There is a great deal of interest in microbial processes in anaerobic sediments. Most of the Earth\'s surface is covered by sediment, and the amount of biomass sequestered there rivals the amount of biomass in the oceans above it. It is known that in the transition zone from methanogenesis to sulfate reduction (Fig. [1](#fig1){ref-type="fig"}), methane from methanogenesis disappears. Below this depth, in the deeper anoxic sediments, the feeding chain starts with fermentation and is short (Parkes, Cragg and Wellsbury [@bib72]). Most of the available organic matter in deep sediments is microbial necromass (Lomstein *et al*. [@bib59]), and most of the fermentable necromass is in the form of proteins and amino acids because cells are mostly protein by weight.

![Physiological considerations of cell mass conversions in anoxic subseafloor sediment. Most of the fermentable substrates are amino acids from dead cells, and the resulting fermentation products result in an upper boundary limit for mass carbon conversion to methanogens of 100:1 (from acetate). Lipids are not fermentable, but degradation products such as fatty acids can be fermented in syntrophic secondary fermentations. Fermentable substrates are shown in pink colors. SMTZ: sulfate methane transition zone.](fuaa004fig1){#fig1}

Oxidants such as Fe\[III\], Mn\[IV\], SO~4~^2--^ or NO~3~^--^ that could serve as terminal acceptors for microbes to drive the oxidation of reduced carbon compounds are generally thought to be depleted below the sulfate methane transition zone (SMTZ; Bowles *et al*. [@bib12]). In anoxic marine sediments, these oxidants are typically used up (reduced) closer to the seafloor surface, whereas deep below the SMTZ in the subseafloor, they are lacking because these settings are insulated from nutrient exchange with ocean water or hydrothermal systems (Parkes, Cragg and Wellsbury [@bib72]). In such systems, extreme energy limitation is thought to play a major role (Hoehler and Jørgensen [@bib40]; Lever *et al*. [@bib54]), and fermentations are dominant (Bowles *et al*. [@bib12]) that require organic compounds of intermediate oxidation states as terminal acceptors.

In subseafloor sediment, there exists a global trend of exponentially decreasing cell densities with depth (Kallmeyer *et al*. [@bib45]). There are some exceptions at redox transition zones (D\'Hondt *et al*. [@bib24]; Parkes *et al*. [@bib73]), but below these zones---in the fermentation zone---the trend of net death continues. The question 'why are the cells always dying faster than they are growing?' concerns the fate of individual cells and their individual adenosine triphosphate (ATP) budgets. Thus, an understanding of the bioenergetic stoichiometry of the relevant necromass fermentations would help to set boundary conditions for production of new cell biomass based on carbon and energy availability. To our knowledge, energetic boundary conditions have not been constrained with ATP consumption and production budgets of relevant fermentation-based metabolisms.

Here, we consider the energetic stoichiometry of the anoxic subseafloor fermentation zone where amino acids are quantitatively the most important substrate for fermentation reactions (Lever *et al*. [@bib54]), the end products of which support methanogens in proximity to the transition zone of anaerobic sediments where methane disappears (Parkes, Cragg and Wellsbury [@bib72]). Our analysis constrains the energetic boundary conditions of this net-death ecosystem with ATP consumption and production budgets of relevant fermentation metabolisms, to obtain an order of magnitude estimate on an upper limit to how much growth in anoxic sediment can result from fermented microbial necromass. The calculations show that amino acid fermentation reactions all yield maximum ATP levels insufficient to result in a net increase in cellular biomass, given the high energetic cost of protein biosynthesis. Our analysis shows that below the SMTZs where fermentation and methanogenesis dominate, fermentation of necromass simply does not provide enough energy to support net growth. Rather, our calculations imply an exponential drop-off in cell biomass over time as has been observed on a global scale (Kallmeyer *et al*. [@bib45]) in deep anoxic subseafloor sediment.

THE FERMENTATION ZONE {#sec2}
=====================

Sediment moves upward with new incoming sediment at the surface. Fermentations occur throughout marine sediment. Our focus in this paper concerns processes in the fermentation zone that begins below the SMTZ. We furthermore focus on isolated sediment that does not receive an influx of nutrients or electron acceptors such as sulfate, Fe oxides or Mn(IV). The fermentation zone below the SMTZ is dominated by fermenters and methanogens. Fermenters live by degrading the cell mass that the overlying aerobes, nitrate respirers and sulfate reducers left behind. Fermentations produce mainly H~2~, CO~2~ and acetate that fuel the methanogens, which produce methane that in turn fuels anaerobic methane oxidation at the overlying SMTZ (Fig. [1](#fig1){ref-type="fig"}). Most of the available organic matter in deep sediments is microbial necromass (Lomstein *et al*. [@bib59]; Braun *et al*. [@bib14]). Most of that necromass is in turn protein, because cells are ∼50% protein by dry weight.

Fermentations do not require a supply of external terminal acceptors; they are disproportionation reactions of organic compounds having an intermediate oxidation state. Fermentations support ATP synthesis by substrate level phosphorylation or chemiosmotic coupling (Buckel and Thauer [@bib18]). Lipids and fatty acids are too reduced for simple fermentations; they need to be degraded by syntrophic associations of fermenting bacteria with methanogenic partners (Fig. [1](#fig1){ref-type="fig"}). In contrast, amino acids, nucleic acid bases and sugars are easily fermentable substrates (Barker *et al*. [@bib9]; Barker [@bib8]; Brasen *et al*. [@bib13]).

In the fermentation zone (below the SMTZ), cell mass is the main substrate for fermenters (Lever *et al*. [@bib54]). By weight, exponentially growing cells are made of roughly 50--60% protein, 20% RNA, 10% lipids, 3% DNA, ∼10% sugars as cell wall constituents and some metabolites (Stouthamer [@bib87]; Neidhardt, Ingraham and Schaecter [@bib68]; Lengeler, Drews and Schegel [@bib51]). Well-fed cells can contain considerable amounts of glycogen, \>10% by weight. Cells in sediments are generally regarded as starved (Hoehler and Jørgensen [@bib40]) and for our purposes can be regarded as having little, if any, glycogen content. Because lipids are degraded relatively slowly (Xie *et al*. [@bib100]), protein, RNA and to a lesser extent DNA, remain as the main substrates. There are also viruses in the fermentation zone (Orsi [@bib70]). They are about two orders of magnitude smaller than cells, but there are about two orders of magnitude more of them (Engelhardt *et al*. [@bib31]) such that they represent a quantitatively similar amount of protein and nucleic acid substrate for fermentations as the cell populations from which they stem. It is well known that starved cells are much smaller than exponentially growing cells (Lever *et al*. [@bib54]), but we will be looking at the issue of how many starving cells can arise from the fermentation products of other starving cells; so, if all cells are small, our conversions should not be heavily affected.

