ANAEROBIC TRANSFORMATION PROCESSES *
A REVIEW OF THE MICROBIOLOGICAL LITERATURE
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ANAEROBIC TRANSFORMATION PROCESSES t
A REVIEW OF THE MICROBIOLOGICAL LITERATURE
by
John E. Rogers
Biology Branch
Environmental Research Laboratory
Ajthens, Georgia 30613
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GEORGIA 30613
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DISCLAIMER
The information in this document has been funded wholly or in part by
the United States Environmental Protection Agency. It has been subject to the
Agency's-peer and administrative review procedure, and it has been approved for
publication as an EPA document. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use by the U.S.
Environmental Protection Agency.
ii
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FOREWORD
As environmental controls become more costly_to implement and the penalties
for judgment errors become more severe,' environmental quality management requires
more efficient management tools based on greater knowledge of the environmental
phenomena to be managed* As part of this Laboratory's research on the occurrence,
movement, transformation, impact and control of environmental contaminants, the
Biology Branch conducts research to predict*the rate, extent, and products of
biological processes that control pollutant fate in soil and water ecosystems*
Techniques for predicting the transport and transformation of toxic
chemicals in the environment require information on microbial degradation
processes. This report presents a literature review of research into the role
of microorganisms in the anaerobic transformation of xenobiotic compounds* In
addition to a compilation of all the available data, this report represents an
attempt to relate the data from tangential areas to the transformation of
xenobiotics in natural environments. Information gathered in the review will
be applied to the Laboratory's research to assess kinetic concepts for describ-
ing the transformation and/or degradation of toxic substances in aerobic and
anaerobic environments.
Rosemarie C. Russo
Director
Environmental Research Laboratory
Athens, Georaia
iii
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ABSTRACT
Ihe purpose of this review is to evaluate an
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TABLE OF CONTENTS
FOREWORD
I
I
ABSTRACT I iv
i
INTRODUCTION 1
CHARACTERISTICS OF ANAEROBIC! MICROORGANISMS 1
i
Photosynthetic bacteria . , 3
i
Anaerobic respiring bacteria 4
Methane-producing consortia ............. 8
Autotrophic acetogehs . . 11
Anaerobic degradation of selected compound classes . » . 12
Hydroxy and methoxy substituted tft&Tlignoaromatic compounds • • 13
Halogenated aromatic compounds 17
;
Halogenated aliphatic compounds ....' 20
Aromatic and aliphatic hydrocarbons . . > 24
Nitrogen containing compounds 25
f
Ether linked compounds ................ . ^ • • • • 27
ENVIRONMENTAL INTERACTIONS i . . . 27
METHODS OF INVESTIGATION! 29
CONCLUSIONS 31.
REFERENCES 33
v
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LIST OF ILLUSTRATIONS
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 3
'. • • .. V.
Figure 10
Figure 11
Figure 12
Figure 13
Proposed Pathways for the Anaerobic Photometabolism of $
Benzoic Acid
Proposed Pathways for the Anaerobic Degradation of Benzoic 7
Acid by Nitrate Respiring Organisms
Proposed Pathway for the Degradation of Benzoic Acid by 10
Methanoge^iic Consortia
i
Proposed pathway for the Degradation of Phenol by a 15
Bacterial.Consortium in the Presence of Nitrate
! ' !
Proposed Pathway for the Degradation of Ferulic Acid and 16
Conifejryl; Alcohol by a Methanoqenic Consortium
Proposed pathway for the Degradation of Syringic Acid by 18
a Methanogenic Consortium
Proposed" Pathway for the Degradation of Phloroglucinol 19
i .. i
Proposed Pathway for the Degradation of 3,5-Dichlorobenzoio *21
Acid by a Methan0g«ni<$ Consortium
Proposed Pathway tlt^ |^^ad«tion of Tetrachloroethylene 23
Mechanism for the K«4UGtl6i1k'.'4# Nitj*o Compounds
Mechanism""foir the Reduction^ of Azo Compounds
Stoichiometry of Anaerobic Indole Degradation
Proposed Pathway for the Degradation of Nicotinic Acid by
Clostridium barker! sp. n.«
TST
26
27
28
Table 1 Comprehensive List of Compounds MXc robially Degraded or
Transformed under Anaerobic Conditions
48
vi
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REVIEW OF ANAEROBIC TRANSFORMATION PROCESSES
INTRODUCTION
The purpose of this review is to evaluate and summarize available informal
tion concerning the role of microorganisms in the anaerobic transformation of j
xenobiotic compounds in natural environments. Unfortunately, information that
relates directly to anaerobic transformation of xenobiotics in the environment;
is limited. A large body of information* however, does exist concerning the
anaerobic transformation (or degradation) of these synthetic chemicals and
related natural compounds by pure organisms, mixed cultures, and adapted and
unadapted sewage sludge and sediment systems. In addition to a compilation
of all the available data, this report represents an attempt to relate the
data from tangential areas to the transformation of xenobiotics in natural
environments.
To focus this review, I have included only information concerning the i
anaerobic transformation of xenobiotio compounds and natural structural analogs.
Naturally occurring compounds are included because it is generally acknowledged
that the closer in structure « xenobiotio compound is to a natural substrate
Stanley Dagley
(1972J stated it
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Progress toward understanding the biochemical details of the anaerobic
degradation of xenobiotics has been slow because of the experimental difficulties
attending the use of communities of microorganisms. For the most part, anaerobic
degradation of xenobiotic compounds generally requires the concerted effort of
several metabolic groups of bacteria. Itie initial group ferments the parent
compound to products such as saturated fatty acids, H2 and CO2. An intermediate
group, the proton-reducing acetogenic bacteria (Bryant 1976, Mclnerney et al.
1979, Mclnerney et'al^ 1981), converts the long chain fatty acids to acetate,
propionate, C02, and H2. The terminal metabolic group includes the many methan-.
ogenic species (Balch et al. 1979, Whitman 1985) and possibly sulfur-reducing
species (Mountfort and Bryant 1982) that catabolize the acetate produced by the
other groups to CO2 atyd CH4, and rapidly use the H2 produced to reduce C02 to
methane. i
1
The integrity and efficiency of anaerobic consortia are maintained in part
by what has been termed "interspecies hydrogen transfer." Biis process was
first described by Bryant et al. <1967). Fermentative bacteria normally reoxi-
dize their reduced coenzymes by transfer of electrons to an organic terminal
electron acceptor such as pyruvate, lactate, acetate, etc. However, .in the
presence of extremelyjlow concentrations of H2* some bacteria Jiave the ability
to reoxidize their reduced coenzymes by proton reduction. Hydrogen concentra-
tions are maintained at a low level by the H2~scavenging activity of methanogenic
bacteria or some sulfate-reducing bacteria. The proton-reducing partner in such
syntrophic associations gains a small energetic increment by H2 .x^pase because
cellular intermediates, which would otherwise be reduced for reoxidiizing reduced
coenzymes, can be further-#Jtfci tired t0 ;<0xtir5mely 1owH2 concentra-
tions {<10-4 atmosphere) that jtormit pi'Otdh tf^dti^ti^ baw been measured in
anaerobic sediments (Winfrey et al* \9l1li 1978jWinfrey«i}df
Zeikur^d) Jones: et al.
Despite the importance of anaerobic consortia, a number of anaerobic ¦
organisms have been shown to grow on substituted aromatic compounds as the soXe
source of carbon. In many cases the substrates are completely mineralized and
the presence of acetogenic or hydrogen utilizing bacteria. Such organisms
include the purple nonsulfur bacteria such as Rhodopseudomonaa palustris and
Rhodopseudomonas gelatlnosa (Evans 1977), nitrate reducing bacteria such as
Pseudooonas (PN1) and Moraxella sp. (N.C.I.B. 11086) (EvttHS 1977) and, sulfate
reducing organisms such as Desulfococcus multivorans, Desulfonema magnum, and
Desulfosarsina varaibllis (Widdel 1980).
The relative importance of each mechanism to the degradation of a
xenobiotic released to a particular environmental niche will depend on the
chemical structure! of the xenobiotic and the physical/chemical characteristics
of the site. Microorganisms active in the degradation of the xenobiotic will
be limited to those 'organisms that have or can produce enzymes and possibly
transport systems that can recognise and act on the xenobiotic. The closer
in structure a xenobiotic compound is to the structure of a common natural
substrate, the more likely that it will be transformed or degraded by a large
variety of microorganisms. These organisms are only active if they are abie
to grow and compete under the physical/chemical constraints of the particular
environment. For example, in estuarine sediments the microbial populations are
2
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generally dominated by sulfate reducing Microorganisms. Alternatively, in
fresh water sediments where the concentration of oxidised sulfur species is low,
methanogenic microorganisms dominate, if the dominating organisms are capable,'
the xenobiotic has a good chance of being rapidly degraded. Unfortunately, if
minor populations, such as methanogenic microorganisms in an estuarine environ-
ment, are the active members of the community, the chemical would most likely be
degraded at a slower, rate.
