen sulfide as reducing equivalent donor), Chloroflexus (hydrogen as reducing equivalent donor)

3. chemolithoheterotrophs obtain energy from the oxidation of inorganic compounds, but can not fix carbon dioxide. Examples: some Nitrobacter spp., Wolinella (with H₂ as reducing equivalent donor), some Knallgas-bacteria

4. chemoorganoheterotrophs obtain energy, carbon and reducing equivalents for biosynthetic reactions from organic compounds. Examples: most bacteria, e. g. Escherichia coli, Bacillus spp., Actinobacteria

5. photoorganotrophs obtain energy from light, carbon and reducing equivalents for biosynthetic reactions from organic compounds. Some species are strictly heterotrophic, many others can also fix carbon dioxide and are mixotrophic. Examples: Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodomicrobium, Rhodocyclus, Heliobacterium, Chloroflexus (alternatively to photolithoautotrophy with hydrogen)

Heterotrophic Microbial Metabolism

Most microbes are heterotrophic (more precisely chemoorganoheterotrophic), using organic compounds as both carbon and energy sources. Heterotrophic microbes live off of nutrients that they scavenge from living hosts (as commensals or parasites) or find in dead organic matter of all kind (saprophages). Microbial metabolism is the main contribution for the bodily decay of all organisms after death. Many eukaryotic microorganisms are heterotrophic by predation or parasitism, properties also found in some bacteria such as Bdellovibrio (an intracellular parasite of other bacteria, causing death of its victims) and Myxobacteria such as Myxococcus (predators of other bacteria which are killed and lysed by cooperating swarms of many single cells of Myxobacteria). Most pathogenic bacteria can be viewed as heterotrophic parasites of humans or whatever other eukaryotic species they affect. Heterotrophic microbes are extremely abundant in nature and are responsible for the breakdown of large organic polymers such as cellulose, chitin or lignin which are generally indigestible to larger animals. Generally, the breakdown of large polymers to carbon dioxide (mineralization) requires several different organisms, with one breaking down the polymer into its constituent monomers, one able to use the monomers and excreting simpler waste compounds as by-products and one able to use the excreted wastes. There are many variations on this theme, as different organisms are able to degrade different polymers and secrete different waste products. Some organisms are even able to degrade more recalcitrant compounds such as petroleum compounds or pesticides, making them useful in bioremediation.

Biochemically, prokaryotic heterotrophic metabolism is much more versatile than that of eukaryotic organisms, although many prokaryotes share the most basic metabolic models with eukaryotes, e. g. using glycolysis (also called EMP pathway) for sugar metabolism and the citric acid cycle to degrade acetate, producing energy in the form of ATP and reducing power in the form of NADH or quinols. These basic pathways are well conserved because they are also involved in biosynthesis of many conserved building blocks needed for cell growth (sometimes in reverse direction). However, many bacteria and archaea utilise alternative metabolic pathways other than glycolysis and the citric acid cycle. A well studied example is sugar metabolism via the keto-deoxy-phosphogluconate pathway (also called Entner-Doudoroff Pathway) in Pseudomonas] instead of the glycolytic pathway. Moreover, there is even a third alternative sugar-catabolic pathway used by some bacteria, the pentose phosphate pathway. This metabolic diversity and ability of prokaryotes to use a huge variety of organic compounds arises from the much deeper evolutionary history and diversity of prokaryotes, as compared to eukaryotes. It is also noteworthy that the mitochondrion, the small membrane-bound intracellular organelle that is the site of eukaryotic energy metabolism, arose from the endosymbiosis of a bacterium related to obligate intracellular Rickettsia, and also to plant-associated Rhizobium or Agrobacterium. Therefore it is not surprising that all mitrochondriate eukaryotes share metabolic properties with these Proteobacteria. Most microbes respire (use an electron transport chain), although oxygen is not the only terminal electron acceptor that may be used. As discussed below, the use of terminal electron acceptors other than oxygen has important biogeochemical consequences.

