Fermentation

Fermentation, while also degradative, never results in mineralization except for the conversion of
acetate to methane and carbon diaxide by certain methanogens. This is because fermentation does
not involve externally supplied electron acceptors but instead uses organic acceptors generated
intracellularly from the compound being degraded. In a chemical sense, fermentation is a
disproportionation or dismutation. One or more products in fermentations are always partially
reduced organic compounds and are balanced by partially oxidized compounds. Although the
homolactic fermentation of glucose, in which only a single product, lactate, is formed, may not
appear to fit this concept, it does. The lactate is really the result of enzymatic disproportionation of
the intermediate triose phosphate into pyruvate and reduced pyridine nucleotide. The lactate
results from the enzymatic reduction of pyruvate by the reduced pyridine nucleotide. Even though
fermation dose not lead to complete degradation of organic compounds, it may have a very
important role in anaerobic mineralization. In nature many complex organics are mineralized by
consortia in which fermenters convert the complex organics into simpler organic compounds that
other microorganisms subsequently mineralize (see also discussion in Section VII below). Many
anaerobic mineralizers are very restricted in the substrates that they can attack and thus depend on
fermenters to produce these substrates from more complex compounds (25-271). Some anaerobic
bacteria can, however, mineralize complex organic substrates completely. For example,
Desulfobacterium anilini has been shown to mineralize aniline [28].
In aerobic mineralization, organic carbon is always transformed into carbon dioxide, whereas in
anaerobic mineralization, organic carbon is transformed into carbon dioxide or methane, depending
on which external electron acceptors are available. When CH4 is the product, it is frequently
oxidined to co2, and H2O by aerobic methanotrophs if it diffunes from its site of formation into an
aerobic
zone in soil or sediment [29]. However, strong indirect evidence has been obtained for anaerobic
methane oxidation in marine sediments by microbes yet to be isolated and characterized (30-341.
Certain sulfate reducers may be involved in this anaerobic methane oxidation (34-361) although they
may not oxidize the CH4, directly (37).
In aerobic mineralization, organie nitrogen is more likely to accumulate as NO3-, and organic sulfur
as SO42-. whereas in anaerobic mineralization, organic nitrogen accumulates as NH4+ and organic
sulfur as HS- .The higher oxidation state of the mineralized organic nitrogen and sulfur accumulating
under aerobic conditions is the result of cooperation between one or more mineralizers and
ammonia-nitrifying and sulfur-oxidizing bacteria, respectively. The latter two kinds of bacteria are
strict aerobes, except for some sulfur oxidizers.
Aerobically, microbial mineralization of organic matter is dependent on the Krebs tricarboxylic acid
cycle, whereby the acetate to which any organic matter is first converted is oxidized to CO2, and
H2O.
me Ce OX CU ae on er SP ex di CH,COO+H 2H,0 2CO. 20
The aerobic pathways to acetate vary and depend on the nature of the organic substance being
mineralized. Examples of aerobic metabolic pathways leading to acetate from specific organic
compounds are listed in Table 1.
Anaerobically, microbial mineralization of organic matter depends on either the CO/acety1-CoA
pathway (38-42) or the Krebs tricarboxylic acid cycle [43-46] after its initial conversion to acetate.
Which pathway is used depends on the organism involved. co/acetyl-CoA pathway, as proposed by
Spormann and Thauer [41], is summarized in Figure 1. Anaerobic metabolic pathways leading to
acetate from specific organic compounds are listed in Table 1 (see also Paul et al. [47] for pathways
involved in denitrification).
