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A newborn's gut starts off sterile and oxygen-rich, much like any other organ in the body (see Initial colonisation and succession). Initial microbial colonisers can handle the oxygen that is present, and can utilise milk oligosaccharides and other undigested food items. Then something strange happens: the distal gut transforms to an anaerobic environment and encourages mostly anaerobic microbes to colonise and persist. Why is this? Commensals that use oxygen during metabolism are more efficient at generating energy, as will be discussed later. So why does the environment change to support a seemingly less productive ecosystem? It appears as though mutualistic gut microbes have a large repetioire of polysaccharide hydrolase enzymes, and they work together to break down dietary fibre (resistant starches, plant polysaccharides and indigestible oligosaccharides) more efficiently. Metabolites produced by these organisms cross feed one another, and don't allow products to build up which could potentially shift chemical equilibria in an unfavourable direction.
Energy for the microbiome is provided through the diet and by the host. The composition of the microbiome includes organisms that are particularly efficient at breaking down indigestible carbohydrates and proteins to fragments that can be further consumed by other microbes or absorbed by the host (Figure 1).
Figure 1. Microbial communities work together to decay large macromolecules (hydrolytic bacteria) and prevent the buildup of metabolic byproducts (non-hydrolytic bacteria) that may hinder further metabolic activity. Fermentation can produce acetate, formate, lactate and other intermediates, which are quickly utilised by associated organisms. These microorganisms, in turn, can produce byproducts that are utilised by colonocytes, muscle cells or the liver of the host (modified from BernalierDonadille2010).
Metabolism, specifically catabolism, is the process by which cells break down organic compounds to generate energy. One of the key processes in catabolism is oxidation, where organic compounds are oxidised, and electrons are transferred from these compounds to electron carriers, such as nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD). The plasma membrane of respiring bacteria incorporates an electron transport chain (ETC), which consists of a series of these electron carriers that sequentially transfers electrons to the carrier with the next highest reduction potential (being a stronger electron acceptor). Ultimately, electron transfer terminates with the strongest electron acceptor, which is oxygen during aerobic respiration or an inorganic acceptor (e.g., CO2, nitrate) during anaerobic respiration.
As electrons pass through the ETC, the released energy is used to pump protons across the membrane, creating a proton gradient. ATP synthase utilises this gradient by allowing protons to flow back across the membrane, and as they do so, ATP molecules are synthesised from adenosine diphosphate (ADP) and inorganic phosphate (Pi).
For non-respiring gut bacteria, fermentation of organic substrates is used to generate ATP. Fermentation doesn't use an ETC or generate a proton gradient, but instead relies on other electron-transfer processes to generate energy (see below).
As a consequence of the varied energy-generating biochemical strategies of bacteria, the microbiome as a whole is able to exploit the different types of nutrients that are presented to it. An individual microorganism's metabolic process is highly dependent on its relationship with oxygen. As mentioned above, obligate aerobes use oxygen as the terminal electron acceptor in the final catabolic step (Figure 2). These aerobes are rare gut colonisers and are typically transient (e.g. Bacillus species). Other microbes require oxygen, but not in the concentrations typically encountered in the atmosphere; these 'microaerobes' are also scanty in the gut microbiome. Then there are those bacteria that prefer oxygen, but don't need it to grow; these 'facultative' anaerobes play an important role in the microbiome ecology but are still in the minority. The majority of organisms occupying our gut are either strict (obligate) anaerobes that avoid oxygen because it is toxic to them, or aerotolerant species that don't use oxygen but are not bothered by it, either (Figure 2).
Figure 2. An organism's relationship with oxygen is important for its metabolism and for its likely survival in the anaerobic gut environment. The figure above illustrates a well known experiment where tubes containing a thioglycolate growth medium are inoculated with bacteria and allowed to grow. The thioglycolate keeps oxygen levels low, especially at the bottom of the tube, whereas the partially permeable cotton plug allows oxygen to enter the headspace. Organisms requiring or prefering oxygen for their metabolism grow at (or near) the top, whereas strict anaerobes only survive where oxygen is limited. Aerotolerant organisms don't require oxygen but are not harmed by it, either, and are able to grow throughout the entire medium.
The following pages provide a refresher on metabolic processes relevant to gut bacteria, including cellular energy, redox chemical reactions, and anaerobic respiration:
Fermenting bacteria generate their ATP through substrate-level phosphorylation (i.e., PEP and 1,3-BPG) during the catabolic process known as glycolysis (see Figure 3 below), and not by oxidative phosphorylation. Fermentation is a redox process (see section on Oxidation and reduction: Redox reactions) in which organic substrates are partially oxidised without an external electron acceptor (Jurtshuk1996, Jackson2002). It relies on internal electron acceptors derived from the organic substrate itself or intermediates generated during the catabolic process. The amount of free energy and products derived from fermentation depends on the type of substrates and the specific conditions.