Fermentation

Main article: Fermentation (biochemistry)

Fermentation is a specific type of heterotrophic metabolism that uses organic carbon instead of oxygen as a terminal electron acceptor. This means that these organisms do not use an electron transport chain to oxidize NADH to NAD⁺ and therefore must have an alternative method of using this reducing power and maintaining a supply of NAD⁺ for the proper functioning of normal metabolic pathways (e.g. glycolysis). As oxygen is not required, fermentative organisms are anaerobic. Many organisms can use fermentation under anaerobic conditions and respiration when oxygen is present. These organisms are facultative anaerobes. To avoid the overproduction of NADH obligately fermentative organisms usually do not have a complete citric acid cycle. Instead of using an ATPase as in respiration, ATP in fermentative organisms is produced by substrate-level phosphorylation where a phosphate group is transferred from a high-energy organic compound to ADP to form ATP. As a result of the need to produce high energy phosphate-containing organic compounds (generally in the form of CoA-esters) fermentative organisms use NADH and other cofactors to produce many different reduced metabolic by-products, often including hydrogen gas (H₂). These reduced organic compounds are generally small organic acids and alcohols derived from pyruvate, the end product of glycolysis. Examples include ethanol, acetate, lactate and butyrate. Fermentative organisms are very important industrially and are used to make many different types of food products. The different metabolic end products produced by each specific bacterial species are responsible for the different tastes and properties of each food.

Not all fermentative organisms use substrate-level phosphorylation. Instead, some organisms are able to couple the oxidation of low-energy organic compounds directly to the formation of a proton (or sodium) motive force and therefore ATP synthesis. Examples of these unusual forms of fermentation include succinate fermentation by Propionigenium modestum and oxalate fermentation by Oxalobacter formigenes. These reactions are extremely low energy-yielding. Humans and other higher animals also use fermentation to use excess NADH to produce lactate, although this is not the major form of metabolism as it is in fermentative microorganisms.

Special metabolic properties

Methylotrophy

Methylotrophy refers to the ability of an organism to use C1-compounds as energy sources. These compounds include methanol, methyl amines, formaldehyde and formate. Several other, less common substrates may also be used for metabolism, all of which lack carbon-carbon bonds. Examples of methylotrophs include the bacteria Methylomonas and Methylobacter. Methanotrophs are a specific type of methylotroph that are also able to use methane (CH₄) as a carbon source by oxidizing it sequentially to methanol (CH₃OH), formaldehyde (CH₂O), formate (HCOO^-) and finally carbon dioxide CO₂ initially using the important enzyme methane monooxygenase. As oxygen is required for this process, all (conventional) methanotrophs are obligate aerobes. Reducing power in the form of quinones and NADH is produced during these oxidations to produce a proton motive force and therefore ATP generation. Methylotrophs and methanotrophs are not considered as autotrophic, because they are able to incorporate some of the oxidized methane (or other metabolites) into cellular carbon before it is completely oxidised to CO₂ (at the level of formaldehyde), using either the serine pathway (Methylosinus, Methylocystis) or the ribulose monophosphate pathway (Methylococcus), depending on the species of methylotroph.

In addition to aerobic methylotrophy, methane can also be oxidized anaerobically. This occurs by a consortium of sulfate-reducing bacteria and relatives of methanogenic Archaea working syntrophically (see below). Little is currently known about the biochemistry and ecology of this process.

Methanogenesis

Main article: Methanogenesis

Methanogenesis is the biological production of methane. It is carried out by methanogens, strictly anaerobic archaea such as Methanococcus, Methanocaldococcus, Methanobacterium, Methanothermus, Methanosarcina, Methanosaeta and Methanopyrus. The biochemistry of methanogenesis is unique in nature in its use of a number of unusual cofactors to sequentially reduce methanogenic substrates to methane. These cofactors are responsible (among other things) for the establishment of a proton gradient across the outer membrane thereby driving ATP synthesis. Several different types of methanogenesis occurs, which differ in the starting compounds oxidized. Some methanogens reduce carbon dioxide (CO₂) to methane (CH₄) using electrons (most often) from hydrogen gas (H₂) chemolithoautotrophically. These methanogens can often be found in environments containing fermentative organisms. The tight association of methanogens and fermentative bacteria can be considered to be syntrophic (see below) because the methanogens, which rely on the fermentors for hydrogen, relieve feedback inhibition of the fermentors by the build-up of excess hydrogen that would otherwise inhibit their growth. This type of syntrophic relationship is specifically known as interspecies hydrogen transfer. A second group of methanogens use methanol (CH₃OH) as a substrate for methanogenesis. These are chemoorganotrophic, but still autotrophic in using CO₂ as only carbon source. The biochemistry of this process is quite different from that of the carbon dioxide reducing methanogens. Lastly, a third group of methanogens produce both methane and carbon dioxide from acetate (CH₃COO^-) with the acetate being literally split between the two carbons. These acetate-cleaving organisms are the only chemoorganoheterotrophic methanogens. All autotrophicmethanogens use a variation of the acetyl-CoA pathway to fix CO₂ and obtain cellular carbon.