Benzoate is a natural product and a structural Component of a large number
of xenobiotic compounds. Benzoate was also one of the first aromatic compounds
shown to be degraded anaerobically. Tarvin and jBuswell (1934) reported that
benzoate and a number of similar aromatic compounds were completely utilized
with the production of C02 and methane by a sewajge sludge inoculum under strictly
anaerobic conditions. Since this initial discovery, the degradation of benzoate
has been demonstrated in all major environmentally important anaerobic microor-
ganisms. The following summary of anaerobic benjzoate metabolism will ibe used
to introduce these microorganisms.
Photosynthetic bacteria
Scher (1960) and Procter and Scher (1960) Ware the first to demonstrate
anaerobic photometabolism of benzoic acid by showing that Rhodopseudomonas
palustris could grew on benzoate anaerobically in the light. In addition,
cells grown anaerobically on a mineral saltsibenzoate medium in the light was
shown, by monometric techniques, to oxidize benzoate, protocatechuate, catechol,
and a-oxoglutarate in the dark. Unfortunately,-these results led the authors
to Incorrectly place catechol as an intermediate in the metabolic pathway,
Thif» conclueion apparently resulted from a literal interpretatio^"of van Kiel's
general metabolic schemes (van Kiel,- 1941) for' phofcesynthetic Bacteria and '
plant** One of the products of the light reaction was considered to be a
»*iong oxidant 1 "OH"}, which in plants was converted to oxygen and in bacteria
was used to oxidize-substrates. Thus, in this early work, the light-induced
oxidant and molecular oxygen were considered equivalent and the anaerobic path-
way was considered to be the saxte as the pathway reported for aerobic degrada-
tion mechanisms. These experiments, however, could not be repeated. Subee-
~qfl€ntly, Dutton and Evans (1968) were able to show that R. palustris grown
photosynthetically on benzoate was not able to respire in the presence of
benzoates or 3-hydroxybenzoate under kerobic conditions. Later they showed
(Dutton and Evans 1969) that return to anaerobic conditions in the light
permits benzoate utilization^^proceed again.
The dependence on anaerobic conditions strongly suggested that anaerobic
photometabolism of benzoate was a reductive process. Guyer and Hegeman (1969)
concluded this from(mutant studies with R. palustris, and suggested that cyclo-
hexanecarboxylate and eyelotiex-1-enecarboxylate might be metabolic intermediates.
At the same time, Dutton and Evans (1968, 1969), using 14C-labelled benzoate,
determined the key intermediates of anaerobic photometabolism of benzoate in R.
palustris. Degradation proceeded from benzoate to pimelic acid with the inter-
mediate formation of cyclohex-1 -enecarboxylate, 2-hydroxycyclohexanecarboxylate
and 2-oxocyclohexanecarboxylate. Pimelic acid was further degraded by 0-oxidation.
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Evans (1977) postulated that benzoate might be activated to benzoyl
phosphate before ring reduction could occur* Benzoyl phosphate, but not
benzoyl-CoA, was reduced by a washed chromatophore suspension. Conflicting
results have been reported by Whittle et al. (1976) who showed that extracts
from R. palustris cells grown photosynthetically on benzoate are capable of the
thioesterification of benzoate to form benzoyl-CoA. The cofactors for this
reaction were ATP and Mg2+. Furthermore, Whittle et al. (1976) obtained a cell-
free system capable of converting cyclohex-1-enecarboxylate to pimelate. These
reactions required the addition of CoA, ATP, , and HAD+. More recently,
Hutber and Ribbons (1983) found that^fcnaerobic cultures of R. palustris produced
a benzoyl-CoA synthetase activity in response to benzoate. A similar CoA syn-
thetase could be induced by cyclohexane carboxylate under aerobic conditions.
The enzymes necessary for #—oxidation of cyclohexane carboxy 1—CoA appeared to
be constitutive in both aerobic and anaerobic cultures. Harwood and Gibson
(1986) have shown that the uptake of benzoate by R. palustris is not the result
of either passive diffusion or active transport, but that accumulation of ben-
zoate is closely coupled to the formation of benzoyl-CoA. The formation of
benzoyl phosphate could not be ruled out as the rate limiting step in the uptake
of benzoate. These most recent findings provide strong circumstantial evidence
that the substrate for ring reduction and subsequent cleavage, however, is
benzoy 1-Co-A.
A metabolic scheme for the anaerobic photometabolism is shown in Fig. 1 •
The scheme, which incorporates the work of Whittle et al. (1976), Hutber and
Ribbons (1983), and Harwoodand Gibson (1986), is essentially an extension of
the "^iegradative pathway first-described by Dutton and Evans (1969). The con—
<1a^8ion of eyelohexanecarboxy 1-CoA to cyclohex-T-enecarboxyl-CoA has not been
demonstra tedi in ctfude cell extracts and ia included here because of the known
f iavpproteih characfcer of the (I—acyldehydrogenaseft of fatty acid
g-oxidation. If the benroyi-CoA reduction wachanism oould go directly to the
cycloheX-1-eneearbdxyl-43pA (dotted line. Fig. V), the-proposed dehydrogenation
step would be redundant aa a major part of the pathway. The conversion of 2-
oxocyclohexanecarboxy 1—CoA to pimelyl di-CoA has been included because it is
consistent with fatty acid 0-oxidation. Because the reaction leads to ring
cleayage, both CoA groups are on the same dicarboxylic acid.
The ecological significance of anaerobic photometabolism of xenobiotic .
compounds is unclear. Rhodospirillaceae occur in all aquatic environments
(Pfennig 1967) and in soil, where their numbers increase with increasing mois-
ture content (Pfennig and Truper 1973), so they could play a significant role
in the dissimilation of xenobiotics in these environments. Unfortunately,
their photometabolic activity is limited to those areas where anaerobic and
euphotic zones coincide, such as small eutrophic lakes during summer strati-
fication, flooded soils, and anaerobic surface sediments in ponds and ditches.
t
Anaerobic respiring bacteria
The potential for anaerobic degradation of benzoate and other aromatic
compounds by Anaerobic respiration with nitrate as the terminal electron
acceptor exists in a large number of environments. Bacteria capable of nitrate
4
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0
*C-OH
'*C-OH . %-SCoA \-SCoA
<-^0—0
1 11 \
Cytoplasmic \
Membrane \
III
\
\
\
\
^2H
Acetyl CoA-
O^SCoA:
^.SCoA
C=0
VII
\
0^ O ^ n
€-SCoA *C-SCoA C-SCoA
0 H2°
VI
IV
Fig. 1. Proposed pathway for the .anaerobic photometabolism of benzoic acid.
Compounds are: (I) benzoic acid, (II) benzoyl-CoA, (III) cyclohexane-
carboxyl-CoA, (IV) cyclohex-1-one car boxy 1-CoA, (V) 2-hydroxycyclohexane-
carboxyl-CoA, (VI) 2-oxocyclohoxanocarboxyl-CoA, (VII) pimelyl di-CoA.
5
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reduction have been found in all types of soil, in.activated sludge, and in
marine and freshwater sediments (Jeter and Ingraham 1981).
Williams and Evans (1973) isolated a soil microorganism able to grow
anaerobically on benzoate in the presence of nitrate. Isotope-dilution experi-
ments in conjunction with thin layer chromatography and gas chromatography sug-
gested that a reductive mechanism of ring metabolism, similar to that shown in
R. palustris by Dutton and Evans (1969), was operating. In more recent work
this isolate was identified as a Moraxella sp. (Williams and Evans 1975).
Additional intermediates in the ring cleavage reactions were identified, and,
although they were not identical wjith those for photometabolism, the basic
principle of ring reduction preceding ring cleavage under anaerobic conditions
was confirmed. ;
The degradative pathway of bejnzoatie metabolism coupled to nitrate
respiration, as proposed by Evans !(1977), is shown in Fig. 2. Hiis pathway
diverges from the scheme for R. palustris at 2-oxocyclohexanecarboxylate. For
R. palustris a hydrolytic cleavage; occurs to give pimelic acid whereas decar-
boxylation followed by ring cleavajge gives adipic acid for nitrate respiring
organisms. Coenzyme-A is proposed as a cofactor in the conversion of cylohex-
anecarboxylate to the 2-oxo-derivative; however, no direct evidence has been
reported to support this. ; ¦
Interestingly, in some aerobic sediments both oxygen-mediated and nitrate-
dependent aromatic metabolism may occur simultaneously or in concert* Focht
aitff Verstraete f"l977) considered anoxic ml crozones, such as those within par-
ticles, as possible sites of denitrificMlfpiv tW-^efrobic sediments# and Jones
(1979) has suggested that si^lA«? »^oro-^iii\4fon»Bnt« were resj^nsibl^ for
nitrate reductase activi€y~in aerobicffreshwater sediments* Fabig et al.