The energetically most demanding task that a cell does during growth is protein synthesis (Stouthamer [@bib88]), which requires ∼75% of the cell\'s energy budget, with ribosomes constituting ∼40% of the cell by weight. In energy-limited environments like sediments, maintenance and survival are more important than growth (Hoehler and Jørgensen [@bib40]). Cryptic elemental cycles occur in the fermentation zone such as sulfate reduction (Holmkvist, Ferdelman and Jørgensen [@bib42]), but these are predicted to result in limited amounts of activity (Hoehler and Jørgensen [@bib40]). Fermentations are thus the major source of energy below the SMTZ; so, it is worthwhile to obtain an order of magnitude estimate that might put an upper limit to how much growth in the anaerobic sediment column can result from how much fermented biomass.

FERMENTATION PRODUCTS {#sec3}
=====================

While the substrate for fermentations (cell mass) is straightforward, the fermentations themselves are not (LaRowe and Amend [@bib49]). Most of the cellular mass is in the form of protein, and the main organic substrates available for living cells in the subseafloor fermentation zone are other dead cells (Lomstein *et al*. [@bib59]). Amino acids are thus the main fermentable substrates. Typical products of bacterial amino acid fermentations are CO~2~, NH~4~^+^, acetate, butyrate and some H~2~ (Smith and Macfarlane [@bib86]).

Amino acid fermentations entail ATP synthesis by substrate level phosphorylation (SLP) and by ion gradient phosphorylation using chemiosmotic coupling and ATPases at the cytoplasmic membrane. In fermenters studied in detail so far, the quantitative contributions of SLP and ion gradients to ATP synthesis are roughly equal and are often interconnected by flavin-based electron bifurcation (Buckel and Thauer [@bib18]). Flavin-based electron bifurcation is a mechanism of energy coupling in anaerobes that interconverts ferredoxin pools, reduced nicotinamide adenine dinucleotide (NADH) pools and, in cooperation with Rnf complexes (Na^+^/H^+^-pumping NAD ferredoxin reductases), ion gradients at the plasma membrane (Müller, Chowdhury and Basen [@bib67]). Although bifurcation is central to energy conservation in many strict anaerobes, it was discovered only recently (Herrmann *et al*. [@bib38]); hence, complete maps with full stoichiometry, redox and energy balance are available for only a few well-studied fermentation systems.

Well-studied examples of amino acid fermentations are pathways of glutamate fermentation. There are five different pathways of glutamate fermentation (Buckel [@bib15]; Plugge *et al*. [@bib74]). Two of the pathways of glutamate fermentation that have been studied in detail, one from *Clostridium tetanomorphum* and one from *Acidaminococcus fermentans*, produce exactly the same end products in exactly the same amounts with exactly the same energy yields, but entail different chemical intermediates in the fermentation process and unrelated enzymes (Buckel and Barker [@bib16]). The overall reaction is $$\documentclass[12pt]{minimal}
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5\,{\rm{glutamat}}{{\rm{e}}^-} + {\rm{2}}\,{{\rm{H}}_{\rm{2}}}{\rm{O}} + {\rm{2}}{{\rm{H}}^ + } \to {\rm{5}}\,{\rm{N}}{{\rm{H}}_{\rm{4}}}^ + + {\rm{5}}\,{\rm{C}}{{\rm{O}}_{\rm{2}}} + {{\rm{H}}_{\rm{2}}} \nonumber \\
\qquad + {\rm{6}}\,\,{\rm{acetat}}{{\rm{e}}^-} + {\rm{2}}\,\,{\rm{butyrat}}{{\rm{e}}^-},
\end{eqnarray*}$$\end{document}$$with an energy yield of 0.95 ATP per glutamate and a free energy change of Δ*G*~o~′ = --63 kJ per mol glutamate (Buckel and Thauer [@bib18]). Per 5 glutamate, the pathway generates 3 ATP by SLP and 3 ATP by a Na^+^-dependent ATPase, but one pumped Na^+^ is expended for each glutamate imported, hence the non-integer ATP yield (Buckel and Thauer [@bib18]). The soluble enzymes involved in the two pathways are different, the reactions harnessed for pumping are also different and electron bifurcation is centrally involved in the pathway of both fermenters (Buckel and Thauer [@bib18]). A similar situation is encountered for glucose fermentations in *Thermotoga maritima* and *Pyrococcus furiosus*, the pathways and enzymes are different and the overall reactions are the same $$\documentclass[12pt]{minimal}
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{\rm{glucose}} + {\rm{2}}\,{{\rm{H}}_{\rm{2}}}{\rm{O}} \to {\rm{2}}\,{\rm{acetat}}{{\rm{e}}^-} + {\rm{2}}\,{{\rm{H}}^ + } + {\rm{2}}\,{\rm{C}}{{\rm{O}}_{\rm{2}}} + {\rm{4}}\,{{\rm{H}}_{\rm{2}}},
\end{equation*}$$\end{document}$$with an energy conservation of 2--4 ATP per glucose, 1.5 ATP generated by SLP and 1.5 generated by chemiosmotic coupling. At low temperature and low hydrogen pressure, up to 4 ATP can be synthesized (Müller *et al*. [@bib65]).

In terms of sediment fermentation processes, protein and nucleic acids are, by weight, the main substrates; so, we focus on them here. Proteins are broken down extracellularly by proteases; peptides are imported and hydrolyzed to amino acids. In bacteria, amino acids can be fermented by reactions similar to the case of glutamate fermentation above, or by Stickland reactions (Barker *et al*. [@bib9]), in which one amino acid is oxidized while another one is reduced. *Clostridium sticklandii* is a bacterial generalist amino acid fermenter that can metabolize all amino acids in Stickland reactions except Ala and Glu (Fonknechten *et al*. [@bib33]). A key enzyme of Stickland reactions is glycine reductase (Andreesen [@bib3]), a selenoprotein that generates acetyl phosphate for SLP from glycine reduction. However, no glycine reductase was found to be expressed in subseafloor metatranscriptomes (Orsi *et al*. [@bib1_749_1583516775552]) suggesting that alternative mechanisms of amino acid fermentation might takeplace.

In Archaea, amino acid fermentations (Adams and Kelly [@bib1]) typically involve conversion of the amino acids to the corresponding 2-oxoacids by transaminases; the 2-oxoacids are oxidatively shortened by 2-oxoacid oxidoreductases that generate CO~2~, reduced ferredoxin and acyl-CoA. Ferredoxin is typically reoxidized by hydrogenases; acyl-CoA typically yields acyl-phosphate as an intermediate and ATP by substrate level phosphorylation, releasing the corresponding short-chain fatty acid, or volatile fatty acid (VFA). This is evidenced in the accumulation of VFAs in the fermentation zone of anoxic sediments (D\'Hondt *et al*. [@bib23]), and by newly discovered yet uncultivated Archaea living in the fermentation zone of anoxic marine sediment featuring an amino acid fermentation-based metabolism (Lloyd *et al*. [@bib58]). Furthermore, two novel Archaea that were obtained and imaged in enrichment cultures from marine sediment were both amino acid fermenters, '*Korarchaeaon cryptophilum'* (Elkins *et al*. [@bib30]) and '*Prometheoarchaeum syntrophicum'* (Imachi *et al*. [@bib43]). Peptide-fermenting Archaea such as *Pyrococcus furiosus* can possess as many as 10 different 2-oxoacid oxidoreductases that will accept as substrates the 2-oxoacids stemming from deamination of Ala, Thr, Cys, Met, Val, Leu, Ile, Phe, Tyr, Trp, His, Arg, Glu and Gln (Scott, Poole and Adams [@bib83]).