Syntrophy

Syntrophy, in the context of microbial metabolism, refers to the pairing of multiple species to achieve a chemical reaction that, on its own, would be energetically unfavorable. The best studied example of this process is the oxidation of fermentative end products (such as acetate, ethanol and butyrate) by organisms such as Syntrophomonas. Alone, the oxidation of butyrate to acetate and hydrogen gas is energetically unfavorable. However, when a hydrogenotrophic (hydrogen using) methanogen is present, the use of the hydrogen gas will significantly lower the concentration of hydrogen (down to 10^-5 atm) and thereby shift the equilibrium of the butyrate oxidation towards product formation.

Anaerobic respiration

In aerobic organisms, oxygen is used as a terminal electron acceptor during respiration. This is largely because oxygen has a very high reduction potential allowing for aerobic organisms to utilize their electron transport systems most efficiently. In anaerobic organisms, terminal electron acceptors other than oxygen are used. These inorganic compounds have a lower reduction potential compared to oxygen, meaning that respiration is less efficient in these organisms generally leading to slower growth rates compared to aerobes. Many facultative anaerobes can use either oxygen or alternative terminal electron acceptors for respiration depending on the environmental conditions. Most respiring anaerobes are heterotrophs, although some do live autotrophically.

Chemolithotrophy

Chemolithotrophy is a type of metabolism where energy is obtained from the oxidation of inorganic compounds. Most chemolithotrophic organisms are also autotrophic. There are two major objectives to chemolithotrophy: the generation of energy (ATP) and the generation of reducing power (NADH).

Hydrogen Oxidation

Many organisms are capable of using hydrogen (H₂) as a source of energy. While several mechanisms of anaerobic hydrogen oxidation have been mentioned previously (e.g. sulfate reducing- and acetogenic bacteria) hydrogen can also be used as an energy source aerobically. In these organisms hydrogen is oxidized by a membrane-bound hydrogenase causing proton pumping via electron transfer to various quinones and cytochromes. In many organisms, a second cytoplasmic hydrogenase is used to generate reducing power in the form of NADH, which is subsequently used to fix carbon dioxide via the Calvin cycle. Hydrogen oxidizing organisms, such as Ralstonia eutropha, often inhabit oxic-anoxic interfaces in nature to take advantage of the hydrogen produced by anaerobic fermentative organisms while still maintaining a supply of oxygen.

Sulfur Oxidation

Sulphur oxidation involves the oxidation of reduced sulphur compounds (such as sulfide (H₂S), inorganic sulphur (S⁰) and thiosulfate (S₂O₂^2-) ) to form sulfuric acid (H₂SO₄). A classic example of a sulphur oxidizing bacterium is Beggiatoa, a microbe originally described by Sergei Winogradsky, one of the founders of microbiology. Generally, the oxidation of sulfide occurs in stages, with inorganic sulphur being stored either inside or outside of the cell until needed. This two step process occurs because energetically sulfide is a better electron donor than inorganic sulphur or thiosulfate, allowing for a greater number of protons to be translocated across the membrane. Sulphur oxidizing organisms generate reducing power for carbon dioxide fixation via the Calvin cycle using reverse electron flow, an energy-requiring process that pushes the electrons against their thermodynamic gradient to produce NADH. Biochemically, reduced sulphur compounds are converted to sulfite (SO₃^2-) and subsequently converted to sulfate by the enzyme sulfite oxidase. Some organisms, however, accomplish the same oxidation using a reversal of the APS reductase system used by sulfate-reducing bacteria (see above). In all cases the energy liberated is transferred to the electron transport chain for ATP and NADH production. In addition to aerobic sulphur oxidation, some organisms (e.g. Thiobacillus denitrificans) use nitrate (NO₃^2-) as a terminal electron acceptor and therefore grow anaerobically.