BIOENERGETICS OF MINERALIZATION
Microbial mineralization is a form of catabolism. It realeases free chemical energy, some of which is
conserved by the organism through chemiosmosis in oxidative phosphorylation for use in anabolic
processes. Using the mineralization of acetate as an example, free energy yields per mole of acetate
consumed under standard conditions at pH 7 can be calculated from appropriate values of free
energy of formation. Theses values show that the corresponding resctions are thermodynamically
favorable under conditions of
aerobic respiration and several forms of anaerobic respiration. The values in Table 2 show that
anaerobic mineralization of 1 mole of acetate at pH 7.0 by ferric ion [dissolved iron (III)] respiration
and denitrification yields almost as much free energy as aerobic respiration and about 62% as much
by MnO2, respiration. On the other hand, acetate mineralization by sulfate respiration yields only
5.6% and by iron respiration with crystalline Fe(OH)3, only 2.7% as much free energy as does aerobic
respiration. Yet sulfate reduction has been found to be a very effective means of anaerobic
mineralization of organic matter in sulfate-rich, anaerobic environments such as salt marshes or
estuarine sediments. Furthermore, iron respiration on some iron oxides has been found to be very
effective in some anaerobic sedimentary environments low in sulfate content (see Section VI below).
Thus, it is more than just the free energy yield that determines the dominant mineralization process
in a given environment
IV. ENVIRONMENTAL COMPATIBILITY OF DIFFERENT ANAEROBIC MINERALIZATION PROCESSES
An empirical correlation has frequently been noted between stand ard oxidation potentials of
specific reducive half-reactions repre- senting known respiratory processes at neutral pH (Table 3)
and prevailing environmental redox potentials (eg. Refs. 48. 49). Electron activities at standard
conditions [pE(W)] are sometimes used in such correlations instead of oxidation or reduction
potentials (e.g., Refs. 50-52). pE(W) is defined as -log (e 2.3RTF , where EH is the equilibrium redox
potential of the reaction under consideration, referred to the hydrogen scale, R is EH
the gas constant, T is the absolute temperature at which the reaction is run, and F is the Faraday
151]. Thus in a sediment profile, aerobic respirers will be found in the oxidizing zone (upper- most
portion of the profile), whereas nitrate respirers may be found in the uppermost part of the reducing
zone, sulfate res pirers below them, and methanogens at the bottom of the biologically active part
of the sediment profile (e.g., Refs. 52, 53) The environmental redox potentiais of these Zones may
range as follows: for denitrifiers, from +0.665 to -0.205 V; for sulfate re- ducers, from +0.115 to .450
V; and for methanogens, from +0.100 to -0.450 [53,54] Optimal redox conditions are assumed to
exist near the middle of each range. The implication of such correlations is that only respiratory
reactions whose oxidation potentials match the redox potential of a given environmental niche
closely can operate in it. Stated differently, environmental redox potentials determine which
respiratory activities and, therefore, Which organisms capable of them shall operate in the
environment. However, such an inference is incorrect. Environmental redox
potential is a symptom and not the cause of a given respiratory activity that occurs in a particular
redox domain. Environmental redox potentials are only a measure of the dominant chemical
oxidizing and reducing species present that can undergo reversible redox reactions. In biologically
active environments, their redox state is affected by the biological activity.
Although the usual location of the sulfate reducing zone is under the zone of nitrate reduction or
denitrification [55,56] and over the zone of methanogenesis [57-62] in marine sediment profiles,
exceptions occur. Similarly, althogh the usual location of the zone of nitrate reduction/
denitrification is below the zone of aerobic respiration in sediment and soil profiles [63,64],
exception also occour. For example, in some sedimentary environments, extensive methancgenesis
has been observed in the same niche in which sulfate reduction occurs [61,65,66]. Evidence has also
been found that nitrate reduction can occur in the same niche in which aerobic respiration occurs
[2,4.61,65,67-70]. Bacterial MnO2 respiration has been noted in the presence of air [3,6,71-73]. It
must be concluded that it is not environmental redox potential that is the determinant of whether
one or more types of respiratory activity shall occur in a given environment but other chemical.
physical, and biological factors.