In order to maintain redox balance, electrons generated from oxidative steps of amino acid fermentation (deaminations and decarboxylations) have to be excreted, typically in the form of reduced molecules such as H~2~, formate (Zindel *et al*. [@bib102]) or as aliphatic residues in organic acids such as acetate or butyrate. This can involve a variety of reactions including Stickland reactions (Andreesen [@bib3]), reduction of crotonyl-CoA (Buckel and Thauer [@bib18]), reduction of protons using soluble hydrogenases as in *Thermotoga* (Schut and Adams [@bib82]) or membrane-bound hydrogenases as in *Pyrococcus* (Yu *et al*. [@bib101]). Below the STMZ in deep anaerobic sediment, CO~2~ and H~2~ can be used by methanogens for ATP synthesis and carbon assimilation, whereas the other end products of amino acid fermentation, namely acetate, short chain fatty acids and NH~4~^+^ accumulate transiently in the environment (D\'Hondt *et al*. [@bib24]).

GROWTH FROM FERMENTATION PRODUCTS {#sec4}
=================================

Through the archaeal or the bacterial fermentation pathways, each amino acid generates approximately one ATP, one acetate, two H~2~ (including some H~2~ hidden in butyrate), one CO~2~ and one NH~4~^+^. We have neglected the cost of peptide import, which if it lies in the range of one ATP per deca- to pentapeptide via an ABC-type peptide importer would consume 10--20% of the ATP budget; *Acidaminococcus fermentans*, for example, requires 0.25 ATP (one Na^+^) per imported glutamate (Buckel and Thauer [@bib18]). For estimating methanogenesis in the next section, we generously estimate that fermentations can provide two H~2~, CO~2~ and acetate each per amino acid, in addition to one ATP and one NH~4~^+^, plus substoichiometric amounts of other organic acids.

Nucleic acids can also be fermented. Actively growing cells contain ∼20% RNA by weight (Stouthamer [@bib87]; Neidhardt, Ingraham and Schaecter [@bib68]; Lengeler, Drews and Schegel [@bib51]), though this might be somewhat lower in the ultraslow growth state of the deep biosphere (Hoehler and Jørgensen [@bib40]). Known pathways of purine fermentation can yield one ATP per purine (Barker *et al*. [@bib9]), and some cells that can satisfy their carbon and energy needs from purines alone (Hartwich, Poehlein and Daniel [@bib35]). It should furthermore be possible to obtain one ATP per pyrimidine as well using known pathways (Schönheit *et al*. [@bib80]). That leaves ribose. RNA is ∼40% ribose by weight; cells are 20% RNA, hence ∼8% ribose by weight. Ribose is probably the most abundant pure sugar in deep sediment. It can enter catabolic pathways by a number of routes (Brasen *et al*. [@bib13]). A novel pathway is the ribose bisphosphate pathway that seems to be present in subsurface microbial fermenters (Wrighton *et al*. [@bib99]). This pathway involves the once puzzling ribulose-1,5-bisphosphate carboxylase that was found in the genome sequence of many heterotrophic archaea (Aono *et al*. [@bib6]). RNases degrade RNA to ribonucleoside 5′-monophosphates, a phosphorylase cleaves the base, which can be degraded to yield one ATP, from the ribose backbone. This yields ribose-1,5-bisphosphate, which is converted by an isomerase to ribulose-1,5-bisphosphate, the substrate for RuBisCO (Aono *et al*. [@bib6]), which carboxylates the substrate to yield two molecules of 3-phospho-[d]{.smallcaps}-glycerate (3PGA). 3PGA is an intermediate of glycolysis that can yield one ATP at the pyruvate kinase reaction and one more ATP from pyruvate oxidation via the 2-oxoacid oxidoreductase, yielding CO~2~, H~2~ and acetate as above. That yields four ATP per ribose, about one each per base or about five ATP per nucleoside; we subtract one ATP per rNMP for import, yielding about four ATP plus two H~2~, two acetate and one CO~2~ per base.

Homoacetogenesis combines the fermentation of sugars with H~2~-dependent CO~2~ reduction by acetogenic microbes and may also be a key form of metabolism in deep anoxic subseafloor sediment (Lever *et al*. [@bib55]; Lever [@bib53]; He *et al*. [@bib36]; Martin *et al*. [@bib60]; Sewell, Kaster and Spormann [@bib84]). Indeed, homoacetogenic fermentation of glucose has the highest ATP yield of any sugar fermentation reaction yet studied, 4.3 ATP/mol glucose fermented (Müller [@bib66]). The widespread nature of homoacetogenic metabolism in Chloroflexi (Kaster *et al*. [@bib46]; Sewell, Kaster and Spormann [@bib84]), 'Atribacteria' (Nobu *et al*. [@bib69]; Katayama *et al*. [@bib47]) and Archaea (He *et al*. [@bib36]; Martin *et al*. [@bib60]; Orsi *et al*. [@bib71]) that are ubiquitous in subseafloor anoxic sediments indicates that homoacetogenesis is a common form of fermentation in these settings. Most likely, because it is a relatively energy efficient anaerobic metabolism (Schuchmann and Müller [@bib81]), it is apparently selected for under these extremely energy limited conditions (Orsi *et al*. [@bib71]). Remarkably, one of the most ubiquitous groups in anoxic subseafloor sediments, the candidate Phylum 'Atribacteria' (Orsi [@bib70]), was recently isolated in pure culture and was found to have a hydrogenogenic fermentative metabolism (Katayama *et al*. [@bib47]) confirming earlier metagenomic predictions (Nobu *et al.*[@bib69]).

SYNTROPHY AND SECONDARY FERMENTATIONS {#sec5}
=====================================

Three observations indicate that amino acid fermentations are ongoing in deep sediment. Amino acid concentrations decrease from micromolar concentrations at the sediment surface down to nanomolar concentrations in the fermentation zone in sediments with ages over one million years old (Braun *et al*. [@bib14]). Genes involved in amino acid metabolism have been found in multiple anoxic subseafloor settings to have a relatively high expression level in the fermentation zone (Orsi *et al*. [@bib1_749_1583516775552]; Bird *et al*. [@bib2_672_1583517258157]). Ammonium (D\'Hondt *et al*. [@bib24]), short chain fatty acids (D\'Hondt *et al*. [@bib23]) and the amino acid fermentation product γ-amino butyric acid (Møller *et al*. [@bib62]) accumulate in the fermentation zone of anoxic sediments. Under anoxic conditions, fatty acids are degraded by syntrophic associations of fermenting bacteria with methanogenic partners (Fig. [1](#fig1){ref-type="fig"}; Schink [@bib77]). Syntrophy is important for survival in deep anoxic subseafloor sediment (Parkes, Cragg and Wellsbury [@bib72]), because it links fermentation to methane production (Schink [@bib77]).