(1980) observed that. In the presence of trace amounts of oxygen, benzoate
.degradation and nitrate reduction occurred simultaneously in either an enriched
culture or a pure culture of P. aeruginosa. Both cultures were capable of
ortho-ring fission. On the other hand, Fabig et al«j (1980) found^that denitri-
fying bacteria, such as a Acinetobacter sp., and a Moraxella sp., that did not
have oxygenase activity were unable to use benzoate under microaerophilic
conditions. The authors speculated that under "anaerobic" conditions trace
amounts of oxygen were required for ring cleavage, and subsequently the ali- -
phatic products were metabolized by nitrate respiration. Similar results were
observed by Sleat (1981) in studies of the growth of nitrate respiring faculta-
tive anaerobes capable of aerobic but not anaerobic utilization of j>—hydroxy ben-
zoate. He found that growth of the organisms on jg-hydroxybenzoate was increased
by the presence of nitrate wheh oxygen was supplied at trace levels.
i
The number of obligately anaerobic microorganisms reported to grow on
benzoate or other aromatic compounds in the presence of inorganic terminal
electron acceptors is limited. A few sulfate reducing bacteria have been
reported to utilize benzoate (Widdel I960). Organisms from three distinct
bacterial genera, Pesulfococcus, Pesulfonema and Desulfosarclna, able to
metabolize benzoate with sulfate as the electron acceptor were isolated.
However, no information on the. degradative pathways has been reported.
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0*C-OH °*c-OH °*C-OH
6H
v
««
O
CO
s*
VI
and
0
VII VIII
7"
Bulyrafe 2H
XI
II HI
C-OH
.OH
IV
HQHoh__ (3'° H°(J-
IX
¦|2H
HO-C O r'Pn
Acetate / nw JZh*
C-OH C-OH
a
Pig. 2. Proposed pathway for the anaerobic degradation of benzoic acid by
nitrate respiring organisms. Compounds are: (I) benzoic acid, (II)
cyclohexanecarboxylic acid, (ill) cyclohex-1-enecarboxylie acid, (IV)
2-hydroxy cyclohexanecarboxy lie acid, (V) 2-oxocyclohexaneCarboxylic
acid, (VI) cyclohexanone, (VII) 1 *2—diliydroxycyclohexane', (VIII) 2—
hydroxycyclohexanone, (IX) 6-hydroxyhexanoic acid, (X) adioic acid
semialdehyde, (XI) adipic acid, _ P
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Methane-producing consortia
Anaerobic fermentation of xenobiotic compounds generally requires the
concerted effort of several metabolic groups of bacteria to degrade a compound
to CO2 and CH4. The initial group ferments the parent compound to products
such as saturated fatty acids, H2# and CC>2«
A variety of bacterial consortia have been described and a number of these
have been shown to anaerobically degrade benzoate and other aromatic compounds
(Tarvin and Buswell 1934, Clark-and Fina 1952, Fina and Fiskin 1960, Nottingham
and Hungate 1969, Ferry and Wolfe 1976, Shlomi et al. 1978). Attests to
isolate component members of the consortia have produced a number of methane-
producing and nonmethanogenic bacteria.
Sufficient evidence exists to suggest that both autotrophic.and acetoclastic
methanogiens function in benzoate degrading consortia. Ferry and Wolfe (1976)
isolated Methanobacteriure formicicum and Methanosplrlllum hungatei from anaerobic
benzoate enrichments but were unable to Isolate an acetate utilizing methanogen.
Methanobrevibacterlum rumjnantium and Methanosplrillum hungatei were tentatively
identified in enrichments by Balba and Evans (1977). Unfortunately, only cir-
cumstantial evidence supports a role for acetate utilizing methanogens in ben-
zoate degrading consortia. Shlomi et al. (1970) identified a gram-negative rod
that formed long chains, in a benzoate-methanogenic enrichment. Healy et al.
(1980) observed similar long chains of rods in a ferulic acid enrichment. The
organisms were thought to be identical to the acetate utilizing methanogen
Methanobacteriuw soehngenji (Zehnder et al. 1980, Iiuser et al. 1982). The
organism forms very long flexible filaments that tend to aggregate in bundles.
Microscopic observations revealed the presence of characteristic bundles in the
methanogenlc-benzpate enrichments of Sleat and Robinson (1983). Methanpthrix
•soehnqenii can use acetate but not methanol, methylamine, formate or for
growth and methane production (Huser et al. 1982).
The isolation of nonmethanogenic organisms from stable consortia has had
limited success. Ferry and Wolfe (197©) isolated a bacterium similar in
appearance to Pseudomonas PN-t (Taylor et al. 1970) that grew aerobically but
not anaerobically on benzoate. A facultative, gram-negative bacterium that
grew aerobically on j>-hydroxybenzoate and anaerobically on benzoate in the
presence of nitrate was isolated by Balba and Evans (1977). Attempts to recon-
stitute a benzoate-methanogenic consortium with M. hungatei were unsuccessful
(Balba 197B). Abram and Hedwell (1978a) were somewhat more successful in
reconstituting a benzoate degrading consortium. They found that recombination
of a gram-negative pleomorphic rod and a Methanococcus sp., both isolated from
the same benzoate enrichment, resulted in methanogenesis from benzoate in 3 of
20 tries. ,
Mclneroey et al. (1979, 1981) and Boone and Bryant (1980) have described
the isolation of aliphatic acid metabolising bacteria in syntrophic'association
with either sulfate reducing or methanogenic bacteria* To isolate a bacterium
in coculture with H2 utilising organisms, aliquots of serial 10-fold dilutions
of enrichments were transferred to roll tubes containing 4 ml of a basal growth
medium plus 0.5 ml of an actively growing culture of the H2 utilizing organism.
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The basal growth medium did not support growth of the ^-utilizing organism or
the organism to be cocultured. Positive cultures generally contained a syn-
throph that degraded aliphatic acids to H2, C02 and acetate in addition to the
H2 utilizing bacterium. Mountfort and Bryant (1982) have used this technique
to isolate a benzoate catabolizing, gram negative, nonsporing, rod-shaped bac-
terium that grows in anaerobic syntrophic association with hydrogen utilizing
Desulfovibrio sp. or with Methanospirillum hungatel. More recently, Barik et
al. (1985) were able to isolate two bacterial strains, P-2 and PA-1, that could
grow syntrophically with Wollinella succlnogens on phenol and phenylacetate,
respectively.
Evans (1969) has reported that Soehngen observed methane production from
aromatic compounds as early as 1906. The earliest published report appears to
be that of Tarvin and Buswell (1934). They demonstrated that a range of aromat-
ics were metabolized to methane and carbon dioxide with an efficiency of greater
than 90%. Many subsequent studies emphasize the biochemical mechanisms of
anaerobic aromatic metabolism. Although ring reduction preceded ring cleavage,
the reduction intermediates varied depending on the enrichment. Hie metabolic
scheme for benzoate degradation by inethanogenic consortia is shown in Fig. 3.
The scheme is a consolidation of published results from a number of laboratories.
Shlomi et al. (1978) described a consortium from enrichments prepared from
black mud from a small polluted river that degrades benzoate by the reductive
pathway reported for R. palustris (Dutton and Evans 1969). Intermediates con-
sistent with the reductive pathway, trans-2-hydroxycyclohexanecarboxylate. 2-
oxo-cyclohexanecarboxylate, pimelate, caproate, butyrate^ and acetate were
identified from pyridine extracts of freeze-dried samples of actively growing
cultures. Jfr a separate study, Palba and Evans (1977), using isotope trapping
techniques, detected the fon«ti?n of adipio acid fron benzoate in methanogenic
cultures obtained from sewagesludge* Tfie presence of adipate was indicative
of decarboxylation before ring cleavage aa found for the nitrate respiring
Moraxella sp. investigated by Williams and Evans (1975). Keith et al. (1978)
proposed that benzoate is reduced step-wise to cyclohexanecarboxylate followed
by a reductive cleavage of the Cj - C2 bond to give heptanoate* Heptanoate
would be degraded to propionate by ((-oxidation. ; Ti>ey identified cyclohexane-
carboxylate, eye lohex-1-enecar boxy late, heptanoate, valerate, butyrate,
propionate and acetate in benzoate-methanogenic cultures.
Grbic-Galic and Young (1985) isolated.a number of intermediates from
benzoate degrading methanogenic consortia treated-y^th bromoethanesulfonic acid
(BES) to inhibit methane formation. The major intermediates isolated included
cyclohexanecarboxylate, cyclohexanone, 1-methylcyclohexanone, pi me late, adipate
and heptanoate. The identification of pi me late is consistent with the photosyn-
thetic pathway (Evans 1977); the intermediates, cyclohexanone and adipate, are
consistent with decarboxylation before ring cleavage as reported for nitrate
respiring organisms (Evans 1977). The formation of heptanoate was suggested to
result from ring cleavage of 2-methylcyclohexanone in contrast to the.reductive
cleavage of cyclohexanecarboxylate proposed by Keith et al. (1978). Direct
confirmation of .either mechanism will await isolation and charaterizatiOn of
the appropriate enzyme systems.