As a generalization, respiratory activities with significantly diferent standard oxidation potentials can
occur in the same redox niche when the two respiratory activities do not compete for the same
electron donor (e.g.. Ref. 65). Furthermore, respiratory activities with different standard oxidation
potentials can occur in the same redox niche if the electron donor common to both respiratory
processes, each in a different type of organism, is present in sufficient excess to eliminate
competition for it. This can be inferred from the work of Kristjansson et al. 174. Lupton and Zeikus
[75], and Schoenheit et al. [76] it was experimentally demonstrated by Lovley et al. [77] in a case of
concurrent meth anogenesis and sulfate reduction with hydrogen in a eutrophic lake sediment and
by Lovley and Phillips (78] in experiments with river sedimen. In both studies, the sediment was
supplied with excess hydrogen as electron donor for concurrent methanogenesis and Fe(III) and
sulfate reduction. In the chase of Thiosphera pantotropha, which can perform nitrate respiration in
air, a process that in Pseudomonas stutzeri is repressed by oxygen in excess of 5 mg L 179], the
compatibility with air seems to reside in the fact that nitrate reductase of this organism is not
repressed or denatured by oxygen (4,5]. Oxygen is a repressor in most other nitrate reducers [80].
Recently, aerobic, i.e., oxygen-compatible, dissimilatory sul fate reduction has been detected during
oxygenic photosynthesis in a hypersaline microbial mat dominated by the cyanobacterium
Microcoleus chthonoplastes [81]. Based on oxygen microsensor meansurements in the mat,
anaerobic microniches in the mat for this reduction were considered to be very unlikely sites for this
activity by the investigators. The organisms responsible for the sulfate reduction has not yet been
isolated and characterized. These observations are reminiseent of reports by Shturm [82] in 1948
and by Bromfield [83] in 1953. Shturm noted aerobic production of sulfide from sulfate by
Pseudomonas zelinskii and Bromfield by Bacillus megaterium. It is not known whether the sulfide in
these instances was produced by assimilatory sulfate reduction, which occurs in aerobes and
anaerobes, or by dissimilatory sulfate reduction, which is thought to occur only anaerobically. If the
sulfide was produced by dissimilatory sulfate reduction, an enzyme system with unusual tolerance
for oxygen or lack of competition with aerobic respirers for electron donors, or both, could be the
reason why the process can occur aerobically.
Spatial separation of different redox processes in a sediment or soil column may result from
spontaneous oxidation of a particular electron donor at a more oxidized potential or from
spontaneous reduction of an electron acceptor at a more reduced potential.
Strict anacerobes do not function in aerobic environments because oxygen may be toxic to them or
may repress their respiratory metabolism.in the presence of an excess of an elektron donor common
to several organisms in a redox profile, spatial separation of different respiratory process may be
related to energy yield from the different processes. with the process having the higher yield
outcompeting a process with a lower yield. On this basis, a sequence of anaerobic respiratory
reactions on acetate in a reducing sediment profile can be predicted from the free energy changes
listed in Table 2, assuming an abundance of acetate and appropriate electron acceptors and absence
of chemical side reactions, such as the reduction of nitrate to nitrite 1841, NO2-to N2O, [85] or
MnO2, to Mn2+ by Fe2+ (86,87]. Another interfering side reaction can be the reduction of Mno2, by
H2S ( 88] such as sequence of respiratory reactions, in which the electron acceptors are consumed in
descending order of the energy yield from the oxidative eactions into which they enter, has been
observed in a natural environment containing a mixed population of anaerobic respires, an excess of
organic matter, and a mixture of different terminal electron acceptors [89]
Thus, the order in which different respiratory activities occur in a soil or sediment profile may
depend on (1) the types of organisms present: (2) available electron donors and the affinity of the
respiratory system of different organisms for them; (3) the repressibility of an anaerobic respiratory
pathway by oxygen: (4) the stability of an anaerobic respiratory reductase in air: (5) the relative
abundance of different environmentally available electron
acceptors: (6) an extracellular, abiotic reaction between the product of respiration of one organism
and the electron acceptor of another, e.g.. the reaction between Fe3+ formed in iron(III) respiration
and MnO2:, the electron acceptor in Mn (IV) respiration [86) and (7) other environmental
determinants such as the toxicity of a terminal electron acceptor or respiratory product to some
organisms.