Most syntrophic reactions are secondary fermentations in which organisms ferment the fermentation products of other anaerobes. Amino acid fermentations yield butyrate:acetate ratios of roughly 1:3 (Schink [@bib77]). The concentration of butyrate is 100 times lower than acetate in anoxic subseafloor sediments (Glombitza *et al*. [@bib34]), indicating microbial consumption of butyrate via secondary fermentation reactions. Butyrate can be consumed via secondary fermentation, producing acetate and H~2~, and providing ∼0.3 ATP per butyrate fermented (Schink [@bib77]). $$\documentclass[12pt]{minimal}
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{\rm{Butyrat}}{{\rm{e}}^-} + {\rm{2}}\,{{\rm{H}}_{\rm{2}}}{\rm{O}} \to {\rm{2}}\,{\rm{C}}{{\rm{H}}_{\rm{3}}}{\rm{CO}}{{\rm{O}}^-} + {{\rm{H}}^ + } + {\rm{2}}\,{{\rm{H}}_{\rm{2}}}
\end{equation*}$$\end{document}$$

This reaction is energetically favorable only in the presence of a H~2~-consuming syntrophic partner, e.g. a methanogen (Schink [@bib77]). The low concentrations of butyrate relative to acetate (Glombitza *et al*. [@bib34]) thus suggest that active syntrophic consortia of butyrate fermenting cells and H~2~-oxidizing partner cells exist in the fermentation zone of deep anoxic sediments. In a similar manner, long-chain fatty acids from lipid degradation are fermented to acetate and H~2~ in the presence of H~2~-oxidizing partners. The short-chain fatty acids butyrate (C4), caproate (C6) and caprylate (C8) can furthermore be produced from exergonic 'chain elongation' reactions involving ethanol and acetate, which yields ∼1 ATP in *Clostridium kluyveri* (Angenent *et al*. [@bib5]). A recent study (Glombitza *et al*. [@bib34]) analyzing volatile fatty acids in anoxic subseafloor sediment showed that there is an order of magnitude more propionate compared to butyrate, and thus we also considered production and consumption of propionate. Propionate is produced under anoxic conditions---among others---via lactate fermentation (Lens, Dijkema and Stams [@bib52]), and the resulting propionyl-CoA is used to convert lactate to lactyl-CoA, which is dehydrated to acrylyl-CoA and reduced to propionyl-CoA (Buckel and Thauer [@bib19]) such that no ATP is generated.

THERMODYNAMICS AT LOW SUBSTRATE CONCENTRATIONS {#sec6}
==============================================

We used thermodynamics to predict the favorability of certain fermentation reactions in anoxic sediments. The standard term Δ*G*°′ is defined as the enthalpy of a reaction under standard conditions: all reactants = 1.0 M, except \[H~2~O\] = 55.6 M, pH = 7.0; \[gases\] = 100 kPa (1 atm); and *T* = 298 K. The Δ*G*°′ values are calculated from the Δ*G*°~f~ values of the formation of the reaction partners from the elements (Thauer, Jungermann and Decker [@bib92]; Buckel and Miller [@bib17]). Δ*G*′ is defined as the enthalpy of a reaction under any condition using the following equation: $$\documentclass[12pt]{minimal}
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\Delta G {\prime} = \Delta G^{\circ \prime} + {\rm{RT}}\,\,{\rm{In}}\,\Pi \left[ {{\rm{Products}}} \right] \times \Pi {\left[ {{\rm{Substrates}}} \right]^{-1}} = \Delta G^{\circ \prime} \nonumber \\
+ {\rm{5}}{\rm{.7}}\,\,{\rm{log}}\,\Pi \left[ {{\rm{Products}}} \right] \times \Pi {\left[ {{\rm{Substrates}}} \right]^{-1}}{\rm{kJ}}/{\rm{mol}}\,\,{\rm{at}}\,{\rm{298}}^{\circ} {\rm{K}}
\end{eqnarray*}$$\end{document}$$

The expected maximum ATP yield in a fermentation, either by SLP (substrate level phosphorylation) or ETP (electron transport phosphorylation) can be calculated as Δ*G*′/(−76 kJ/mol) (Thauer, Jungermann and Decker [@bib92]). For example, oxidation of glucose (C~6~H~12~O~6~) with 6 O~2~ to 6 CO~2~ + 6 H~2~O (ΔG°′ = −2870 kJ/mol) in a cell with mitochondria yields up to 38 ATP. Hence, −2870/38 = −76 kJ/mol from the fermentation is the minimum requirement for the synthesis of 1 mol ATP. For more than four decades, this value has provided a good estimate of ATP yield in most fermentations, especially since it has become evident that anaerobes conserve energy via not only substrate phosphorylation but also electron transport (ion gradient) phosphorylation. Under standard equilibrium conditions, however, hydrolysis of ATP to *P*~i~ and ADP yields ΔG°′ = −32 kJ/mol. But in a cell, at 1 mM concentrations, the free energy falls to ΔG′ = ΔG°′ + 5.7 log \[ADP\] × \[*P*~i~\] × \[ATP\]^−1^ = --32 + 5.7 × (--3) = --49 kJ/mol. The remaining --76 -- (--49) = --27 kJ/mol is due to irreversible heat-releasing processes that occur in all fermentations.

In pore waters of anoxic freshwater lake sediments, we find fatty acids and acetate at concentrations of few micromolar, and H~2~ at 10 to 1 Pa (Montag and Schink [@bib63]). These concentrations find their lower limits in the range of ∼10 μM and 1 Pa because only above these thresholds will their conversion to methane yield sufficient energy to keep the respective methanogens running (Schink [@bib77]). In marine sediments, H~2~ concentrations are lower in the nM to µM range, and correlate positively with organic matter concentrations (Lin *et al*. [@bib56]). On the other hand, concentrations of amino acids and sugars in deep sediments can vary widely from site to site and are somewhere in the nanomolar to micromolar ranges but generally decrease in concentration with depth below the seafloor (Burdige [@bib20], [@bib21]; Lomstein *et al*. [@bib59]). Thus, exact calculations of energy yields of fermenting bacteria that capture the full range of *in situ* substrate concentrations are difficult, but one can estimate ranges.

In Table [1](#tbl1){ref-type="table"}, we examined the changes in free energy, designated as Δ*G*′^1^ where the concentrations of all reactants are 1000 times lower (1 mM, gases at 100 Pa = 10^−3^ atm) and Δ*G*′\* at the more realistic concentrations (10 µM, gases at 10 Pa). We used previously studied fermentation reactions for glutamate (Buckel and Thauer [@bib18]), alanine (Buckel and Thauer [@bib19]), glucose (Buckel and Thauer [@bib18]) and lactate (Buckel and Thauer [@bib19]) in order to calculate the Δ*G*′^1^and Δ*G*′\* values, all of which result in a more favorable free energy and higher net ATP yield compared to Δ*G*°′ (Table [1](#tbl1){ref-type="table"}). The increased efficiency at lower concentrations is due to a smaller number of substrates compared to products (5 glutamates give 19 products). Any fermentation processes producing more product molecules than consuming substrate molecules will become more exergonic at lower concentrations of the reaction partners (Le Châtelier\'s principle; Thauer, Jungermann and Decker [@bib92]). Moreover, already at 1 mM glutamate, a much simpler pathway of glutamate fermentation could be possible where direct production of hydrogen from reduced ferredoxin, formed by pyruvate-ferredoxin oxidoreductase, could be coupled to ATP synthesis, resulting in ∼1 ATP/glutamate (Table [1](#tbl1){ref-type="table"}). In this much simpler pathway, electron bifurcation and energy conservation via ΔμNa^+^ (generated by Rnf, a ferredoxin:NAD reductase) would not be required. In addition, the production of ammonia helps to drive the reaction forward, because fermentations of carbohydrates such as lactate yield water instead. This additional push, however, comes to effect only at very low concentrations. The fermentations with higher H~2~ production reveal smaller efficiencies under standard conditions (Δ*G*°′) but increase at lower concentrations (Table [1](#tbl1){ref-type="table"}).