Ferrous Iron (Fe²⁺) Oxidation

Ferrous iron is a soluble form of iron that is stable at extremely low pHs or under anaerobic conditions. Under aerobic, moderate pH conditions ferrous iron is oxidized spontaneously to the ferric (Fe³⁺) form and is oxidized abiotically to insoluble ferric hydroxide (Fe(OH)₃). There exists, therefore, three distinct types of ferrous iron-reducing microbes. The first are acidophiles, such as the bacteria Acidithiobacillus ferooxidans and Leptospirrillum ferrooxidans, as well as the archaeon Ferroplasma. These microbes oxidize iron in environments that have a very low pH and are important in acid mine drainage. The second type of microbes oxidize ferrous iron at neutral pH along oxic-anoxic interfaces. Both these bacteria, such as Gallionella ferruginea and Sphaerotilus natans, and the acidophilic iron oxidizing-bacteria are aerobes. The third type of iron-oxidizing microbes are anaerobic photosynthetic bacteria such as Chlorobium, which use ferrous iron to produce NADH for autotrophic carbon dioxide fixation. Biochemically, aerobic iron reduction is a very energetically poor process which therefore requires large amounts of iron to be oxidized by the enzyme rusticyanin to facilitate the formation of proton motive force. Like during sulphur oxidation reverse electron flow must be used to form the NADH used for carbon dioxide fixation via the Calvin cycle.

Nitrification

Nitrification is the process by which ammonia (NH₃) is converted to nitrate (NO₃^-). Nitrification is actually the net result of two distinct processes: oxidation of ammonia to nitrite (NO₂^-) by nitrosifying bacteria (e.g. Nitrosomonas) and oxidation of nitrite to nitrate by the nitrite-oxidizing bacteria (e.g. Nitrobacter). Both of these processes are extremely poor energetically leading to very slow growth rates for both types of organisms. Biochemically, ammonia oxidation occurs by the stepwise oxidation of ammonia to hydroxylamine (NH₂OH) by the enzyme ammonia monooxygenase in the cytoplasm followed by the oxidation of hydroxylamine to nitrite by the enzyme hydroxylamine oxidoreductase in the periplasm. Electron and proton cycling are very complex but as a net result only one proton is translocated across the membrane per molecule of ammonia oxidized. Nitrite reduction is much simpler, with nitrite being oxidized by the enzyme nitrite oxidoreductase coupled to proton translocation by a very short electron transport chain, again leading to very low growth rates for these organisms. In both ammonia- and nitrite-oxidation oxygen is required, meaning that both nitrosifying and nitrite-oxidizing bacteria are aerobes. As in sulphur and iron oxidation, NADH for carbon dioxide fixation using the Calvin cycle is generated by reverse electron flow, thereby placing a further metabolic burden on an already energy-poor process.

Anammox

Anammox stands for anaerobic ammonia oxidation and is a relatively recently (late 1990’s) discovered process. It occurs in members of the Planctomycetes (e.g. Candidatus Brocadia anammoxidans) and involves the coupling of ammonia oxidation to nitrite reduction. As oxygen is not required for this process these organisms are strict anaerobes. Amazingly, hydrazine (N₂H₄-rocket fuel) is produced as an intermediate during anammox metabolism. To deal with the high toxicity of hydrazine, anammox bacteria contain an hydrazine-containing intracellular organelle called the anammoxasome surrounded by highly compact (and unusual) ladderane lipid membrane. These lipids are unique in nature, as is the use of hydrazine as a metabolic intermediate. Anammox organisms are autotrophs although the mechanism for carbon dioxide fixation is unclear. Because of this property, these organisms have been applied industrially to remove nitrogen in wastewater treatment processes. Anammox has also been shown have widespread occurrence in anaerobic aquatic systems and has been speculated to account for approximately 50% of nitrogen gas production in some marine environments.

Phototrophy

Many microbes are capable of using light as a source of energy (phototrophy). Of these, cyanobacteria and algae are particularly significant because they are oxygenic, using water as an electron donor for electron transfer during photosynthesis. Along with plants these microbes are responsible for all biological generation of oxygen on Earth. In a certain manner of speaking, all biological oxygen generation descends from microbes because chloroplasts were derived from a lineage of the Cyanobacteria. Thus the general principles of metabolism in Cyanobacteria can also be applied to chloroplasts. In addition to oxygenic photosynthesis, many bacteria can also photosynthesize anaerobically, typically using sulfide (H₂S) as an electron donor to produce sulfate. Inorganic sulfur (S⁰), thiosulfate (S₂O₃^2-) and ferrous iron (Fe²⁺) can also be used by some organisms. Phylogenetically, all oxygenic photosynthetic bacteria are Cyanobacteria, while anoxygenic photosynthetic bacteria belong to the purple bacteria (Proteobacteria), Green sulfur bacteria (e.g. Chlorobium), Green non-sulfur bacteria (e.g. Chloroflexus) or the heliobacteria (Low %G+C Gram-positive). In addition to these organisms, some microbes (e.g. the archaeon Halobacterium or the bacterium Roseobacter, among others) can utilize light to produce energy using the enzyme bacteriorhodopsin, a light-driven proton pump. This type of metabolism is not considered to be photosynthesis but rather photophosphorylation, since it generates energy, but does not directly fix carbon.