9
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Not surprisingly, several unusual reduced compounds (toluene, benzene,
cyclohexane, and methylcyclohexane) were detected in BES-inhibited consortia
and nonmethanogenic mixed cultures derived from the inhibited consortia. It
was suggested that these compounds were formed when the normal flow of elec-
trons and hydrogen was interrupted with BES. For similar complex systems
(Wolin 1974? Wolin 1976; Bryant 1979), in the absence of methanogenesis,
reduced products such as ethanol, propionate, butyrate, lactate, succinate, or
adipate are formed. It is interesting to speculate that some of the variability
in intermediates (Fig. 3) observed for uninhibited methanogenic consortia may
result from a decrease in normal level of "interspecies hydrogen transfer,"
which modulates the efficient conversion of organic compounds to CO2 and CH4.
i
Methanogens and sulfate 1 reducing bacteria have been detected in a number
of ecosystems that include mud, sediment, subsurface materials and flooded
soils of both marine and fre^hwatfer environments (Zeikus 1977). Methanogenic
activity dominates in environments (freshwater) having low concentrations of
oxidized sulfur species and,.conversely, sulfate reducing activity dominates
in environments (marine) that contain high concentrations of oxidized sulfur
species. Sulfate reducing bacteria have been shown to outcompete methanogenic
bacteria in the presence of Sulfate (Abram and Nedwell 1978a,b; Lovley and Klug
1982; Winfrey and Zeikus 1977). Sleat and Robinson (1983) examined the methan-
ogenic fermentation of benzoate in anaerobic freshwater mud and showed that
carbon dioxide was the only significant end-product of benzoate fermentation
in the presence of sulfatle, whereas in the absence of sulfate both methane and
carbon dioxide were formed. Itie apparent inhibition of methanogenesis by sul-
fate reducing bacteria has been attributed to thermodynamic principles (Winfrey
and Zeikus 1977, Zehnder 1978) or kinetic competition for substrates (Abram and
Nedwell 1978, Kristj^nsroi! Schooheit et al. 1982, Winfrey and
Zeikus 1977). Xovley (1985) has reemphasized that sulfate-reducing bacteria
may outcompete methanogenic bacteria by maintaining the hydrogen partial pres-
sure below a minimum threshold; necessary for-mpthane production (Lovley et al.
1982). He recently demonstrated that methanogenic*isolates cannot consume)
hydrogen below a partial pressure of 6.5ra. In contrast, in the absence of
sulfate some sulfate-reducing bacteria (Fiebig and Gottschalk 1983, Traore et
al. 1983, Phelps et al. 1985) are acetogenic proton reducers. Thus, in the
absence of sulfate, they function syntrophically and produce substrates for
methanogenic bacteria.
Autotrophic acetogens
A fourth class of organisms known to utilize H2 in anaerobic environments
is the acetogenic bacteri'a (Ljungdahl and Wood 1982). Unlike the methanogenic
and sulfate reducing bacteria that utilize both H2 and acetate, the major
carbon intermediate, the acetogens utilize H2 and produce acetate from CC^.
Braun et al. (1979) reported viable counts of acetogens to be ca. 1% of those
of methanogens in sludge and lake sediments. It also has been estimated
(Lovley and Klug 1983) that acetogens account for ca. 5% of H2 consumption in
the sediments of hypereutrophic Wintergreen Lake. In the profundal sediments
of Blelham Tarn (English Lake District), where anaerobic degradation processes
can be limited by_the input of organic carbon, hydrogen consumption by acetogens
can be as high as 50% of the methanogenic rate (Jones and Simon 1985). Apparently
10
-------
0
C-OH
C-OH
C-OH
C-OH
C-OH
\ HO-C O
C-OH
VIII
Fig. 3- Proposed pathway for the degradation of benzoic acid by methanogenic
consortia. Compounds arc: (I) benzoic acid, (II) 2-oxocyclohexanecar-
boxylic acid (III) 2-methylcyclohexanone, (IV) heptanoic acid, (V)
pimelic acid, (VI) cyclohexanone, (VII) caproic acid, (VIII) adipic
acid.
11
-------
the acetogenic bacteria may have a larger role in the mesotrophic to oligotrophia
lakes as opposed to the more eutrophic lakes.
Although acetogens have not been demonstrated to function in the degradation
of benzoate, other than acting as a hydrogen sink, acetogens have been shown to
demethylate methoxylated aromatic compounds to yield the hydroxy derivative,
carbon dioxide and acetic acid. Using anaerobic enrichments with methoxylated
aromatic compounds as substrates, Bache and Pfennig (1981) isolated two bacter-
ial strains, NZva16 and NZva24, which were recognized as Acetobacterium woodii.
Both isolates and the type strain of Acetobacterluro strains were able to grow
with aromatic compounds containing methoxyl groups. Product analysis revealed
that the aromatic ring was not cleaved and that the corresponding hydroxy
derivatives of the methoxylated compounds were formed. For example, vanillic
acid was transformed to protocatechuic acid and syringic acid was converted to
gallic acid. Recently, a novel anaerobic gram-negative rod bacterium that
utilized o-methyl substituents of monoaromatic acids as a sole source of carbon
was isolated from municipal sewage sludge by Frazer and Young (1985). The
organism transformed syringic acid to gallic acid, vanillic acid to protocate-
echuic acid, and ferulic acid to hydrocaffeic acid. Frazer and Young (1986)
postulated that the carboxyl carbon of the product acetate is derived from CO2
and the methyl carbon is derived from the o-methyl substituent of vanillate.
In support of this hypothesis they showed that, for every o-methyl carbon of
vanillic acid converted to acetate, two were converted to carbon dioxide.
ANAEROBIC DEGRADATION OF SELECTED COMPOUND CLASSES
Progress in understanding the anaerobic degradation of xenobiotic compounds
has been slow primarily because of the experimental difficulties in using bac-
terial consortia that are extremely sensitive to oxygen. However, in recent:
years, a number of aromatic and aliphatic compounds have been shown to be
degraded by anaerobic microorganisms and consortia. For example, the lignoaro-
—matic compounds—ferulic acid, vanillin, cinnamic acid, protocatechuic acid,
and catechol—can be converted to methane by anaerobic bacterial consortia
(Healy and Young 1978, 1979). Suflita et al. (1982) demonstrated that anaerobic
microorganisms in lake sediments and sewage sludge degrade halogen—substituted
benzoic acid^bf eliminating halide ion to produce benzoate, which can then be
metabolized to methane. Anaerobic dehalogenation may be more important than
the corresponding aerobic process. Recent work by Grbic-Galic and Vogel (1986]
has shown that benzene and toluene can be degraded toTlfethane and carbon dioxide
by a ferulic—acid—adapted anaerobic -tenrichment. These compounds were generally
believed to be resistant to microbial attack in the absence of oxygen.
A comprehensive list of compounds known to be transformed or degraded
under anaerobic conditions is presented in Table 1. The table provides a list
references for each compound, the source of the microorganisms for the .
individual studies, and an indication of the analytical methodology used to
measure degradation or transformation. The remainder of this section will be
concerned with the anaerobic degradation and transformation of a number of
compound classes.
12
-------
Hydroxy and methoxy substituted and lignoaromatic compounds
Numerous reports have appeared concerning the degradation of hydroxy- and
metiioxy-substituted benzenes and lignoaromatic compounds. Chemielowski and
coworkers described the methanogenic fermentation of phenol in 1964. Since
that time Bakker (1977) has demonstrated the anaerobic degradation of phenol to
CC>2 in the presence of high concentrations of nitrate. Healy and Young (1978)
and Balba and Evans (1979) confirmed the anaerobic degradation of phenol to CO2
and methane by methanogenic consortia; Healy and Young (1978) extended their
studies to demonstrate the degradation of catechol as well. Subsequently,
Healy and Young (1979) investigated the anaerobic degradation of eleven aromatic
compounds, which included vanillin, vanillic acid, ferulic acid, cinnamic acid,
benzoic acid, catechol, protocatechuic acid, phenol, £-hydroxybenzoic acid,
syringic acid, and syringaldehyde. Methanogenic populations acclimated to a
particular aromatic substrate were simultaneously acclimated to some, but not
all, of the aromatic substrates investigated. Boyd et al. (1983) investigated
the anaerobic degradation of phenol and the ortho, meta, and para isomers of
methoxyphenol and methylphenol (cresol) initially at 50 ppm in diluted municipal
sewage sludge. Only ^-cresol was not degraded to CO2 and methane by the end of
the 8-week incubation period. The degradation of phenol and seven alkyl-phenols
(o-, m-, and £-cresol, 2,5-, 2,6-, 3,4-, and 3,5-dimethylphenol) at concentra-
tions ranging from 100 to 2000 ppm was examined (Fedorak and Hrudey 1984) in
anaerobic domestic sludge. Only the lower concentrations of phenol (500 ppm)
and p-cresol (200 and 400 ppm) were degraded to methane. At 500 ppm for 2,5-,
-3,4-, and 3,5-dimethylphenol, the rate and amount of methane produced was below
"unamended sludge samples. Phenol did not inhibit methane production until
concentrations reached 2000 ppm or more.
