Currently, most information on fermentation metabolism in algae comes from studies of Chlamydomonas (Gfeller and Gibbs, 1984, 1985; Kreuzberg, 1984; Gibbs et al., 1986; Ohta et al., 1987; Hemschemeier and Happe, 2005; ; Mus et al., 2007; Hemschemeier et al., 2008; Dubini et al., 2009; Philipps et al., 2011; Burgess et al., 2012; Catalanotti et al., 2012; Magneschi et al., 2012). Genes encoding proteins associated with a diverse set of fermentative pathways have been identified on the Chlamydomonas genome, while a number of biochemical studies have revealed various fermentation circuits. The flexibility among the different pathways for catabolism of stored carbon under dark, anoxic conditions has been demonstrated through analyses of various mutants perturbed for these pathways (Mus et al., 2007; Dubini et al., 2009; Philipps et al., 2011; Burgess et al., 2012; Catalanotti et al., 2012; Magneschi et al., 2012). This flexibility allows Chlamydomonas to satisfy its energy requirements as O₂ from the surrounding environment is depleted.

Over the course of the day there is a natural cycle for storage and utilization of fixed carbon. In phototrophic organisms, polysaccharides (sometimes lipids) accumulate in cells during daylight hours when photosynthetic CO₂ fixation is a dominant metabolic process. During the evening, much of the starch reserve can be hydrolyzed to sugars by amylase activity (Ball, 1998; Dauvillee et al., 2001a,b; Zabawinski et al., 2001) and then, through the activity of glycolysis, be converted to pyruvate (Figure 2). As in bacteria, pyruvate fuels fermentation processes, serving as substrate for pathways that generate various organic acids, acetyl-CoA, alcohols, CO₂, and H₂. Chlamydomonas has multiple pathways for converting pyruvate to acetyl-CoA (Hemschemeier and Happe, 2005; Atteia et al., 2006; Grossman et al., 2007; see Figure 2 for details). Three enzymes involved in these pathways are pyruvate formate lyase (PFL1), pyruvate ferredoxin oxidoreductase (PFR, often referred to as PFOR), and the pyruvate dehydrogenase (PDH) complex. As PDH generates NADH, a product that must be re-oxidized to sustain fermentation metabolism, it is presumed that PFL1 and PFR are the favored pathways for pyruvate catabolism in hypoxic/anoxic cells (Figure 2). While PFL1 catalyzes the conversion of pyruvate to acetyl-CoA and formate, in the PFR reaction pyruvate is oxidized to acetyl-CoA and CO₂ with the concomitant generation of reduced ferredoxin (FDX). FDX can then pass reducing equivalents to hydrogenases to generate H₂ (Happe and Naber, 1993; Ghirardi et al., 1997, 2000, 2007; Melis et al., 2000; Melis and Happe, 2001; Müller, 2003). However, the reduced FDX can also serve as a substrate for nitrite and sulfate/sulfite reductases (Ghirardi et al., 2008).

The acetyl-CoA produced by PFL1 and PFR reactions is either reduced to ethanol by alcohol/aldehyde dehydrogenase 1 (ADH1; Hemschemeier and Happe, 2005; Atteia et al., 2006; Dubini et al., 2009), or metabolized to acetate by the PAT–ACK (Atteia et al., 2006). An alternative pathway for ethanol production may be direct decarboxylation of pyruvate to CO₂ and acetaldehyde through the activity of pyruvate decarboxylase (PDC3). The acetaldehyde generated in this reaction can be reduced to ethanol by ADH (either the same enzyme that catalyzes acetyl-CoA reduction or a distinct enzyme, e.g., ADH2). While the conversion of acetyl-CoA to ethanol by ADH1 oxidizes two NADH molecules, only a single NADH is oxidized in the PDC pathway.

Mutants in specific branches of fermentative metabolism have proven extremely valuable for elucidating the various routes of fermentation metabolism in Chlamydomonas, which are shown in Figure 2.