The standard oxidation potentials at pH 7.0 for various respiratory half-reactions are listed in Table
3. Special notice should be taken of the great difference in oxidation potentials of the Fe3+/Fe2+,
the Fe(OH)3,/Fe2+, and the Fe2 O3/Fe2+ couples. The oxidation potential of crystaline feeric
hydroxide is very close to that of sulfate reduction, whereas the oxidation potential of fe3+ is very
close to that of oxidant. Inspection of tabel 2 show that when acetate oxidation is coupled to these
reactions, three to four times as much energy is released when ferric ion is the oxidant than when
sulfate or crystalline ferric hydroxide (or some other solid ferric oxide) is the oxidant. On the basis of
the oxidation potentials of the reactions in Table 3. an anerobic sediment profile may exhibit iron
reduction in a zone overlying a methanogenic zone if soluble ferric iron is an acceptor, but it may
coincide with or underlie a methanogenic zone if a crystalline iron oxide is the acceptor. as observed
in nature (90) Measured environmental potentials would be only a reflection of these respective
activities and not their determinant (see discussion above). Although the oxidation potentials in
Table 3 also predict that iron respiration of a crystalline oxide and sulfate reduction could occur in
the same niche, this would not occur as the H2S from sulfate respiration would tend to reduce
chemically the ferrie iron in the oxide and thus interfere with its bacterial reduction. hydrogen
sulfide can also have this effect on the reduction of MnO2. [88].
V. A BRIEF SURVEY OF BACTERIA RESPONSIBLE FOR ANAEROBIC MINERALIZATION OF ORGANIC
MATTER
The bacteria that contribute to anaerobic mineralization are a very diverse group. Those currently
recognized are mostly gram-positive and gram-negative eubacteria, but some archaebacteris are
also able to mineralize organic matter anaerobically. Table 4 lists some examples of bacteria capable
of anaerobic mineralization of organic matter.
As noted in Section 11, the capacity to respire anaerobically should not be equated with a capacity
to mineralize organic matter anaerobically, as a number of bacteria respire anaerobically but
are unable to oxidize the organic carbon completely to CO2, classic example of such an organism is
Desulfovibrio desulfuricans which oxidizes Inctate to acetate and CO2, with SO42-,as the termial
electron acceptor (91). Another example is Shewanella (formerly Alteromonas putrefaciens), which
converts lactate and pyruvate to acetate and CO2, with ferric iron as the terminsnl electron acceptor
(92) both organism appear to lack a mechanims for oxidizing acetate to CO2, such as an
anaerobically operating tricarboxylic scid (TCA) cycle or an acetyl-CoA/CO dehydrogenase pathway.
By contrast, strain GS-15 oxidizes acetate by the TCA cycle with Fe(III) or Mn (IV) as the terminal
electron acceptor (93). and De sulfotomaculum acetoxidans oxidizes acetate by the acetyl-CoA pathway with SO42- AS the terminal electron acceptor 138]. Table 5 lists some representative types of
organisms that are able to respire anaerobically but that cannot oxidize the organic matter com
pletely.
Some extremely thermophilie archaebacteria seem to respire organic matter anaerobically by using
elemental sulfur as terminal electron acceptor, but they do not do so. Exsamples of such organisms
are Pyrococcus furiosus, Thermotoga thermarum, Thermo toga neopolitana, and Thermococcus
litoralis (94-96). They convert organic carbon that they use as an energy source to CO2, and H2, and
detoxify the H2. whose accumulation is inhibitory to them. by oxidizing it with the reduction of So to
HS- . No energy seems to be gained in the sulfur-reducing step (94). clear how these organisms can
completely mineralize organic carbon. since the primary process by which they catabolize organic
matter appears to be fermentation and not respiration.