###### 

Free energies and estimated net ATP yields from fermentation reactions at different substrate and product concentrations except for hydrogen as indicated, 55 M water and pH 7.0. The equations in bold letters below the table are experimentally established reactions, whereas the others, which produce more hydrogen, most likely are used at concentrations around 1 mM and lower. The ranges of amino acid and sugar substrate concentrations cover those measured in marine sediments (Burdige [@bib20]), which can vary widely from site to site but generally decrease in concentrations with increasing depth below the seafloor (Burdige [@bib21]; Lomstein *et al*. [@bib59]). Concentrations of H~2~ in anoxic sediments also vary from nM to μM concentrations but tend to co-vary with concentrations of organic matter that are the source of H~2~ from fermentations (Lin *et al*. [@bib56]). The mechanistically calculated ATP yields are given as mol ATP/mol substrate. A \"cannot proceed\" means that the reaction cannot proceed as indicated. At lower substrate/product concentrations, the ATP yield is probably the same as that at the higher concentration. Alanine and lactate are fermented either via acrylyl-CoA or via methylmalonyl-CoA, which yield 0.33 or 0.67 ATP/alanine and lactate, respectively (Buckel and Thauer [@bib19]). The maximum ATP/substrate yield can be calculated from Δ*G*°′/substrate divided by −76.

                    1 M substrate 100 kPa H~2~   1 mM substrate 100 Pa ~H2~   10 µM substrate 10 Pa ~H2~                              
  ----------------- ---------------------------- ---------------------------- ---------------------------- ---------------- --------- ----------------
  **Glutamate 1**   −319 kJ                      0.9                          −547 kJ                      cannot proceed   −712 kJ   cannot proceed
  Glutamate 2       −43 kJ                       cannot proceed               −111 kJ                      1.0              −151 kJ   1.0
  **Alanine 1**     −162 kJ                      0.3--0.7                     −230 kJ                      cannot proceed   −299 kJ   cannot proceed
  Alanine 2         −143 kJ                      0.7                          −280 kJ                      0.3--0.7         −411 kJ   0.3--0.7
  **Lactate 1**     −168 kJ                      0.3 − 0.7                    −185 kJ                      0.3--0.7         −197 kJ   cannot proceed
  Lactate 2         −98 kJ                       cannot proceed               −184 kJ                      0.3--0.7         −223 kJ   0.3--0.7
  **Glucose**       −216 kJ                      cannot proceed               −353 kJ                      4.0              −450 kJ   4.0
  Pyruvate          −52 kJ                       cannot proceed               −86 kJ                       1.0              −103 kJ   1.0

**Glutamate 1: 5 Glutamate^−^ + 6 H~2~O + 2 H^+^ → 5 NH~4~^+^ + 5 CO~2~ + H~2~ + 6 Acetate^−^ + 2 Butyrate^−^**

Glutamate 2: Glutamate^−^ + 2 H~2~O → NH~4~^+^ + CO~2~ + H~2~ + 2 Acetate^−^

**Alanine 1: 3 Alanine + 2 H~2~O → 3 NH~4~^+^ + CO~2~ + Acetate^−^ + 2 Propionate^−^**

Alanine 2: 3 Alanine + 4 H~2~O → 3 NH~4~^+^ + 2 CO~2~ + 2 Acetate^−^ + Propionate^−^ + 2 H~2~

**Lactate 1: 3 Lactate^−^ → CO~2~ + Acetate^−^ + 2 Propionate^−^ + H~2~O**

Lactate 2: 3 Lactate^−^ + H~2~O → 2 CO~2~ + 2 Acetate^−^ + Propionate^−^ + 2 H~2~

**Glucose: Glucose + 2 H~2~O → 2 CO~2~ + 2 Acetate^−^ + 2 H^+^ + 4 H~2~**

Pyruvate: Pyruvate^−^ + H~2~O → CO~2~ + Acetate^−^ + H~2~

LIVING FROM FERMENTATION LEFTOVERS {#sec7}
==================================

For the constituents of dead cells to generate a live cell, if we look just at the main components, it is advisable to recycle amino acid residues after partial degradation, i.e. deamination and decarboxylation, as this was shown for anaerobic microbial communities in rumen contents (Allison and Bryant [@bib2]). Complete degradation of amino acids yields ∼2 ATP per amino acid at best, but to make a new cell that contains the same amount of protein we need to expend 4 ATP per peptide bond at translation, plus occasional error correction or ∼5 ATP per amino acid polymerized. Five amino acids need to be consumed to make one new peptide bond (an additional, 6th amino acid), such that even if we import amino acids for \<0.25 ATP per amino acid, from the amino acid standpoint alone, one generation of cells gives rise to roughly 1/6 (maximally 1/3) the number of progeny on a per-weight basis, no maintenance energy, protein unfolding or uncoupling considered. In terms of what is possible converting one cell to (what fraction of) another, the curve falls quickly, much more steeply than 1/6, 1/36, 1/216 in deep anoxic sediment settings where the resources are finite and consumed. This steep downward trend toward chemical equilibrium (what life tries to avoid) is exactly what Finkel ([@bib32]) found for starving cells, albeit in the presence of oxygen. We arrive at the same general conclusion from stoichiometric conversion rates of the main substance of cells (amino acids) for making protein out of protein in heterotrophic survival, that is, without coupling it to ATP formation in methanogens.

The carbon conversion ratio for RNA is similarly low, because ribonucleoside monophosphates need to be activated (2 ATP) for polymerization, in addition to processing and post-transcriptional modifications that need to be performed, and every monomer incorporated is one that the cell cannot use for energy conservation. Starved cells are much smaller than exponentially growing ones, indicating simply that staying alive as opposed to growing is a successful strategy in low-energy environments (Lever *et al*. [@bib54]).