Formate was demonstrated to be the dominant, secreted organic acid synthesized by Chlamydomonas maintained in anoxic conditions at near neutral pH in dark (Kreuzberg, 1984; Gibbs et al., 1986). The synthesis of formate by PFL1 uses a free-radical mechanism to catalyze the homolytic cleavage of pyruvate into formate and acetyl-CoA. This reaction depends upon a radical S-adenosyl methionine-dependent activating enzyme, designated PFL-AE (Atteia et al., 2006; Hemschemeier et al., 2008), which is usually present as an inactive form in aerobic cells, and is allosterically activated by pyruvate. In Chlamydomonas, PFL1 appears to be located in both mitochondria and chloroplasts (Kreuzberg et al., 1987; Atteia et al., 2006).

Algal strains deficient for PFL1 activity were isolated by independent groups (Philipps et al., 2011; Catalanotti et al., 2012) using different strategies (Burgess et al., 2012). The elimination of PFL1 activity in Chlamydomonas led to a marked accumulation of extracellular lactate, elevated pyruvate decarboxylation, and extracellular ethanol accumulation (Figure 2). The accumulation of lactate in the medium of pfl1 mutants allows for recycling of NADH as a consequence of pyruvate reduction by LDH. Catalanotti et al. (2012) also demonstrated that the pfl1 mutant accumulates elevated intracellular levels of lactate and alanine. Additionally increased intracellular levels of succinate, malate, and fumarate were observed, suggesting operation of the left branch of the reverse TCA reactions to recycle NADH.

Acetyl-CoA produced by PFR/PFL1 activities can be metabolized to generate ATP by conversion to acetate or to help maintain redox balance by conversion to ethanol (Mus et al., 2007). Chlamydomonas possesses three distinct enzymes potentially important for ethanol production when the cells become anoxic: ADH1 (putative dual-function alcohol/acetaldehyde dehydrogenase; Mus et al., 2007; Hemschemeier et al., 2008; Magneschi et al., 2012), and two other putative alcohol dehydrogenases that were identified based on protein homology, designated ADH2 (Augustus version 5.0 protein identifier 516421) and ADH3 (Augustus version 5.0 protein identifier 516422). ADH1 has been localized to chloroplasts (Terashima et al., 2010).

A Chlamydomonas mutant devoid of ADH1 was unable to synthesis either ethanol or CO₂ when the cells were transferred to anoxic conditions (Magneschi et al., 2012). The inability of the adh1 mutant to accumulate ethanol and CO₂, while synthesizing low levels of formate, suggests that the acetaldehyde synthesized by PDC3 and the acetyl-CoA synthesized by PFL1 and PFR cannot be rapidly reduced in the mutant. These findings also indicate that ADH1 is the only acetaldehyde-alcohol dehydrogenase in Chlamydomonas capable of reducing acetyl-CoA or acetaldehyde to ethanol under the conditions used in this study. Interestingly, the adh1 strain was able to compensate for its inability to reduce acetyl-CoA or acetaldehyde to ethanol by reducing a significant amount of pyruvate to lactate. This elevated lactate accumulation was not as high as the level measured in pfl1 (Philipps et al., 2011; Burgess et al., 2012; Catalanotti et al., 2012). However, the adh1 mutants also accumulated high extracellular and intracellular levels of glycerol relative to anoxic wild-type (WT) cells. This acclimation response removes a significant amount of the C3 metabolites at the dihydroxyacetone phosphate (DHAP) step of the glycolytic pathway, which is prior to the reduction of NAD⁺ to NADH; the DHAP is then used as a substrate to re-oxidize NADH in the synthesis of glycerol (Figure 2).

The acetyl-CoA that is produced by PFL1 or PFR activities can be converted to acetate by PAT and ACK (Mus et al., 2007). Two parallel pathways have been identified in Chlamydomonas; PAT1–ACK2 appear to be mitochondrial while PAT2–ACK1 are in the chloroplast (Atteia et al., 2006, 2009; Terashima et al., 2011; Figure 2). Interestingly, the PAT2 and ACK1 genes are contiguous on the genome while PAT1 and ACK2 are far apart on the same chromosome (http://genome.jgi-psf.org/Chlre4/Chlre4.home.html).