VI. 1N SITU STUDIES
A global assessment of the contribution that anaerobie mineralization of organic matter makes to
the carbon economy in marine sediments was presented by Henrichs and Reeburgh (30]. They con
cluded that anaerobic mineralization rates are intrinsically similar to serobic ones. However, because
much of the organic carbon is consumed in the aerobic zone before it reaches the anaerobic zones,
aerobic rates are greater near the sediment surface than anaerobic rates at depth. They estimated
that global anaerobic carbon minerelization in marine sediments represents only 9% of the global
aerobic mineralization, 1.e., an average of 150 Tg C yr-1 Exceptions do occur on a local scale.
Jorgensen (197] found that in coastal sediments of the Kattegat and Skagerrak at the entrance to the
Baltic from the North Sea and in some Danish fjords, sulfate reducers mineralized as much organic
matter as did all the aerobic respirers, but the contribution of the SO42-, reducers to total
mineralization of organic matter decreased to one-third toward the edge of the shelf area.
In contrast to marine sediments, which contain some persistent anaerobic regions, soil is generally a
predominantly aerobic environment where mineralization of organic matter is the result of
aerobically respiring bacteria and fungi [49,98] neverthelees, anaerobic microhabitais exist even in
these soils. Water-saturated soil crumbs larger than 6 mm in diameter are anaerobic at their center
(Ref. 99 as cited in Ref. 100) denitrification nitrate ammonification (reductoin of nitrate to
ammonia), and methanogenesis occur in these anaerobic microhabitats. evidence for denitrification
in anaerobic zones adjacent to aerobic zones in the rhizosphere of rice growing in wetland soil has
been described by Reddy et al. [101]. NO3- reduced in this process originated from bacterial NH3
nitrification in the aerobic zones, and some of it diffused into the anaerobic zones. Oxygen transport
through the stems and roots of the rice into the rhizosphere was responsible for the aerobic zones
around the roots.
Although aerobic soils predominate globally, anaerobic soil environments such as those of rice
paddies, swamps, and salt marshes that are extensive and predominate in some areas should not be
ignored In Asia, rice paddies constitute a significant portion of agricultural soil. Anaerobic
mineralization in these soils, when
flooded, appears to be dominated by nitrate respiration and methanogenesis, although iron
respiration may be equally or more important [90]
Sulfate respiration is rare in most soils because sulfate is generally not abundant in them. Salt
marshes sre an exception, as they contain abundant sulfate that had its origin in seawater [102-104].
This does not mean, however, that SO42-,rcducers cannot exist in soils with a low sulfate
concentration. A few species of Desulfovibrio and Desulfotomaculum and Desulfobacterium
catecholicum are able to use nitrate in place of sulfate as terminal electron acceptor. They reduce
the nitrate to ammonia by nitrate ammonification [64, 105-107] Such activity has been observed in
organic-rich, reducing sediments [109,110]. Clostridium butyricum is another organism that reduces
NO3- to NH4+ when growing under conditions of NO3-, [111]. The extent to which nitrate
ammonification occurs
in more typical anerobic soils has not yet been determined. alternatively. some So42-,reducers have
the capacity to live in association with methanogens in the absence of sulfate. The methanogens
serve as scavengers of the H2 produced by the SO42- reducers in interspecies H2 transfer [112-114].
Accumulation of the H2 produced by the sulfate reducers in the absence of sulfate is inhibitory to
them. Such consortia could exist in some anaerobic soil. However, they do not necessarily have to
cause complete degradation of the organic matter attacked by the sulfate reducers.