What about the H~2~, CO~2~ and acetate that arise from fermentation? How much methanogenesis can they support? Each amino acid in the dead cell has on average five carbon atoms and generates one CO~2~ in fermentation. Methanogens, unless they supply themselves with partly degraded amino acids from fermenting bacteria (Allison and Bryant [@bib2]), generate their amino acids from acetate and CO~2~. The growth yield of 1.6 g of cells (dry weight) per mol of CH~4~ produced as reported for *Methanobacterium thermoautotrophicum*, a methanogen that lacks cytochromes, grown on H~2~ and CO~2~ (Schönheit, Moll and Thauer [@bib79]) delivers an estimate of one molecule of carbon being fixed for every 18.5 molecules of CH~4~ produced. Rounding that corresponds to ∼20 molecules of CO~2~ that go through the cell to be reduced to methane for every CO~2~ that is incorporated into cell mass. For acetogens (Daniel *et al*. [@bib26]) the reported ratio is 24:1. For a methanogen growing on H~2~ and CO~2~, ∼21 fermented amino acids (20 CO~2~) are required to supply one CO~2~ for methanogen cell mass; ∼100 fermented amino acids are required to make one average methanogen amino acid with 5 carbons (Fig. [2](#fig2){ref-type="fig"}). Nitrogen is not limiting, as NH~4~^+^ is abundant in deep anoxic sediment (D\'Hondt *et al*. [@bib24]). Those 100 fermented amino acids deliver 100 H~2~, which is the amount required to make only 25 methane (Fig. [2](#fig2){ref-type="fig"}) according to the methanogenic reaction $$\documentclass[12pt]{minimal}
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{\rm{4}}\,{{\rm{H}}_{\rm{2}}} + {\rm{C}}{{\rm{O}}_{\rm{2}}} \to {\rm{C}}{{\rm{H}}_{\rm{4}}} + {\rm{2}}\,{{\rm{H}}_{\rm{2}}}{\rm{O}}{\rm{.}}
\end{equation*}$$\end{document}$$

![Fermentation of 105 bacterial amino acids (C5) to synthesize one peptide unit (C5) in a methanogenic archaeon. Red lines: fermentations, blue lines: methanogenic activities.](fuaa004fig2){#fig2}

Starting from H~2~ and CO~2~, in order to make 100 methane to support the synthesis of one average methanogen amino acid, a fermenting community has to disproportionate 200 amino acids (without H~2~ loss to the environment). Add to that the ATP that is required to make a peptide bond. We can estimate a methanogen requirement of 5 ATP per peptide bond, four at translation plus one for nutrient import, misincorporation, misfolding and the like (Fig. [2](#fig2){ref-type="fig"}). ATP is provided by methanogenesis at 0.5 ATP per CH~4~ (Thauer *et al*. [@bib93]); 5 ATP require 10 CH~4~ which in turn requires 40 H~2~ or 20 amino acids if each amino acid supplies two H~2~, partly through secondary fermentations.

Thus, for a methanogen growing on H~2~ and CO~2~ and synthesizing all amino acids from CO~2~, fermenters would need to disproportionate 220 amino acids. For an idealized methanogen growing on H~2~ and CO~2~ and synthesizing no amino acids at all, importing and assimilating all amino acids from the environment while obtaining ATP from methanogenesis, fermenters would need to disproportionate 20 amino acids for each methanogen peptide bond (Fig. [2](#fig2){ref-type="fig"}), but they would also need to leave a substantial proportion of their growth substrate (amino acids) untapped to enable methanogen amino acid assimilation. These values (∼200:1 and ∼20:1) probably represent upper and lower bounds on carbon conversion rates for conversion of fermented cell mass into methanogen protein, the main component of cell mass, in sediment where no other nutrients such as sulfate or nitrate enter the system. Just looking at protein, which is likely to be the limiting factor for survival, the conversion of C from fermented dead cell protein into C of methanogen protein via H~2~ and CO~2~ occurs at a ratio of ∼200:1 in terms of net *de novo* protein synthesis ('growth', which is not the norm for energy-limited environments) if growth is supported by diffusing gasses (H~2~ and CO~2~) or ∼20:1 if methanogens are efficiently competing with fermenters for amino acids.

If methanogenesis is being fueled by acetate instead of H~2~ and CO~2~, and if we assume that the fermenters are generating two H~2~, CO~2~ and acetate per degraded amino acid, the picture is less bleak by about a factor of 2. That is because methanogenesis from acetate (aceticlastic methanogenesis) converts acetate to methane $$\documentclass[12pt]{minimal}
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{\rm{C}}{{\rm{H}}_{\rm{3}}}{\rm{CO}}{{\rm{O}}^--} + {{\rm{H}}^ + } \to {\rm{C}}{{\rm{H}}_{\rm{4}}} + {\rm{C}}{{\rm{O}}_{\rm{2}}},
\end{equation*}$$\end{document}$$yielding ∼0.3 ATP per acetate consumed (Schlegel *et al*. [@bib78]; Welte and Deppenmeier [@bib98]). The growth yields (grams of cells per methane) for methanogens growing on acetate are in the range of 1.5--3 g per mol methane produced (Welte and Deppenmeier [@bib98]), similar to those for growth on H~2~ and CO~2~ (Thauer *et al*. [@bib93]). Evidence exists that in deep subseafloor fermentation zones, some acetate carbon may be mineralized first to CO~2~, prior to being reduced to methane with H~2~ (Beulig *et al*. [@bib10]).

*Methanosarcina* species have a lower affinity for acetate in the environment because they use a different system of acetate activation involving acetate kinase, which has a ∼100-fold higher K~m~ for acetate than the acetyl-CoA synthase (AMP-forming) used by *Methanosaeta* species (Welte and Deppenmeier [@bib98]). The consequence is that *Methanosarcina* species require ∼1 mM acetate for growth, whereas *Methanosaeta* species can survive with 7--70 µM acetate (Welte and Deppenmeier [@bib98]). That might be relevant in deep sediment, where acetate levels have been measured at the low micromolar range (D\'Hondt *et al*. [@bib23]). However, acetate activation in *Methanosaeta* species is energetically more costly, so that the growth yield is lower, on the order of 1.5 g per mol of acetate consumed. The similar growth yields per methane from acetate vs H~2~ and CO~2~ mean that also for growth on acetate, about one CO~2~ is fixed as cell mass per 20 methane produced. Instead of two fermented amino acids to make 4 H~2~, one acetate generates one methane, so that ∼100 amino acids (rather than 200) would need to be fermented to make one new methanogen amino acid. We generously assume growth yields similar to those for exponentially growing cells in lab cultures and no product losses, such that these carbon conversion rates represent best-case scenarios.

Our calculations indicate that cell division is probably not the primary mode of substrate turnover. Replacing old proteins with new ones within the same cell (reusing one\'s own amino acids) is a far more likely fate for amino acids within the methanogens, because the average sized 5-carbon amino acids are already within the cell. Such processes of replacing old or damaged proteins might constitute a good portion of maintenance, a still poorly understood component of long-term survival (Hoehler and Jørgensen [@bib40]; Lever *et al*. [@bib54]), but one that is vital when there is insufficient energy for growth.