While PAT–ACK activities comprise the predominant pathways for acetate formation under dark anaerobiosis, other enzymes are present on the Chlamydomonas genome that may play a role in acetate synthesis. Four genes encoding homologs of acetyl-CoA synthase (ACS) and eight genes encoding homologs of aldehyde dehydrogenase (ALDH) have been identified on the Chlamydomonas genome (Kirch et al., 2004, 2005; Brocker et al., 2012). The ACSs catalyze the putatively reversible conversion of acetate to acetyl-CoA (dash line in Figure 2). The ALDH reaction produces NAD(P)H during the conversion of acetaldehyde to acetate, therefore it is unlikely that these enzymes are active in fermentative metabolism when the cells require regeneration of reducing power (Kirch et al., 2004, 2005; Brocker et al., 2012). To date, there is no biochemical evidence to demonstrate that these alternative pathways for acetate generation are active in Chlamydomonas. In bacteria, the two pathways active under aerobic conditions that generate acetate are the Pat–Ack pathway, which is active in exponentially growing cells, and the PoxB pathway, which dominates during late exponential and stationary phase (Dittrich et al., 2005). It is uncertain whether or not similar regulatory features occur in Chlamydomonas.

The presence and/or production of acetate as Chlamydomonas cells become anoxic was found to be critical for maintenance of anoxic conditions in the light since acetate assimilation promotes O₂ utilization (Kosourov et al., 2007; Morsy, 2011). The level of acetate accumulation during fermentative metabolism has proven to be difficult to predict, probably because it can also be used for the biosynthesis of key metabolites in anoxic cells, provided sufficient ATP and NAD(P)H is available. The adh1 mutant exhibits a higher ratio of acetate production under anoxic conditions compared to WT cells, which reflects the elimination of ethanol production from the acetyl-CoA that is generated by PFL1 and PFR activities in the mutant strain; glycerol and lactate production serve as the primary NADH re-oxidation mechanisms in this mutant (Magneschi et al., 2012). In contrast, the pfl1 mutant strains exhibit strongly reduced acetate accumulation (Burgess et al., 2012; Catalanotti et al., 2012); this decrease is likely due to a diminished intracellular acetyl-CoA pool.

The mitochondria of cells maintained in aerobic conditions use PDH to convert pyruvate to acetyl-CoA; the acetyl-CoA generated can be metabolized to CO₂ by the TCA cycle. In some animals PDH can function under anaerobic conditions (reviewed by Tielens and van Hellemond, 1998; Tielens et al., 2002; Hoffmeister et al., 2005; Tucci et al., 2010; Atteia et al., 2013). However, in many prokaryotes and eukaryotes, pyruvate oxidation in the absence of O₂ is typically mediated by PFR. PFR belongs to a large family of thiamine pyrophosphate (TPP)-dependent enzymes. It catalyzes the oxidative cleavage of the carbon–carbon bond of the carboxyl group of pyruvate to liberate CO₂ and reducing equivalents, with the attachment of the resulting acetyl group to CoA. However, unlike PDH, PFR can also function in the reverse direction catalyzing the production of pyruvate from CO₂ and acetyl-CoA (Evans et al., 1966; see below), with FDX or flavodoxin serving as electron donors (Charon et al., 1999; Ragsdale, 2003; Figure 2). In Chlamydomonas, the reduced FDX generated from pyruvate oxidation by PFR activity can be re-oxidized by hydrogenases, generating H₂ (Müller, 2003), or by reactions that enzymatically reduce nitrite and sulfate/sulfite. Hydrogenases are widespread among prokaryotes, whereas they are not as common among eukaryotes, and are restricted to a subset of unicellular eukaryotes, including photosynthetic algae (Meuser et al., 2011; Müller et al., 2012). Chlamydomonas hydrogenases belong to the class of [FeFe]-hydrogenases in which a [4Fe4S] cluster is linked through a cysteine residue to a 2Fe- cluster (Peters et al., 1998; Mulder et al., 2011).