There is a significant energy cost for not using an external oxidant with a high oxidation potential. For example, a classic and well-studied fermentation process involving the conversion of glucose to lactate by lactic acid-producing bacteria (LAB) highlights the difference between glucose fermentation and its complete oxidation: fermentation produces about -185 kJ/mol of free energy, or two ATP molecules for each glucose (2 x 76 kJ/mol) with a bit of energy left over, while eukaryotic aerobic oxidation produces 2872 kJ/mol of energy, or 38 ATPs/glucose. There is clearly a huge discrepancy, so how do anaerobes prosper in the absence of potent inorganic electron acceptors?
Figure 3. The glycolysis pathway is the main method of generating energy in anaerobic gut bacteria, but is also used by aerobic microorganisms as the entry point for oxidative metabolism. Two pathways to the production of pyruvate are used, including the traditional Emden-Meyerhof-Parnas (EMP) pathway that nets 2 ATP molecules for each glucose consumed and two lactate molecules produced, and the Entner-Doudoroff (ED) pathway, which is more exogonic but yields only a single ATP for each glucose consumed. About 45% of aerobic bacteria measured can use the ED pathway compared to only 3% for anaerobes (Flamholz2013). Abbreviations: Edd, phosphogluconate dehydratase; Eno, enolase; Fba, fructose-bisphosphate aldolase; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; G6pd, glucose 6-phosphate dehydrogenase; Hxk, hexokinase; Kdpga, kdpg aldolase; Ldh, lactate dehydrogenase; Pfk, 6-phosphofructokinase; Pgi, phosphoglucose isomerase; Pgk, phosphoglycerate kinase; Pgl, 6-phosphogluconolactonase; Pgm, phosphoglycerate mutase; Pyk, pyruvate kinase; Tim, triose-phosphate isomerase; (modified from Flamholz2013).
The fermentation process can be made more efficient if enzyme reaction products are rapidly removed or consumed. This can be achieved by a combination of high enzyme concentrations and the excretion of fermentation end products. Although enzymes don't directly shift the reaction equilibrium, increasing catalyst (enzyme) concentrations or locating related enzymes close together to increase local concentrations of reactants can have an indirect effect on equilibria if end products are efficiently removed as well (e.g., by excretion from the cell). A number of steps in the traditional Emden-Meyerhof-Parnas glycolysis pathway (Figure 3), for instance, are readily reversible because they can also be used for gluconeogenesis (i.e., it is an amphibolic pathway because it is anabolic as well as catabolic). This would otherwise be a potential bottleneck if the system operated under standard conditions. However, fermenting bacteria invest heavily in enzyme production and rely on end-product excretion (e.g., lactate) to drive the catabolic process (Flamholz2013).
While the excretion of fermentation byproducts can be sufficient to allow a microorganism to grow, in reality it is rare to see a buildup of these waste compounds in situ. High concentrations of excreted products can be toxic (e.g., ethanol) or change ecosystem physiology (e.g., pH). As such, the microbiome is very efficient at utilising byproducts and very few actually end up being excreted by the host (exceptions include excess H2, CO2 and CH4).
Many gut microbes can efficiently use fermentation end products of other microorganisms in a metabolically synergistic process. This 'cross-feeding' allows an organism to achieve chemical reactions that, on their own, would be energetically less favourable. The extreme form of this type of mutualistic metabolism is known as syntrophy, where at least one organism is obligately reliant on the metabolites produced by another (Morris2013). For example, the rare gut coloniser, Syntrophomonas zehnderi, is obligately syntrophic with hydrogenophilic archaea, which are responsible for removing hydrogen gas produced by S. zehnderi during the endogenic conversion of butyrate to acetate (Sousa2007). Because the concentration of hydrogen is substantially lowered, Le Chatelier's principle explains a shift towards acetate production (normal butyrate oxidation has a ΔGº’= +48.2 kJ/mol, but is lowered to ΔG' = -8.9 kJ/mol at 10−5 atm H2 levels)(see Wikipedia entry).
There are a number of other energy-generating fermentation pathways stemming from pyruvate that lead to a variety of end products. Pathways to excreted metabolites can be used to 'burn off' NADH and regenerate its oxidised form (NAD+). Energy in the form of ATP can also generated through substrate-level phosphorylation of ADP when acetate or butyrate are produced from their corresponding phosphate esters (Figure 4). Acetate fermentation of glucose produces more ATP than lactate or ethanol fermentation. Some homoacetogens, such as Eubacterium limosum, Blautia producta, Blautia coccoides, Blautia hydrogenotrophica, and Streptococcus mitis, use hydrogen and carbon dioxide to produce acetate.