IMPLICATIONS FOR ANAEROBIC METHANE OXIDATION {#sec8}
============================================

The methane produced by methanogens disappears in the SMTZ (Egger, Riedinger and Mogollón [@bib28]), where the energy yield from anaerobic oxidation of methane (AOM) is much lower than that from methanogenesis (Valentine [@bib94]). The reaction is thus thought to be energetically coupled to a syntrophic partner, such as a sulfate reducer, in cases studied so far (Holler *et al*. [@bib41]). But if the conversion of fermented cell mass to methanogen cell mass occurs at a ratio of 100:1 (20:1 if unfermented amino acids are assimilated with H~2~-dependent ATP synthesis), the subsequent anaerobic conversion of methane to cell mass via AOM can hardly be better. We can briefly consider the possibility of biomass accumulation via anaerobic methane oxidation. Unfortunately, at the time of writing, balanced stoichiometric reactions including a carbon balance for cells that employ anaerobic methane oxidation are not available. However, in one example where anaerobic methane oxidation was studied in laboratory cultures, the methane oxidizing cells were acting as autotrophs, incorporating labeled CO~2~, as opposed to incorporating labeled methane (Milucka *et al*. [@bib61]) or amino acids. The energy yield for AOM is lower than that for methanogenesis (Holler *et al*. [@bib41]). That means that the biomass conversion ratio for AOM cannot be higher than for methanogenesis, placing a very generous upper bound on the carbon conversion ratio at one CO~2~ fixed as cell mass per 20 methane oxidized (the methanogen ratio). The ∼10--100-fold longer doubling times for AOM, combined with the circumstance that the ATP costs for amino acid biosynthesis will be the same for autotrophic growth during AOM (Milucka *et al*. [@bib61]) as for methanogenesis, suggest that the overall carbon conversion ratio from methanogenesis to AOM will be on the order of 100 methane oxidized per carbon fixed as anaerobic methane oxidizing archaeon (ANME) cell mass. This corresponds to a cell mass conversion ratio from fermentation to ANME via methanogenesis on the order of 10 000:1 (Fig. [3](#fig3){ref-type="fig"}). This probably explains why the activity and abundance of ANME populations is so low in subseafloor settings, compared to methane seeps at the surface (Orsi [@bib70]).

![Estimated carbon conversion factors for metabolic feeding chains in subseafloor anoxic sediments. Cartoon schematic showing the decreasing carbon conversion from the base of the microbial food chain (fermentation) to methanogenesis, and AOM. This explains the global exponential decline in biomass with increasing depth in anoxic sediment (Fig. [3](#fig3){ref-type="fig"}). Photographs of methanogens and the single AOM (ANME) cell are modified from Karl Stetter, and Lloyd, Alperin and Teske ([@bib57]), respectively. The image of the fermenters is the author\'s (W. Orsi) own work.](fuaa004fig3){#fig3}

Preserved consortia of anaerobic methane oxidizing ANME cells with sulfate-reducing bacteria in close physical contact (that is, cells that were evidently growing together) are typically enriched in methane seeps, like those on the continental shelf break and in the Black Sea (Knittel *et al*. [@bib48]). However, in deep anoxic sediment where the SMTZ can lie down to 30 m below the seafloor (D\'Hondt *et al*. [@bib23]), our calculations suggest that anaerobic oxidation of methane does not represent a successful strategy for growth, because it has a carbon conversion factor from fermentation at roughly 1:10 000 (Fig. [3](#fig3){ref-type="fig"}). This fits with the observations from sampling of such deep biosphere environments, as, to our knowledge, no photographic evidence of preserved ANME-sulfate reducing bacteria consortia similar to those observed at methane seeps have been reported from deep anoxic sediments, specifically in the deep SMTZs. Rather, the ANME cells observed are often found in isolation (Lloyd, Alperin and Teske [@bib57]). In deep anoxic sediment, it is thus possible that the ANME are living as methanogens, rather than as anaerobic methane oxidizers (Lloyd, Alperin and Teske [@bib57]; Beulig *et al*. [@bib11]).

Recent studies point to the possibility of iron reduction coupled to the anaerobic oxidation of methane in anoxic sediments. At cold methane seeps, ANME cells can oxidize methane with Fe oxides at the sediment surface (Scheller *et al*. [@bib76]). Vigderovich *et al*. ([@bib96]) reported iron reduction in methanogenic sediment down to a depth of 5 m in the Mediterranean. Bar-Or *et al*. ([@bib7]) reported slurry cultures that reduce Fe oxides and oxidize methane, but the partners and pathways involved have not been characterized. Egger *et al*. ([@bib29]) suggested that iron oxide reduction might affect sediments subject to changes in organic matter loading, but very deep sediment that we are discussing here is isolated from changes in organic matter loading. Riedinger *et al*. ([@bib75]) suggested that methane oxidation coupled to Fe oxides and Mn(IV) reduction at depths down to 8 m are important in sediments characterized by rapid deposition and that are rich in manganese and iron oxides in the methanogenic zone. Slomp *et al*. ([@bib85]) also suggested a role for methane oxidation coupled to Fe oxides and Mn(IV) reduction in surface sediments down to a depth of 40 cm. The biological process of iron oxide-dependent methane oxidation is thought to require 8 atoms of Fe oxides for each methane oxidized (Riedinger *et al*. [@bib75]). Although it cannot be excluded that Fe oxides and/or Mn(IV)-dependent methane oxidation occurs in deep sediments, we note that the activity and abundance of ANME populations is very low in subseafloor settings, as opposed to shallow sediment or methane seeps at the surface where ANME tends to be more abundant with higher activity (Orsi [@bib70]). Accordingly, it is unlikely that ANME-mediated Fe oxides and Mn(IV)-dependent methane oxidation play a substantial role in deep sediment carbon conversion.

EVIDENCE FOR A LOW-TEMPERATURE LIFE--DEATH TRANSITION ZONE (LDTZ) {#sec9}
=================================================================

Our analysis suggests that the steadily decreasing ATP budgets and subsequent carbon transfer efficiencies for protein biosynthesis that result primarily from amino acid fermentations should eventually represent an insurmountable barrier for life in ultradeep subseafloor anoxic sediment at low temperatures (\<120°C). This potential LDTZ would represent a transition from the last biologically catalyzed life processes to catalyzed processes that are no longer coupled to life. While a low-temperature (\<120°C) LDTZ has never been observed in anoxic sediment, the deepest report of subseafloor life is 2.5 km below the seafloor where the detection limit for life was already close to being reached at roughly one cell per cubic centimeter of sediment or below detection (Inagaki *et al*. [@bib44]; Fig. [4](#fig4){ref-type="fig"}). The temperatures at the deepest depths were between 40 and 60°C (Heuer *et al*. [@bib39]), and thus the apparent lack of subseafloor life at 2500 m below seafloor (mbsf) cannot be explained by high temperature. Thus, Inagaki *et al*. ([@bib44]) apparently sampled the beginnings of a low-temperature LDTZ that our physiological calculations predict.