Hydrogen production in algae is likely to have significant impacts on redox poising, photoprotection, and fermentative energy metabolism. Hydrogen production is coupled to cellular metabolism in a variety of ways, all of which are associated with O₂ limitation: (i) direct biophotolysis, (ii) indirect biophotolysis, and (iii) dark fermentative metabolism (Figure 3). Direct biophotolysis involves light-dependent oxidation of water by photosystem II (PSII), the transfer of electrons from PSII to photosystem I (PSI), light-dependent excitation of PSI with the concomitant reduction of FDX and the subsequent transfer of electrons from FDX to hydrogenase (Benemann et al., 1973; Greenbaum, 1982; Happe and Naber, 1993; Miura, 1995; Ghirardi et al., 2007). During direct biophotolysis, the O₂ generated by PSII must be reduced in order to prevent the accumulation of O₂ to levels that would inhibit the hydrogenase. Indirect biophotolysis involves non-photochemical reduction of the PQ pool by NAD(P)H generated as a consequence of catabolic metabolism, followed by light-dependent FDX reduction by PSI and the subsequent transfer of electrons from FDX to hydrogenase (Cournac et al., 2000; Kosourov et al., 2003; Mus et al., 2005; Chochois et al., 2009). In the third H₂-production pathway, starch catabolism provides electrons to the hydrogenases under dark fermentative conditions (Gfeller and Gibbs, 1984; Kreuzberg, 1984; Ohta et al., 1987; Happe et al., 1994; Ghirardi et al., 1997; Melis and Happe, 2001; Posewitz et al., 2004; Mus et al., 2007; Dubini et al., 2009).

Hydrogenases also function in H₂ uptake, with two distinct uptake pathways described in Chlamydomonas (Gaffron, 1944; Kessler, 1974; Maione and Gibbs, 1986a,b; Chen and Gibbs, 1992a; Figure 3). In the first pathway, H₂ oxidation and cyclic PSI activity in the light are linked to RuBisCO-mediated anaerobic CO₂ fixation. Electrons from H₂ are used to reduce FDX, which then reduces FDX-NAD(P) oxidoreductase (FNR), leading to the generation of NAD(P)H which, along with the ATP generated by cyclic electron flow, can be used to fix CO₂. This pathway requires the absence of O₂ evolution from PSII. In the second pathway, termed the oxyhydrogen reaction, H₂ oxidation occurs concomitantly with the uptake of low levels of O₂ in a process that can be coupled to CO₂ fixation (Gaffron and Rubin, 1942; Gaffron, 1944; Russell and Gibbs, 1968; Kessler, 1974; Chen and Gibbs, 1992a). Although not well characterized, it is posited that H₂ oxidation provides the reducing equivalents for CO₂ fixation and that the low levels of O₂ present are respired to provide ATP (Gaffron and Rubin, 1942; Gaffron, 1944; Maione and Gibbs, 1986a; Chen and Gibbs, 1992a).

Recently, mutants were obtained in each of the two HYDA genes of Chlamydomonas, HYDA1 and HYDA2 (Meuser et al., 2012). The phenotypes of the single (hydA1 and hydA2) and double (hydA1–hydA2) mutants were analyzed under both light and dark anoxic conditions. Both single mutants could catalyze H₂ production from reductant generated from either fermentative or photosynthetic metabolism. However, the contribution of the HYDA2 enzyme to H₂ photoproduction under the conditions tested was approximately 25% of that of HYDA1 (Godman et al., 2010; Meuser et al., 2012).

The impact of the hydEF-1 lesion on fermentation is more interesting since it demonstrates the flexibility of Chlamydomonas anaerobic metabolism (see below). This mutant is unable to assemble the inorganic constituents of the hydrogenase active site, and consequently cannot catalyze H₂ synthesis (Posewitz et al., 2004).

Anoxic cultures of the Chlamydomonas hydEF-1 mutant exhibit lower CO₂ evolution and reduced extracellular formate, acetate, and ethanol accumulation. Interestingly, the mutant synthesizes elevated levels of extracellular succinate (Dubini et al., 2009; Figure 2), indicating activation of a fermentative pathway that is not operating at significant levels in WT cells. Microarray data and metabolite analyses suggest that carboxylation of pyruvate in the hydEF-1 mutant leads to the synthesis of either malate or OAA (or both), which is subsequently converted to succinate via reverse reactions of the TCA cycle. Activation of the reductive TCA branch as a means of recycling NADH was previously observed in anaerobic bacteria (Gray and Gest, 1965; Schauder et al., 1987; Buchanan and Arnon, 1990; Beh et al., 1993; Yoon et al., 1999), in the green alga Selenastrum minutum (Vanlerberghe et al., 1989, 1990) and in vascular plants (Sweetlove et al., 2010), but was not known to occur in Chlamydomonas.