Ehrlich et al. 1982»haves examined the anaerobic degradation of phenolic
compounds contaminating an aquifer at St. Louia Park, Minnesota. Analysis o£
the in situ concentration of phenols in a series of we lis indicated) that the
.concentration of the phenolic compounds was decreasing faster than epuld be
accounted for by dispersion* Sorption of the phenolic compounds was negligible}
thus anaerobic degradation was considered the controling factor. The key role
of anaerobic bacterial activity Was supported by the observations that methane
was only found in water samples from the contaminated zone, that me thane-producing
bacteria were only found in waters from the contaminated zone, and that methane
was produced in laboratory cultures of
-------
Bakker (1977) has suggested that phenol is reduced to cyclohexanone followed
by hydrolytic conversion of the ring to give n-caproic acid, which can then
undergo 0-oxidation to yield acetate (Fig. 4). As evidence for this pathway,
Bakker pointed to the identification of trace concentrations of caproate in
mixed bacterial cultures that were able to convert (U-^C) phenol to *4C02
and radioactive cell material in a nitrate-mineral salts medium. No methane
was formed, presumably because of the presence of high nitrate concentrations.
Bakker's pathway was later partially confirmed by Balba and Evans (1979), who
were able to isolate the metabolic intermediates cyclohexanone, adipate, suc-
cinate, propionate, and acetate from a phenol—adapted methanogenic fermenta-
tion. In this case the identification of adipate^suggests an alternative path-
way (Fig. 4) for the cleavage of cyclohexanone, which could be similar to the
pathway observed for the nitrate-respiring Moraxejla sp. (Fig.2).
I
The degradation of the lignoaromatic compound, ferulic acid, has been
shown to occur anaerobically by a reductive pathway (Fig. 5) that overlaps the
previously reported pathways for benzoic acid (Fig. 3) and phenol (Fig. 4).
Evidence in support of this pathway has been provided by Healy et al. (1980),
Grbic-Galic and Young (1985), and Grbic-Galic (19$5). Healy et al. (1980)
observed that ferulic acid was biodegraded to C02jand methane under strictly
anaerobic conditions by a bacterial consortium similar to that observed for
benzoate (Shlomi et al. 1978). By incubating the cultures with BES to inhibit
methanogenesis, intermediates of phenylacetate, cinnamate, 3-phenylpropionate,
benzoate, cyclohexanecarboxylate, adipate, andpimelate were detected. A reduc-
tive catabolic pathway was described that,, in ||>art# supports the pathway shown
in Fig. 5. The pathway merged with the benzoate pathway suggesting that a
portion of the pathway may be common to the degradation of many ligoaromatic
compounds. In a subsequehfstudy (Grbic-Galic aittTYoung 1985), two additional
intermediates, caffeate and j?-hydroxycinnamate, were identified in BES-inhibited,
^?51^c"ac^",a^aP^e'' consortiaj the intermediates cyclohexanone, methylcyclohex-
anone, and isocaproate were identified in BES-inhibited, benzoic-acid-adapted
consortia. These results complimented the previous results of Healy et al.
(.^BOJj^and the combined efforts are responsible for the degradative! pathway
showrfin Fig. 5.
In further investigations of the degradation of" ferulic acid, Grbic-Galic
(1985) isolated a facultatively anaerobic, gram—negative, nonsporeforming,
rod—shaped bacterium from a bacterial consortium degrading ferulic acid.
The organism degrades ferulic acid completely under aerobic conditions and
partially transforms i* under strictly anaerobic conditions, without the addi-
tion of exogenous electron acceptors. Anaerobically, ferulic acid was demeth-
oxylated and dehydroxylated with subsequent si^e chain reduction to yield
phenylpropionate and phenylacetic acid. All intermediates were members of the
proposed degradative pathway for ferulate (Fig; 5).
As further evidence for the commonalty of.these pathways Grbic-Galic
(1983) has suggested that the degradation of coniferyl alcohol overlaps with
t^ie ferulic acid pathway (Fig. 5). Coniferyl alcohol, was shown to be com-
pletely biodegraded to carbon dioxide and methane under strictly anaerobic
conditions and ferulic acid-and phenylpropionic acid were identified in active
cultures. Similarly, results of Taylor (1983) reasonably support a degradative
14
-------
Fig. 4. Proposed pathway for the degradation of phenol by a bacterial
consortium in the presence of nitrate. Compounds are: (I) phenol,
(II) cyclohexanol, (III) cylohexanone, (IV) c^proic acid.
15
-------
>0<
HO-^ ^-CH = CH-CH2OH
h3co*
II
HO-/ \cH = CHCOO*
H3CO
•1
HO-f \ch2 = CH-COO* ~ HO ~C VCH = CHCOO"
O
HO
IV
III
CH = CHCOO"
VI
COO"
VII
VIII
Figure 3
IX
Fig. 5, Proposed pathway for the degradation of ferulic acid and coniferyl
alcohol by a methanogenic consortium. Compounds are: (I) ferulic
acid, (II) coniferyl alcohol, (III) caffeic acid, (IV) p-hydroxy-
cinnamic acid, (V) cinnamic acid, (VI) phenylpropionic acid, (VII)
benzoic acid, (VIII) phenylacetic acid, (IX) cyclohexanone.
16
-------
pathway for vanillic acid that Involves sequential elimination of substituents
to generate benzoate, which could then be anaerobically degraded by the reduc-
tive pathway for aromatic metabolism. Taylor found that vanillic acid supported
the growth of Pseudomonas sp. strain PN-1 when nitrate was used as the electron
acceptor. Protocatechuate, m-hydroxybenzoate, p-hydroxybenzoate, and benzoate
were immediately metabolized by such cultures, which suggested that they were
intermediates in the degradation of vanillic acid.
Some hydroxybenzene and methoxybenzene derivatives may be anaerobically
degraded by alternative methods. Kaiser and Hanselmann (1982) prepared an
enriched bacterial consortium that degraded syringic acid to carbon dioxide and
methane. The authors proposed the degradative pathway shown in Fig. 6. Gallic
acid and pyrogallol were rapidly degraded by active cultures and 3,4-dihydroxy-
5—methoxybenzoic acid was identified in well—grown cultures. The authors pos-
tulated that syringic acid was demethylated and decarboxylated to pyrogallol,
which was then reduced by a mechanism analogous to benzoate degradation (Evans
1977). No evidence was presented for the reductive pathway. Strains of Strep-
tococcus bovis and Coprococcus isolated from bovine rumen that were shown (Tsai
and Jones 1975, Patel et al. 1981) to anaerobically degrade phloroglucinol to
acetate were unable to metabolize benzoate. Schink and Pfennig (1982) isolated
five Pelobacter acidigallici strains capable of metabolizing a variety of
trihydroxybenzenes to acetate. These strains, which were isolated from both
marine and freshwater sediments, produced acetate stoichiometrically from
gallic acid, pyrogallol, 2,4,6-trihydroxybenzoate and phloroglucinol. They
also converted syringic acid to acetate in co—culture with flcetobacterium
woodii. None of the strains wereable to metabolize benzoic acid or cyclo-
hexanecarboxylic acid. Whittle et al. (1976) reported that Rhodopseudomonas
gelatinosa Is able to grow anaerobically in the light with phloroglucinol a®.—
the sole source of carbon. Cell—free extracts of the organism were able fc©'
reduce phloroglucinol to dihydrophloroglucinol in the dark. The cell-free
systems contained cysteine and NADPH(H ) as reductants. Reactions beyond this
point were unclear; however, mass spectral data (Dutton and Evans 1978) did
indicate the intermediate formation of 2—oxo—4—hydroxyadipate. These results
are summarized in the tentative pathway shown in Fig. 7. Apparently reduction
with subsequent ring cleavage can occur for some hydroxylated and methoxylated
lignoaromatic compounds.
Halogenated aromat^-g, compounds
Reductive dehalogenation appears to be the general mechanism for initiating
the degradation of a number of chloro—, bromo— and iodo—substituted aromatic
compounds in anaerobic environments. Ide et al. (1972) and Murthy et al. (1979)
observed that, in flooded soils, pentachlorophenol was converted to a number of
lesser substituted phenolic products. Reductive dechlorination of a tetra-
chlorophthalamic moiety was shown by Kirkpatrick et al. (1981) to be the initial
step in the degradation of Techlofthalam [N-(2,3-dlchlorophenyl)-3,4,5,6-tetra-
chlorothalamic aci4]„in flooded rice paddy soil stored in laboratory flasks.
Boyd et ,al. (1983) examined the degradation of ortho-, meta-, anj jpara—chloro—
phenol in dilute anaerobic sewage sludge. Only p-chlorophenol was not signifi-
cantly degraded during an 8-week incubation period; o—chlorophenol was metabol-
ized to phenol, which indicated that reductive dechlorination was the initial
17
-------
• C0°H H20 CH3OH COOH H?0 CH 0H C00H
CH3O
OCH,
1 —I !1
CH3O HO'^^OH
0H OH
II
CH20M
6H
J \
HO-^Ss
HO^Sc^COOH -CHaCOOH
hoh2c -
COOH
VIII
ch3cooh
Fig. 6. Proposed pathway for the degradation of syringic acid by a methanogenic
consortium. Compounds are: (I) syringic acid, (II) 5-methoxy-3,4-di-
hydroxybenzoic acid, (III) gallic acid, (*V) pyrogallol, (VI) 1,2,3-
trihydroxycyclohexane, (VI) 2,3-dihydroxc^clohexanone, (VII) 5,6-di-
hydroxyhexanoic acid, (VIII) 6-hydroxy-2-oxohexanoic acid.