Figure 4. Microorganisms can change their metabolic pathways to suit conditions and prevent the problematic buildup of toxic or acidic metabolites. SCFA, alchohols, and lactate are common fermentation products made from pyruvate. Abbreviations: Acat, acetyl-CoA acetyltransferase (thiolase); Aldh, acetaldehyde dehydrogenase; Acr, acetoin reductase; Adc, acetoacetate decarboxylase; Adh, alcohol dehydrogenase; Aldc, α-acetolactate decarboxylase; Als, α-acetolactate synthase; Ak, acetyl kinase; Bdh, butanol dehydrogenase; Bcd, butyryl-CoA dehydrogenase; Bydh, butyraldehyde dehydrogenase; Bhbd, β-hydroxybutyryl-CoA dehydrogenase; Buk, butyrate kinase; Cro, crotonase; Fhl, formate-hydrogen lyase; Frd, fumarate reductase; Fh, fumarase; iPdh, isopropanol dehydrogenase; Ldh, lactate dehydrogenase; Mdh, malate dehydrogenase; Pep-Pts, phosphoenolpyruvate phosphotransferase system; Pfl, pyruvate-formate lyase; Pfor, pyruvate-ferredoxin oxidoreductase; Pta, phosphotransacetylase; Ptb, phosphotransbutyrylase; Thl, thiolase; (modified from Willey2013).
Butyrate production from the fermentation of hexoses has been adopted by a number of important gut mutualists. Louis and Flint (Louis2009) have proposed a generalised scheme for butyrate biosynthesis from glucose that is flexible and adaptive, and depends on the nutrients available and the microorganism's phylogeny (Figure 5). The biosynthesis of butyrate typically comes from two pathways: via butyrate kinase, which produces 3 ATP per glucose consumed (Figure 4), or via disproportionation of crotonate (Figure 5), which yields ~2.5 ATP / glucose in addition to energy from an increased proton-motive force (Louis2009). Analysis of key butyrate-producing gut anaerobes has shown that most butyrate is produced using the latter pathway (Louis2009). Prominant butyrogens include Anaerobutyricum hallii, Butyrivibrio crossotus, Clostridium symbiosum, Coprococcus eutactus, Faecalibacterium prausnitzii, Eubacterium rectale and Roseburia faecis.
Figure 5. Intestinal butyrate-producing microorganisms are very important for host homeostasis. Prominant gut bacteria from Eubacterium, Roseburia, Clostridium, Coprococcus and Faecalibacterium genera produce butyrate as a metabolic byproduct and most of these bacteria use disproportionation of crotonate rather than butyrate kinase to produce the metabolite. Abbreviations: Ak, acetyl kinase; Bhbd, L-(+)-β-hydroxybutyryl-CoA dehydrogenase; But, butyryl CoA:acetate CoA transferase; Bcd, butyryl-CoA dehydrogenase; Buk, butyrate kinase; Cro, crotonase; Etf, electron transport protein; Fd, ferredoxin (oxidised and reduced forms); Ldh, lactate dehydrogenase; Pfor, pyruvate-ferredoxin oxidoreductase; Pta, phosphotransacetylase; Thl, thiolase; (modified from Louis2009).
Energy can also be obtained from the fermentation of amino acids. Lysine, for example, can lead to the generation of SCFAs and ATP while utilising much of the catabolic machinery used to ferment carbohydrates. For example, Bui and coworkers proposed a mechanism for the degradation of fructoselysine, a byproduct of the browning process during cooking. Fructoselysine is first degraded to fructose and lysine afterwhich two catabolic pathways lead to butyrate (Bui2015). A human strain of Intestinimonas butyriciproducens (strain AF211) was shown to convert lysine stoichiometrically into butyrate and acetate (Figure 6, Bui2015).
Figure 6. Proposed model for the breakdown of L-lysine, fructose and fructoselysine by Intestinimonas butryiciproducens. Lactate production is dependent on the redox state: in the presence of exogenous acetate, which is typical in the human colon, fructoselysine is converted to approximately three butyrate, with no lactate is produced. Abbreviations: AtoC-A, Member of the two-component regulatory system AtoS/AtoC. In the presence of acetoacetate, AtoS/AtoC stimulates the expression of the atoDAEB operon, leading to short chain fatty acid catabolism (String-db.org); AtoD-A, Acetate CoA-transferase subunit alpha; Fd, ferredoxin; Kal, 3-aminobutyryl-CoA ammonia lyase; KamA, L-lysine-2,3-aminomutase; KamD,E, L-lysine-5,6-aminomutase; Kce, 3-keto-5-aminohexanoate cleavage enzyme; Kdd, L-erythro-3,5-diaminohexanoate dehydrogenase; Ppase, pyrophosphatase; Rnf, proton pumping Rnf cluster;. See Figure 4 for other relevant abbreviations; (modified from Bui2015).
The majority (~70%) of the top 500 gut microbiome species - and up to 99% in terms of abundance (Nagpal2017) - are anaerobic and can't use oxygen as a terminal acceptor during metabolism. Instead, these organisms rely on fermentation of the abundant indigestible carbohydrate and protein fibres leftover from the host's digestive processes. Fermentation generates much less energy than respiration but can be made efficient when waste products are removed and consumed by the host or other commensal microorganisms. Metabolic flexibility is key for members of the microbiome. Many organisms are able to adjust their metabolism to suit the types of substrates available and produce different waste products.
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