The alternative pathway suggested by Dubini et al. (2009) not only explains succinate accumulation under anaerobic conditions, but also raises the possibility that Chlamydomonas could potentially operate a complete reverse TCA cycle. This would require that PFR functions in the direction of pyruvate synthesis under the appropriate metabolic conditions. Various researchers have suggested that PFR could function in the synthesis of pyruvate in Chlamydomonas (Chen and Gibbs, 1992b; Melis et al., 2007; Terashima et al., 2011; Figure 2). Chen and Gibbs (1992b) detected ATP-citrate lyase, as well as PFR and α-ketoglutarate synthase activities in Chlamydomonas cell extracts, speculating that the existence of these three key enzyme activities indicated that the reverse TCA cycle could operate in Chlamydomonas. These authors showed that a Chlamydomonas mutant with a compromised Calvin–Benson cycle takes up CO₂ in the dark under minimal aerobic conditions (1% O₂), and that the CO₂ uptake is coupled to H₂ oxidation (Chen and Gibbs, 1992a), suggesting that the reverse TCA cycle could be a significant pathway for CO₂ assimilation when the Calvin–Benson cycle is compromised (Chen and Gibbs, 1992b). Hence, under these conditions H₂ oxidation would provide the reducing equivalents to drive the reverse TCA cycle and to allow PFR to synthesize pyruvate, leading to the accumulation of an array of biosynthetic precursors. The possibility of PFR-dependent synthesis of pyruvate has also been observed in many hydrogenosome-containing eukaryotic organisms experiencing anaerobic conditions (Lindmark and Müller, 1973). Furthermore, in the unicellular microaerophilic eukaryote Trichomonas vaginalis, MME and PFR are central to carbohydrate metabolism in the hydrogenosomes (Müller, 1993). In addition, PFR and MME activities have been linked to malate production in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, also suggesting reductive carboxylic acid cycle activity (Fukuda et al., 2005). The association of PFR and MME with pyruvate metabolism in hydrogenosome-containing anaerobic eukaryotes, the findings that a similar set of anoxic-induced proteins are associated with Chlamydomonas chloroplasts, and the metabolite data obtained with various Chlamydomonas strains exposed to anoxic, reductant-rich conditions, all suggest that the TCA cycle may operate in the reverse direction in Chlamydomonas chloroplasts in anoxic cells that have sufficient reducing equivalents and ATP.

Glycerol and lactate are usually minor end products of green algal fermentation (Gfeller and Gibbs, 1984; Kreuzberg, 1984). Glycerol is synthesized from DHAP, and its synthesis results in recycling of one NADH. The reaction precedes the formation of pyruvate and the C3 oxidation (NADH formation) step in glycolysis. Hence, glycerol and lactate production in the adh1 mutant would allow for efficient recycling of NADH, maintenance of redox balance and sustained glycolytic production of ATP even though the cells are unable to reduce acetaldehyde or acetyl-CoA to ethanol (Magneschi et al., 2012; Figure 2). The pfl1–1adh1 double mutant cannot synthesize either formate or ethanol (Catalanotti et al., 2012; Figure 2). This strain, like pfl1, secretes significant levels of lactate, however, like the adh1 mutant, it also synthesizes and secretes high levels of glycerol and acetate. Hence, this mutant exhibits a complete rerouting of glycolytic carbon to lactate and glycerol, transforming Chlamydomonas cells from a formate/acetate/ethanol to a glycerol/lactate fermenter (Catalanotti et al., 2012; Figure 2).