Anaerobic iron and manganese respiration must now be added to denitrification, nitrate
ammonification, and methanogenesis as processes that contribute significantly to anaerobic
mineralization of organic matter in soils and sediments (88,115,116]. Indeed, becsuse of the general
abundance of Fe(III) and Mn(IV) in many soils and sediments, anaerobic mineralization based on
Fe(111) and/or Mn(IV) respiration may predominate in them (90] However, if iron and manganese
oxides occur together, each in significant quantities, iron respiration may be the dominant process.
This because the ferrous iron produced in the respiratory process is likely to reduce the manganese
oxide by a nonbiological chemical reaction (86] anaerobic mineralization in a soil by seo42- is
globally unimportant, but it may be significant in some special localities from a bioremedial
standpoint [15,16,56].
Microbial consortia can make important contributions to anaerobic mineralization of simple and
complex erganic matter and xenobiotics in soils or sediments. Wolin and Miiler [1171 have described
the activity of some consortia of fermenting and methanogenic bacteria in converting organic matter
to methane in sulfate-deficient anaerobic environments. The Such consortia my be based on
interspecies H2, transfer, in which fermenters transform their substrate to acetate, H2 and CO2.
Methanogens may then generate methane from the acetate and from the H2 and Co2 le.g.. 118].
Such consorita may also be besaed on acetate formation form substrates such as glucose or fructose
by acetogens, which methanogens then convert into methane by hydrogen reduction [119] or
fermentation [120] terrestially, rice paddies and swamps area important sites of this methanogenic
activity.
Mineralization by sulfate respiration often involves consortia, some of which may be consortia of
different SO42-, reducers. For instance, Desulfovibrio desulfuricans and Desulfotomaculum
nigrificans oxidize lactate only as far as acetate with SO42- as the terminal electron acceptor. This is
as previously explained because they have neither the enzymes for an anaerobic tricarboxylic acid
cycle nor the enzymes for the acetyl CoA/CO dehydrogenase pathway. For complete mineralization
of lactate, these organisms need to associate with acetate-oxidizing sulfate reducers such as
Desulfotomaculum acetoxidans. Desulfobacter postgatei, Desulfobacter
hydrogenophilus, or other acetate-mineralizing bacteria (see Tables 4 and 5) for other organic
compound different consorita of SO42-,reducers or of fermenters plus SO42- reducers may be
required [98].
Young and Fraser [121] have discussed the evidence for the coupling of anaerobic degradation of
lignin and lignin-derived compounds to denitrification and methanogenesis. an activity that is very
important in the soil environment, where lignin from woody plants is a major source of organic
matter. This anaerobic degradation of lignin may involve microbial consortia.
Lovley and Phillips [122] have described microbial associations in which iron respiration formed the
terminal step in organic matter mineralization. They found that in sediments that support iron
respiration, glucose was first fermented to fatty acids. then oxidized to CO2, by way of iron
respiration. This phenome- non is likely to apply to some iron oxide-rich soils as well, as suggested by
early observations by Bromfield [123] iron minerals such as amorphous ferric oxides, lepidocrocite
(gamma- FeOOH). hematite (Fe2O3) and goethite (alpha-FeOOH), that may occur in some soils can
serve as terminal electron acceptors in anaerobic iron respiration processes [124-126].
VII CONCLUSIONS
Anaerobic mineralization of organic matter is important in the carbon cycle because it prevents
relatively permanent anoxic environments in the biosphere from becoming permanent sinks for
organic Instances in which organic matter. Instance in wich organic matter is not completely
degraded in anoxic environments and thus accumulates, as in organic soils, may be due to limited
availability of external electron acceptors, the relative refractoriness of some of the organic matter,
an accumulation rate of organic matter that is significantly greater than the rate of mineralization, or
a combination of these.
ACKNOWLEDGMENT The author is greatly indebted to Derek R. Lovley for a critical reading of a
draft of this chapter and for several valuable sugges- tions for improvement. REFERFNCES