1«
-------
OH
0
2H
HO
OH
HO
OH
h2o
0
II
HO>
HO-^ ^OH
III
HOOC
HOOC
OH
IV
Fig. 7. Proposed pathway for the degradation of phloroglucinol. Compounds are:
(I) phloroglucinol, (II) dihydropliloroglucinol, (III) 2,3,5-trihydroxy--
cyclcohexanone, (IV) 2-oxo-4£|iydroxyadipic acid.
19
-------
degradation step. The anaerobic degradation of various chlorophenols was
studied in groundwater aquifer microcosms (Suflita and Miller 1985). Micro-
organisms in actively methanogenic aquifer material initiated the degradation
of chlorinated phenols by reductive dechlorination. Complete removal of the
chlorine moieties occurred before the phenol Intermediate could be mineralized
to methane and carbon dioxide. Methanogenic activities were apparently required
for the loss of chlorine because the chlorinated phenols persisted in anoxic
microcosms containing material from the same aquifer bat from a nonmethanogenic
site. The reductive dehalogenation of chloro-, bromo- and iodo-benzoates was
demonstrated in methane-producing fresh water lake sediment (Horowitz et al.
1983). Dehalogenation occurred after a lag period which lasted from 1 week to
more than 6 months, depending on the chemical.
f
Horowitz et al. (1983) have suggested that acclimation of sediments to
dehalogenation and mineralization of the aromatic ring may be independent
processes. They reached t^iis conclusion from three lines of evidence. Firstly,
acclimation was observed regardless of whether the parent substrate was eventu-
ally mineralized to carbon; dioxide and methane. Secondly, the pattern of dehal-
ogenation of consortia acclimated to different halogenated benzoates was unaf-
fected by prior acclimatiop of the sediment to benzoate. Lastly, acclimation
to dehalogenation was not poncurrent with acclimation to benzoate mineraliza-
tion. This last piece of evidence also tends to support a degradative mechanism
(Fig 8). where dehalogenation is required before a substrate can be degraded to
carbon dioxide and methane.
t
Such a pathway may not be restricted to a single oUsq of.^oingQundiu Boyd
and She 1 ton ~i 1-904) observed the sequential dehalogenation diohiprirt-*
ated phenols, and an anaerobic consortium was shown tQ 4#$-tricblorcH
phenoxy }aoetio acid to (2,3-dichloro|>Jtert<}xy ) jlcfdfeici:
In much earlier studies, pentachlorophenol was shown to be degraded (ifde *fc A1
1972, Murthy et al. 1979) in anaerobic soil to isomeric mixtures of partially
dehalogenated phenolic intermediates, and Kijkpatrick"et al. (1901) showed that
the bactericide N-<2,3-dichlorophenyl)-3,4,5;6,-tetrachlorophthalamic acid was
metabolized to two and possibly more monodechlorinated products. Apparently
for halogenated benzoates and other compounds, dehalogenation and mineralization
of the benzoate ring are, likely, independent processes and dehalogenation
occurs before degradation of the rest of the compound.
Not all chlorinated compounds are dechlorinated as the initial step in
anaerobic degradation. Chloroguaiacols and chloraveratroles have recently been
reported (Remberger et al. 1986) to be de-O-methylated to the-corresponding
chlorocatechols. The f£te of the chlorocatecols was not resolved.
Halogenated aliphatic compounds
The initial step in the degradation of halogenated aliphatic compounds is
reductive dehalogenation. Unlike halogenated aromatic compounds, however,
halogenated aliphatics can lose one or two halides during the reductive process.
The mechanism for the loss of two halogens involves the removal of two vicinal
halogens with the formation of a double bond (Kray and Castro 1964, Be land et
al. 1976). The reductive loss of a single halide is the same for both aromatic
20
-------
COOH
COOH
COOH
Figure 3
Fig. 8. Proposed pathway for the degradation of 3,5-dichlorobenzoic acid by a
methanogenl8c~fconsortiura. Compounds ares (I) 3,5-dichlorobenzoic acid,
(II) 3-chlorobenzoic acid, (III) benzoic acid.
21
-------
and aliphatic compounds, involving the replacement of the halogen with a hydrogen
atom (Essac and Matsumura 1980).
Vicinal dehalogenation of several potentially hazardous compounds has been
reported. Castro and Belser (1968) found that flooded soil cultures stoichiomet—
rically converted ethylene dibromide (1,2—dibromoethane) to ethylene. They
also were able to demonstrate the conversion of 2,3-dibromobutane to 2,3-butene.
Examination of the products of the debromination of the d, 1— and me so—isomers
indicated trans elimination of the two bromide ions. The vicinal dechlorination
lindane to 3,4,5,6—tetrachlorocyclohexene has been demonstrated in sewage
sludg5% of
added lindane), 2,3,4,5,6-pentachlorocyclohexane (2%) and trace amounts of
1«2,4—trichlorobenzene and 1,2,3,5— and/or 1,2,4,5—tetrachlgrobenzene.
Reductive dehalogenation by replacement of a single halogen with a hydrogen
atom also has a prominent role in the degradation of halogenated aliphatic
compounds. For example, ! DDT [1,1,1-trichloro-2,2-bis (parachlorophenol) ethane}
is converted to DDD [ 1,1 r-dichloro—2,2-bis (parachlorophenol) ethane] in flooded
soil or in moist soil incubated anaerobically (Spencer 1967, Hill and McCarty
1967, Guenzi and Beard 1968, Ko and Lockwood 1960, Kearney et al. 1969, Castro
a°d Yashida 1971). Chloroform has been observed, as an intermediate in the
degradation of carbon tetrachloride under nitrifying conditions (Bouwer and
McCarty 1983) and in anaerobic subsurface aquifer microcosms (Parsons et al.
1985, Parsons and Lage 1985). Parsons and Lage (1985) also reported that
11-trichloroethane was converted to 1,1—dichloroethane. Data reported from
a number of laboratories indicates that tetrachloroethylene is sequentially
deahlorinated under anaerobic conditions by a series of one chlorine extractions
to yield vinyl chloride, which is then degraded to CO2 (Fig. 9). Bouwer and
McCarty (1983) showed that, in methanogenic bacterial cultures, the initial step
in the transformations of tetrachloroethylene and 1,1,2,2-tetrachloroethane to
nonchlorinated end products appeared to be reductive dechlorination to trichlor-
oethylene and 1,1,2-trichloroethane. In later studies. Parsons et al. (1984)
reported that depletion of tetrachloroethylene and appearance of cis— and
trans-1,2-dichloroethylene and vinyl chloride were observed following incubation
of tetrachloroethylene in microcosms containing material from an aquifer recharge
basin. Kleopfer et al. (1985) observed the reductive dechlorination of
22
-------
CU xCI
c = c
cT ^ci
^ H
^-Cl"
II
cr
ci
H
ct
H
Cf
H
c = c
CI
IV
111
cr
cr
H
H
C = C VII
H C\
VI
i
i
Fig. 9. Proposed pathway for the degradation of tetrachloroethylene by anaerobic
bacterial consortia. Compounds are: (I) tetrachloroethylene, (II) tri-
chloroethylene, (III) 1,1-dichloroethylene, (IV) trans-dlchloroethylfin»t
(V) cis-dichloroethvlene. (VI) vinyl-chloride; (VII) chloroethane.
23
-------
trichloroethylene to 1,2-dichloroethylene in anaerobic soil samples. No 1,1-
dichloroethylene was observed. The degradation of els- and trans-dichloroethy-
lene was examined in anaerobic subsurface aquifer materials (Barrio-Lage et al.
1986). Vinyl chloride was produced in all amended microcosms and was not
observed in sterile or unamended controls. Chloroethane was produced only in
microcosms amended with the cis isomer. Vogel and McCarty (1985) found that
24% of the tetrachloroethylene was mineralized in a continuous-flow fixed-film
methanogenic column study. Trichloroethylene was the major intermediate, but
traces of dichloroethylene isomers and vinyl chloride also were found. Under
a different 6et of methanogenic conditions, nearly quantitative conversion of
tetrachloroethylene to vinyl chloride was observed.
Efficient evidence exists to suggestj that some anaerobic dehalogenations
are not strictly biological, but are catalyzed by free iron, other metal ions
or iron porphyrins in reducing environments. Glass (1972) postulated that
reduction of DDT to DDD in soils is mediated by, the iron (Fe+2/Fe+3) couple.
Hie reduction of DDT in an aqueous system Jcontaining hematin and sodium dithion-
ate has been demonstrated by Miskus et al.! (1965), and Zoro et al. (1974) has
extended this work to the degradation of DDT by iron porphyrins. Zoro et al.