When O₂ in the environment is depleted, plants can use PDC to convert pyruvate to acetaldehyde, which is metabolized to ethanol by ADH (Gibbs and Greenway, 2003; Bailey-Serres and Voesenek, 2008). The ethanol generated in plant roots can rapidly diffuse into the rhizosphere, which limits its toxicity. Plants can also synthesize lactate under conditions of low O₂. The transition from lactic to ethanolic fermentation appears to be controlled by the pH of the cytoplasm of the cell. A ~0.6 unit decrease in cytosolic pH favors PDC activity, which promotes ethanol production and limits lactate synthesis (reviewed by Bailey-Serres and Voesenek, 2008). This lactic to ethanolic switch is critical for maintaining cytosolic pH (Roberts et al., 1989). In addition to eliciting metabolic changes, low O₂ can trigger alterations in plant morphology which include petiole or internode elongation, altered anatomy and cell ultrastructure in leaves and roots, development of lateral or adventitious roots and the formation of aerenchyma cells (Bailey-Serres et al., 2012).

Ethanol, lactate, and glycerol are common end products of fermentative metabolism in many organisms. The synthesis and excretion of ethanol allows carp to survive anaerobiosis for up to ~5 months (van Warde et al., 1993) and goldfish to withstand anoxia for several weeks (van den Thillart et al., 1983). It is notable that the fermentation pathways used for these reactions appear to have their origins in a typical yeast-type PDC and ADH (van Warde et al., 1993; Figure 4). The fungi, a highly diverse group, can also ferment carbohydrates to lactate, glycerol, and ethanol. Glycerol acts as a redox valve under anaerobic conditions since it enables re-oxidation of NADH that is generated during the conversion of sugars into biomass. While fungi may also excrete organic acids, the levels are generally low; these acids include formate, acetate, lactate, and succinate (Figure 4). Formate production is not uncommon in fungi as a result of the activity of a cytosolic (and hydrogenosomal) PFL, which provides the acetyl-CoA for ethanol production (Boxma et al., 2004; Figure 4).

Finally, pathogenic amoebozoa such as Entamoeba histolytica often experience anaerobic conditions; their main end products of anaerobic energy metabolism are alanine, CO₂, ethanol, and acetate (Müller et al., 2012). The enzymes responsible for generating these products are exclusively in the cytosol (Müller, 2003). The initial reactions of the pathway involve conversion of PEP to pyruvate by pyruvate orthophosphate dikinase (PPDK; Reeves, 1968; Figure 5A), which also generates ATP. The pyruvate is then oxidized via PFR to CO₂ and acetyl-CoA, with the latter converted into a mixture of acetate and ethanol (Figure 5A). Alternatively the PEP can be carboxylated to OAA by PEP carboxytransferase, reduced to malate by malate dehydrogenase (MDH) and the malate then converted to pyruvate by the malic enzyme (ME; Figure 5A; as reviewed by Müller et al., 2012). Entamoeba possesses a bifunctional aldehyde/alcohol dehydrogenase (ADH), which represents a fusion protein that contains an N-terminal aldehyde dehydrogenase domain and a C-terminal alcohol dehydrogenase domain. This enzyme regenerates 2 NAD⁺, is present in many eukaryotes, and has been found to be highly expressed in Chlamydomonas (Mus et al., 2007; Catalanotti et al., 2012; Magneschi et al., 2012), other green algae and protozoan parasites such as Giardia intestinalis, Trichomonas, and euglenids.

Trichomonas vaginalis synthesizes ethanol from pyruvate in the cytosol via PDC and ADH. However, the main end products of T. vaginalis fermentative metabolism are distributed between the cytosol (glycerol, lactate, and ethanol) and the hydrogenosome (CO₂, H₂, and acetate; Figure 5B). Similarly, Chlamydomonas can synthesize glycerol from DHAP, which is catalyzed by glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, and LDH can catalyze lactate accumulation. However, while both reactions in T. vaginalis occur in the cytosol, their locations in Chlamydomonas are not clear.