![Global distribution of microbial biomass in subseafloor sediments. Data are replotted from Kallmeyer *et al*., with cell counts from the more recent Shimokita (Inagaki *et al*. [@bib44]) and the Baltic Sea (Andren *et al*. [@bib4]) deep drilling expeditions. Note that the global regression line changes at ∼500 mbsf for continental margins, and at ∼75 mbsf for equatorial Pacific sites, whereby the rate of cell death increases after dropping below 10^5^ cells cm^−3^. Mbsf: meters below seafloor.](fuaa004fig4){#fig4}

Below subseafloor depths of 2 km, life was below detection except for intervals that had elevated organic matter contents from ancient coal (Inagaki *et al*. [@bib44]). Thus, organic matter availability is indeed a most critical factor that constrains the depth of a potential low-temperature LDTZ. This fits with our physiologically based estimate that posits fermentation of organic substrates ultimately to control the size of the deep anoxic subseafloor biosphere. Geochemical data from samples taken in the deepest deep biosphere suggested that hydrogenotrophic CO~2~-reducing methanogenesis was the predominant pathway for methane formation, as opposed to acetoclastic methanogenesis (Inagaki *et al*. [@bib44]). This is also consistent with our calculations, which indicate that the biomass conversion from hydrogenotrophic methanogenesis is two times lower than that for acetoclastic methanogenesis.

Looking at the compiled data (Fig. [3](#fig3){ref-type="fig"}), an inflection point is reached when the cell counts drop below a critical cell concentration of 10^5^--10^6^ cells cm^−3^ (Fig. [4](#fig4){ref-type="fig"}) indicating that the distance between cells results in an increased rate of cell death. Indeed, because the time required for chemical diffusion is proportional to the square of diffusion distance, the metabolization rate must decline exponentially with increasing distance between sources and sinks of organic matter (D\'Hondt *et al*. [@bib25]): dead and living cells, respectively. This increase in cell death rate below a cell concentration of 10^5^ cells cm^−3^ can be partly explained by the physiological limits to life that we calculate here. Since the primary fermentable substrates are necromass (amino acids) from other microbes, once the cell density of microbes reaches a critical threshold of \<10^5^ cells cm^−3^ the rate of cell death increases (Fig. [4](#fig4){ref-type="fig"}), presumably due to less organic matter from recently deceased cells becoming available to living fermenters.

With the exception of hydrothermal sediments, the geothermal gradient within the upper 100 km of the Earth is usually ∼30°C/km (Earle [@bib27]). However, the slope of the geothermal gradient is controlled by the heat flux from the underlying crust, the temperature at the sediment--water interface and the thermal conductivity of the sediments (Stranne and O\'Regan [@bib89]). Thus, the geothermal gradient can in some instances be as low as 15°C/km (Earle [@bib27]) such as in the Gulf of Mexico where sediments are 10 km thick (Straume *et al*. [@bib90]) and exhibit a wide range of thermal gradient strength from 15 to 50°C/km (Christie and Nagihara [@bib22]). If the sediments with a 15°C/km geothermal gradient also exhibit an LDTZ below ∼2500 mbsf as indicated in Japanese shelf sediments (Inagaki *et al*. [@bib44]), the 'low-temperature' (40--120°C) abiotic zone could extend from 2.5 km to the 120°C isotherm several kilometers deeper below. This abiotic zone would not be due to high temperature, but rather to the depletion of fermentable organic matter to concentrations that no longer meet the minimal energy requirement of a cell.

The total volume of subseafloor sediment habitats cooler than 120°C is immense and estimated to be 2.6 × 10^8^ km^3^ (LaRowe *et al*. [@bib50]; D\'Hondt *et al*. [@bib25]). However, if 80°C is taken as the major thermal barrier for subseafloor life, then this habitable area would be restricted (LaRowe *et al*. [@bib50]). The high-temperature limit to oil and gas biodegradation (Head, Jones and Larter [@bib37]) is 80--90°C, which is considerably lower than the high-temperature limit to life determined in experiments using methanogenic archaea grown under energy- and nutrient-rich conditions (Takai *et al*. [@bib91]) at 122°C. Thus, the high-temperature limit to life may vary from one environment to another as a function of bioavailable energy flux or nutrients. The high-temperature limit to life in the subseafloor is currently unknown but is an area of active investigation through the recent IODP Expedition 370 'Temperature Limit of the Deep Biosphere off Muroto' (Heuer *et al*. [@bib39]). The results of that expedition will surely have important implications for the effect of high, and potentially low-temperature LDTZs.

POTENTIAL CHEMICAL REACTIONS BELOW AN LDTZ {#sec10}
==========================================

Where life tapers out, the only possible chemical reactions that remain are abiotic. The most likely chemical reactions no longer coupled to life below the LDTZ are those that are exergonic with a non-zero rate (Fig. [5](#fig5){ref-type="fig"}). These include peptide hydrolysis and nucleic acid hydrolysis whereby RNA would be more affected than DNA. Additional purely chemical reactions would be amino acid racemization and sugar decay. Bacterial lipids are mainly esters of long chain fatty acids, the ester bonds of which are readily hydrolyzed in marine sediments (Xie *et al*. [@bib100]). Archaeal lipids on the other hand are ethers that are much better preserved in anoxic marine sediments (Xie *et al*. [@bib100]), and should be more stable below the LDTZ. Radiolysis of water results in the abiotic formation of H~2~ in deep marine sediments that could be a potential energy source (Dzaugis *et al*. [@bib3_367_1583518460582]). Cellular activity might result from H~2~ derived purely from radiolysis of water. Genes encoding hydrogenase proteins involved in the potential oxidation of H~2~ have been found in deep subseafloor clays supporting a possible utilization of H~2~ derived from radiolysis by subseafloor life (Vuillemin *et al*. [@bib4_120_1583518837626]). Abiotic radiolysis of water might also play a role in controlling the depth of the LDTZ by supplying H~2~ as an energy source to H~2~-utilizing microbes (methanogens) above the LDTZ.

![Potential abiotic chemical reactions taking place below the LDTZ.](fuaa004fig5){#fig5}

CONCLUSION {#sec11}
==========

Fermentation in anoxic sediment is not primary production; it is terminal disproportionation. The aim of this work is not to calculate exact biomass conversion rates, but to get an order of magnitude estimate on their trends based on the estimated availability of relevant substrates in the methane zone of anoxic subseafloor sediment. Our analysis indicates that fermentation in deep anoxic sediment is a kind of swan song---a farewell to the active biosphere and a transition into the death and long-term stationary phases of microbial life. From the standpoint of metabolism, life likely started on Earth as H~2~-CO~2~ dependent reactions giving rise to autotrophic acetogens and methanogens as the first free living cells (Weiss *et al*. [@bib97]; Varma *et al*. [@bib95]; Muchowska, Varma and Moran [@bib64]). Fermentations of amino acids and bases were probably the first form of heterotrophic metabolism on Earth because cell substance is an excellent growth substrate and because carbon compounds from space are mostly unfermentable and structurally too heterogeneous to support a metabolism (Schönheit *et al*. [@bib80]). Looking forward in time, fermentations in anoxic sediments might end up being the last form of metabolism on Earth. By injecting carbon source substrates and terminal acceptors into sediment and monitoring growth or gene expression, it should be possible to identify factors limiting life near the LDTZ.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) projects OR 417/1-1 and Emergence of Life initiative TRR235 (Project-ID 364653263), both granted to WDO.

***Conflicts of Interests.*** None declared.