(1974) also observed that, when dithionate; was added to sterilized sewage
sludge or yeast suspensions, dehalogenatioh activity was either restored or
enhanced. These authors have suggested that under anaerobic conditions in the
environment, cell-free reduced iron porphyrins (released from decaying organ-
isms) are responsible for the reductive dechlorination of DDT. Presumably,
the active organisms participated in tl\e degradation process by maintaining a
strongly reducing environment.and providing a source of iron porphyrins.
Nonspecific reactions are not limited to DDT. Subsequent studies by
Holmstead (1976) and Khalifa et al. (1976) have demonstrated the reductive
dehalogenation of toxaphene and mirex by iron porphyrins. More recently,
Klecka and Gonsior (1984) observed that iron (II) porphyrins;reduced carbon
tetrachloride, chloroform, and 1,1,1-trichloroethane to lower' chlorinated
homologs. Solutions of chromium salts have been reported (Kfay and Castro
1964) to catalize dehalogenation of vicinal halides to olefins. The membrane
fraction of cell-free extracts of Clostridium sphenoides catalyzed the vicinal
dechlorination of lindane to 3,4,5,6—tetrachloro—1—cyclohexepe if reduced
glutathione was added to the reaction mixture (Heritage and MacRae 1977b).
Bouwer et al. (1981) found that brominated trihalomethanes were degraded
rapidly in both sterilized and active mixed methanogenic cultures.
Aromatic and aliphatic hydrocarbons
Although a limited number of reports describing the degradation of
hydrocarbons have appeared in the literature, these pollutants are generally
believed to be resistant to degradation! in the absence of oxygen. Unfortun-
ately many of the early reports suffered from inadequate anaerobic techniques,
poorly defined culture conditions, mixed cultures of aerobic and anaerobic
bacteria, and substrates potentially contaminated with readily degradable
compounds (Novell! and Zobell 1944, Rosenfeld 1947, Muller 1957, Davis and
Yarbrough 1966). Within the last year, however, two definitive studies have
24
-------
appeared describing the degradation of both aliphatic (Schink 1985) and
aromatic (Grbic-Galic and Vogel 1966) hydrocarbons.
Schink (1965) investigated the degradation of hydrocarbons in anaerobic
enrichments using mineral media inoculated with sewage sludge or sediment
samples of limnic or marine origin. Complete degradation of 1-hexadecene was
observed; squalene was only partially degraded to carbon dioxide and methane.
Methanogenic enrichments were maintained on either squalene or 1-hexadecene.
No indication of methanogenic degradation was obtained with either n-hexane,
ji-hexadecane, n-heptadecane, 1-hexene, cis-2-hexene, trans-2-hexene, isoprene,
1-hexine, benzene, toluene, xylene, cyclohexene, cycloheptatriene, cyclopenta-
diene, styrene, naphthalene, azulene, or p-carotene. Schink concluded that,
under his assay conditions, a terminal double bond can be sufficient to allow
methanogenic degradation of hydrocarbons, whereas branching and terminal ring
closures may significantly contribute to hydrocarbon stability in anoxic sedi-
ments. Although no degradation of linear saturated hydrocarbons was observed
in this study, Giger et al. (1980) observed the degradation of heptadecane in
anoxic lake sediment samples and Hambrick III et al. (1980) observed the
degradation of octadecane in anaerobic sediment—water suspensions.
Only recently has there been an indication that aromatic hydrocarbons can
be degraded under anaerobic conditions. Two reports (Batterman and Werner
1984, Kuhn et al.^1985) have appeared decribing the degradation of aromatic
compounds under denitrifying conditions. Batterman and Werner (1984) were able
to effect anaerobic degradation of—toluene, benzene, and xylenes, tn a contamin-
ated aquiffer by injecting nitrate. Kuhn et al. (1985) observed' anaerobic
degradation of xylenes under denitrifying conditions in the field and in labora--
tory aquifer columns simulating a river water/groundwater infiltration system.
«nd m-xylene isomer* toer6 degraded at similar rates and significantly
faster than the o—isomer, in an extensive laboratory study using methanogenic
cultures acclimated to the degradation of ferulic acid, Grbic-Galic and Vogel
(1986) demonstrated that benzene, toluene and o-xylene were first oxidized to
phenolic and carboxylic acid intermediates before the compounds could be
degraded by standard ring reduction pathways (Evans 1977). Using 180 labelled
water, Vogel and Grbic—Galio (1986) demonstrated that oxygen incorporated into
the ring came from water and not methanol (used to add substrates) or molecular
oxygen that may have contaminated the sample.
Nitrogen containing compounds
Nitrogen containing organic compounds can be anaerobically transformed by
a number of reaction mechanisms that include nitro reduction, N-dealkylation,
azo reduction and the reduction of nitrogen heterocycles with subsequent ring
cleavage.
The primary route for anaerobic degradation of nitro substituted xenobiotic
compounds is reduction of the nitro-group to the corresponding amino substituent.
Soleim and Schelini (1972) reported that hydroxyamino substituents nay.be inter-
mediates in the reduction process. They have identified ja-hydoxyaminobenzoate
and £-aminobenzoate in cultures of anaerobic strains of Clostridia, Bacteriodes.
and other anaerobes metabolizing £-nitrobenzoate. McCormick et al. (1976)-
25
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observed that the reduction of several nitro aromatic compounds followed the
mechanism proposed by Yamashina et al. (1954) for the enzymatic reduction of
aromatic nitro, nitroso, and hydroxylamine compounds (Fig. 10). For compounds
that have multiple nitro-substituents, sequential reduction of the nitro groups
occurs. For example, for trifluralin (a,a,a-trifluoro-2,6-dinitro-N,N dipropyl-
£-toluidine) reduction of one of the nitro groups is followed by reduction of
the other nitro group and/or dealkylation of one or both propyl groups (Probst
et al. 1967, Golab et al. 1969, Williams and Fiel 1971, Parr and Smith 1973).
Benifin (N-butyl-N-ethyl-a,a,a-trifluoro-2,6-dinitro-|>-toluidine) is also
degraded in a similar manner (Golab et al. 1970). Hie nitro groups are sequen-
tially reduced to amino groups and one or both of the alkyl groups may be lost.
Substituent specificity also was demonstrated for 2,4,6-trinitrotoluene and
2,4-dinitrotoluene where the 4-nitro group is reduced before the 2-or 6 nitro
groups (McCormick et al. 1976). Little is known of the fate of the amino
intermediates in the degradation of these compounds. In many cases they appear
to be persistent (Williams 1977); however, Braun and Gibson (1984) and Shelton
and Tiedje (1984a) have demonstrated that aminobenzoates can be mineralized
under denitrifying and methanogenic conditions.
h2 h2 h2
R-N02 -R-NO -R-NH0H "•R-NH2
Figure 10. Mechanism for the reduction of nitro compounds.
N-dealkylation by anaerobic ruminal and flooded soil microorganisms has
been reported for trifluralin (Williams and Feil-1971, Parr and Smith 1973) and
for benefin incubated in ruminal solutions IGolab et al, 1970)< Several of the
trifluralin' and benefin metabolites were characterized as compounds lacking one
or both of the N-alkyl groups, and it was postulated that N-dealkylation was a
result of anaerobic microorganisms.
The initial reaction (Fig. 11) in the degradation of azo compounds by
i. anaerobic microorganisms is the four, electron reduction of the azo linkage to
yield two primary amines (Wuhrmann et al. 1980),
(R1-N=N-R2) + 4e~ + 4H+ - Rj-Nl^ + R2~NH2
Figure 11. Mechanism for the reduction of azo compounds.
Although previous studies had indicated that azo compound reduction occurred on
the outside of anaerbbic microorganisms (Dubin and Wright 1975), more recent
results indicate that reduction is an activity internal to the microorganisms
(Wuhrmann et al. 1980). Wuhrmann et al. (1980) found that a series .of azo dyes
having different permeability properties were degraded at similar rates by cell
extracts and were degraded at markedly dissimilar rates by whole cells. These
authors proposed that their results were consistent with the mechanism advanced
26
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by Gingell and WalJcer (1971) which proposed an intracellular, nonenzymatic
reduction of azo compounds by reduced flavin nucleotides.
The number of nitrogen heterocycles known to be degraded anaerobically
include indole (Wang et al. 1984), nicotinic acid (Harary 1957, Stadtman et al.