Fermentation in animals often involves malate dismutation. It is not uncommon for parasitic worms to switch to complete anaerobic metabolism once they are established in the host tissue. In parasitic mode they convert the PEP generated by glycolysis to OAA, which is then reduced to malate via a cytosolic malate dehydrogenase (Figure 6). This reaction results in re-oxidation of one molecule of NADH. The malate is then imported into the mitochondrion where dismutation occurs; a portion of the malate is oxidized to acetate (via pyruvate), and another is reduced to succinate. In the latter reaction, malate is converted to fumarate by FUM and the fumarate is reduced to succinate (Figure 6); this pathway is similar to the alternative fermentation pathway activated in the Chlamydomonas hydEF-1 mutant. Many organisms excrete succinate produced by malate dismutation rather than decarboxylating the succinate to generate propionate plus an extra molecule of ATP (Pietrzak and Saz, 1981; Müller et al., 2012; Figure 6). Interestingly, in the parasite system, fumarate reduction is performed by a membrane-associated, anaerobiosis-specific enzyme (FRD) that is coupled to an electron transport chain that functions specifically under anaerobic conditions. Electrons are transferred from NADH to fumarate via rhodoquinone (RQ; Figure 6) instead of ubiquinone (UQ, which is normally used under oxic conditions); the lower redox potential of RQ (relative to UQ) allows for the thermodynamically favorable use of electrons in the synthesis of succinate by FRD (as reviewed by Müller et al., 2012).

O₂ deficiency is often associated with a wide range of excreted metabolites, but may also trigger more complicated responses involving sequestration of specific end products. Plants experiencing low O₂ accumulate alanine and γ-aminobutyric acid (GABA; reviewed by Bailey-Serres and Voesenek, 2008). Upon re-oxygenation, alanine can be recycled back to pyruvate, and GABA can be converted to succinate. This set of amino acid oxidation reactions may minimize the decline in cytosolic pH and reduce the loss of fixed carbon as ethanol or lactate. Alanine also accumulates in T. vaginalis and in many animals belonging to the Excavata taxa (Edwards et al., 1989) as a minor end product.

A less common fermentation process, but largely used in marine environments, involves opine formation. This pathway is localized to the cytosol and involves pyruvate condensation with an amino acid in a redox reaction that regenerates NAD⁺. A possible advantage of this alternative pathway for balancing cellular redox is that opine is less acidic than lactate. Moreover the process maintains an osmotic equilibrium since one amino acid is consumed per opine synthesized (Ballantyne, 2004).

The capacity for nitrate respiration is widespread among bacteria, fungi, and other eukaryotic organisms (Morozkina and Kurakov, 2007). Details of the denitrification pathway have been studied in fungi and bacteria (see previous paragraphs). On the other hand, the enzymes required for nitrogen metabolism in foraminifera and diatoms are not well characterized, although the occurrence of the pathway was noted (Risgaard-Petersen et al., 2006; Kamp et al., 2011).

Numerous reports have demonstrated the presence of two main pathways for denitrification; one is localized in the mitochondrion and usually occurs under low O₂ conditions, while the other, often referred to as ammonia fermentation, is localized in the cytosol (Zhou et al., 2001; Takasaki et al., 2004; Morozkina and Kurakov, 2007; Figure 7). The latter pathway appears to be activated under strict anoxic conditions and involves reduction of nitrate to ammonia using reductant generated by the catabolic oxidation of ethanol (the donor of electrons) to acetate, which is coupled to SLP. As shown in Figure 7, the ethanol is oxidized to acetaldehyde by an alcohol dehydrogenase (designated Ald), which is converted to acetyl-CoA by acetaldehyde dehydrogenase (AddA). The acetyl-CoA is then converted to acetate and CoA, with the concomitant production of ATP by Ack (Zhou et al., 2001). Under hypoxic conditions the ethanol is oxidized to acetate and the electrons generated in the reaction are used to reduce nitrite to N₂O, which is excreted from cells (Zhou et al., 2001; Figure 7). Nitrate and nitrite reductases catalyze the reduction of nitrogen oxides to ammonia using NADH as the electron donor, and are assimilatory enzymes.

H₂ and CO₂ are generated in an ancestral anaerobic pathway that is present in many green algae. The generation of H₂ in algae often serves as a redox valve. This pathway can be in chloroplasts, as in Chlamydomonas and other algae, in mitochondria-like organelles, as in the Stramenopiles, or in the hydrogenosomes of the amoebozoa, some opisthokonta and Excavata. H₂ production is often associated with PFR activity, which oxidizes pyruvate to acetyl-CoA and CO₂. Reduced ferredoxin transfers electrons to a hydrogenase that can convert protons and electrons into H₂ (Figure 5B).