As befits the large diversity of photosynthetic bacteria, there exist many different mechanisms by which light is converted into energy for metabolism. All photosynthetic organisms locate their photosynthetic reaction centers within a membrane, which may be invaginations of the cytoplasmic membrane (purple bacteria), thylakoid membranes (Cyanobacteria), specialized antenna structures called chlorosomes (Green sulfur and non-sulfur bacteria) or the cytoplasmic membrane itself (heliobacteria). Different photosynthetic bacteria also contain different photosynthetic pigments such as chlorophylls and carotenoids allowing them to take advantage of different portions of the electromagnetic spectrum and thereby inhabit different niches. Some groups of organisms contain more specialized light-harvesting structures e.g. phycobilisomes in Cyanobacteria and chlorosomes in Green sulfur and non-sulfur bacteria, allowing for increased light utilization efficiency.

Biochemically, anoxygenic photosynthesis is very different from oxygenic photosynthesis. Cyanobacteria (and by extension chloroplasts) use the Z scheme of electron flow in which electrons eventually are used to form NADH. Two different reaction centers (photosystems) are used and proton motive force is generated both by using cyclic electron flow and the quinone pool. In anoxygenic photosynthetic bacteria electron flow is cyclic, with all electrons used in photosynthesis eventually being transferred back to the single reaction center. A proton motive force is generated using only the quinone pool. In heliobacteria, Green sulfur and non-sulfur bacteria NADH is formed using the protein ferredoxin, an energetically favorable reaction. In purple bacteria NADH is formed by reverse electron flow due to the lower chemical potential of this reaction centre. In all cases, however, a proton motive force is generated and used to drive ATP production via an ATPase.

Most photosynthetic microbes are autotrophic, fixing carbon dioxide via the Calvin cycle. Some photosynthetic bacteria (e.g. Chloroflexus) are photoheterotrophs, meaning that they use organic carbon compounds as a carbon source for growth. Some photosynthetic organisms also fix nitrogen (see below).

Nitrogen Fixation

Nitrogen is an element required for growth by all biological systems. While extremely common (80% by volume) in the atmosphere, dinitrogen gas (N₂) is generally biologically inaccessible due to its high activation energy. Throughout all of nature, only specialized bacteria are capable of nitrogen fixation, converting dinitrogen gas into ammonia (NH₃), which is easily assimilated by all organisms. These bacteria, therefore are very important ecologically and are often essential for the survival entire ecosystems. This is especially true in the ocean, where nitrogen-fixing cyanobacteria are often the only sources or fixed nitrogen and in soils where specialized symbioses exist between legumes and their nitrogen-fixing partners to provide the nitrogen needed by these plants for growth.

Nitrogen fixation can be found distributed throughout nearly all bacterial lineages and physiological classes but is not a universal property. Because the enzyme nitrogenase, responsible for nitrogen fixation, is very sensitive to oxygen which will inhibit it irreversibly, all nitrogen-fixing organisms must possess some mechanism to keep the concentration of oxygen low. Examples include:

• heterocyst formation (cyanobacteria e.g. Anabaena) where one cell does not photosynthesize but instead fixed nitrogen for its neighbors which in turn provide it with energy
• root nodule symbioses (e.g. Rhizobium) with plants that supply oxygen to the bacteria bound to molecules of leghaemoglobin
• anaerobic lifestyle (e.g. Clostridium pasteurianum)
• very fast metabolism (e.g. Azotobacter vinelandii)

The production and activity of nitrogenases is very highly regulated, both because nitrogen fixation is an extremely energetically expensive process (16-24 ATP are used per N₂ fixed) and due to the extreme sensitivity of the nitrogenase to oxygen.

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