1972, Itnhoff and Andressen 1979, Imhoff-Stuck le and Pfennig 1983), and alkyl
pyridines (Rogers et al. 1985). Wang et al. (1984) demonstrated that indole
was anaerobically degraded to methane, carbon dioxide, and ammonium ion. Based
on the amount of indole added and the stoichiometric .relationship in Fig. 12,
c8H7N + 8h2° »3.5C02 + 4.5CH4 + NH4+ + OH~
Figure 12. Stoichiometry of anaerobic indole degradation.
they were able to recover 84.4% of the methane that would bejproduced theoret-
ically. Harary (1957) was the first to demonstrate anaerobic degradation of a
nitrogen heterocycle. He showed that nicotinic acid was fermented to acetate,
propionate, ammonia, and carbon dioxide. Subsequently, Tsaijet al. (1966)
isolated and charaterized the metabolic intermediates shown in Fig. 13. "The
aerobic and anaerobic degradation of a mixture of alkyl pyridines in a ground-
water sample were investigated by Rogers et al. (1985). A marked difference
was observed between the aerobic and anaerobic degradation rates. Under aerobic
conditions, the residual alkylpyridine concentrations generally approached zero
concentration within 10 to 31 days; whereas under anaerobic conditions the
concentrations of residual alkylpyridines decreased between 40 and 80% over 33
days. Biodegradation rates under aerobic conditions were greatly affected by
the specific ring substitution of structural isomers. A similar effect was not
observed for anaerobic degradation rates.
Ether -jtitnTced compounds
Methanogenic enrichments capable of degrading a series of polyethylene
glycols and ethylene glycol have been isolated from sewage sludge (Dwyer and
Tiedje 1983). The enrichments were shown to best degrade glycols close to the'
molecular weight on which they were enriched. Ethanol, acetate, methane, and
ethylene glycol were detected as products. The sequence of product formation
suggested that the ethylene oxide unit IHO-(CH2-CH2-0-)xH was dismutated to
acetate and ethanol; ethanol was subsequently oxidized to acetate by a
syntrophic association that produced methane.
ENVIRONMENTAL INTERACTIONS
Although the degradation of many hazardous organic chemicals has been
conclusively established in laboratory studies, extrapolation of this work to
the environment is only beginning to be explored. There is a general lack of
information on the concentration and turnover rate of hazardous organic chemi-
cals in anaerobic environments, and, unfortunately, the effect of environmental
parameters on degradative processes also has received little attention.
27
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QCOOH
COOH
COOH
III
/
i /
^^YCOoh Xnh3
HOOC CH^
IV i
ch3
yCH2
~
HC-q
/ \
HOOC COOH
CH3
V3
c =
/
/
,ch3
HOOC COOH
VI
Fig. 13. Proposed pathway for the degradation of nicotinic acid by Clostridium
barkeri sp, n. Compounds are; (I) nicotinic acid, (II) 6-oxonicotinic
acid, (III) 1,4,5,6-tetrahydro-6-oxonicotinic acid, (IV) 2-methylene-
glutaric acid, (VI) methylitaconic acid, (VI) dimethylmaleic acid..
28
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The limited information available is scattered. For example. Raj a ram and
Sethunathan (1975) observed that organic carbon sources accelerated nitro group
reduction, of parathion in flooded soils. Degradation rates followed the order
glucose > rice straw > algal crust > farmyard manure > unamended. Capone et al.
(1983) examined the effects of several metals on microbial methane, carbon
dioxide and sulfide production in sediments from Spartina alternifora communities.
Comparisons were made between control and experimental assays with respect to
initial rates of production (after acclimation) and overall production. Methane
evolution was inhibited by CI^HgCl, ligS, and NaAs02* A period of initial inhi-
bition was followed by a period of overall stimulation with the chlorides of Hg#
Pb, Mi, Cd and Cu, and with Z11SO4, K^rO^j, and ^0^07. Production of CO 2 was
generally less affected by the addition of metals. Inhibition was noted with
NaAs02, CHjHgCl, and Na2Mo04, and minor stimulation of CO2 production occurred
over the long term with chlorides of Hg, Pb, and Fe. Sulfate reduction was
inhibited in the short term by all metals tested and over the long term by all
but FeCl2 and MiCl2« Several studies have indicated that in aerobic sediments
that both oxygen- and nitrate-dependent aromatic metabolism can occur. Fabig et
al. (1980) found that, in the presence of trace amounts of oxygen, benzoate
degradation and nitrate reduction occurred concurrently in either a Pseudomonas
aeruginosa or an undefined mixed culture; they were unable to demonstrate
nitrate-dependent benzoate degradation under strictly anaerobic conditions.
Sleat (1981) demonstrated that the growth of nitrate-reducing facultative
anaerobes was increased by the presence of nitrate when they were grown on
p-hydroxybenzoate under trace oxygen concentrations. Jones (1979) has suggested
that microzones within particles were responsible for nitrate reductase activity
in aerobic freshwater sediments, and Focht and Verstraete (1977) considered such
anoxic microzones as the sites of denitrification in aerobic sediments. -
In examining the anaerobic degradation ofTSe'nzoate in undiluted sediments#
Sleat ai\d Robinson (1983) observed that the addition 10 mM sulfate did not < ...
inhibit the rate of ^ ^C-labelled gas production but did alter the ratio of
methane to carbon dioxide. In the presence of sulfate, only carbon dioxide was
produced and in its absence a mixture of methane and carbon dioxide indicative
of methanogenic activity was observed. These results are consistent with results
of several other investigators (Cappenberg 1974, MacGregor and Keeney 1973,
Winfrey and Zeikus 1977) who have shewn that sulfate inhibits methanogenesis in
freshwater sediments. Greater affinities for H2 or acetate (Abram and Nedweli
1978a,b; Bryant et al. 1977; Mclnerney et al. 1979; Hard and Olson 1980; Winfrey
and Zeikus 1977), the minimum threshold for hydrogen metabolism in methanogenic
bacteria (Lovley et al. 1982; Lovley 1985) and bioenergetic reasons (Zehnder
1978; Lovley et al. 1982) have beeh invoiced to explain the*competitive advan-
tage of sulfidogens over methanogens. Whatever the true mechanism, ^sulfate-
reducing bacteria appear to assume the role of methanogenic bacteria in sulfate-
containing sediments by using methanogenic precursors. Hie fact that gas
production from benzoate was not affected by sulfate in Sleat and Robinson's
work would suggest 'that ring cleavage or the production of methanogenic
precursors was the rate limiting step in the presence and absence of sulfate.
METHODS OF INVESTIGATION
Current methods used to investigate the anaerobic degradation and
trans formation-of potentially hazardous chemicals in the environment range
29
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from the classical Hungate (1969) techniques, using pure bacterial cultures or
suspensions of sewage sludge, sediment, or aquifer materials, to more modern
techniques that attempt to use sediment and aquifer materials in an undisturbed
state.
Classical anaerobic techniques have been the workhorse of countless
scientific investigations since the original decription of the "Hungate"
technique in 1950 (Hungate 1950). Since that time the technique has undergone
numerous modifications and improvements (Bryant 1972, Miller and Wolin 1974,
Balch and Wolfe 1976). This method uses glass anaerobic culture tubes, serum
bottlesj or various glass containers fitted with serum bottle necks that are
tightlyj sealed with rubber stoppers and a crimped metal seal. Neoprene, butyl,
or synthetic, but not gum, rubber stoppers are suitable. If an anaerobic cham-
ber is available the addition of anaerobic sewage sludge, sediment or aquifer
materiajls, hazardous chemicals and other necessary solutions are added in the
chamber and the glass container is sealed before removal. If a chamber is not
available, additions are made to glass containers that are continually gassed
with O2 free gases. The stoppers are inserted as the bottles are withdrawn
from tt|e gassing needles.
Anaerobic techniques of this nature combined with analytical organic
procedures have been used to investigate the fate of hazardous organic chemi-
cals in the environment. Most studies have followed classical microbiological
approaches and have used pure cultures or acclimated or fresh sediment, sewage
sludge, or aquifer material in suspension to investigate the mechanisms of
anaerobic transformations and anaerobic degradative pathways (e.g. Bakker 1977,
Boyd and Shelton 1984, Healy and Young 1979), to identify the microorganisms "
active in the degradative process (Huser et al. 1982, Frazer and Young 1985,
Harwood and Gibson 1986),. and to provide evidence for kinetic mechanisms mathe-
matically describing the jpersistance of these compounds (Suflita et al. 1983>.
*
Shelton and Tiedje (|1984a), for example, have proposed a simple, general
method to test whether an; organic chemical was susceptible to anaerobic degra-
dation to CH4 and 2. The method uses digested sewage sludge diluted to 10%
and incubated anaerobically in 160-ml serum bottles with 50 pg of C per ml
of test chemical. Biodegradation was determined after 8 weeks by the net
increase in gas pressure in bottles with test chemicals over the pressure in
nonamended sludge bottles. Gas pressure could be measured by a pressure trans-
ducer. Alternatively the increase in CH4 or CO2 could be measured in amended
sample by GC. Shelton and Tiedje cautioned that this general screening method
only measures degradation potential. In a particular anaerobic environment,
the' potential may not be expressed if degrading organisms are absent or if
toxic chemicals or other environmental factors limit the organisms activity.
Degradation potential should not be construed as the actual rate that a
compound is degraded if released to the environment.
For many studies a long lag period, where a loss in compound cannot be
detected, precedes degradation of the compound. This time interval has been
called an "adaptation" period (Claik and Fina 1952), a "time lag" (Balba and
Evans 1977) and an "acclimation lag" (Healy and Young 1979). Sleat and Robinson
30
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Robinson (1984) have suggested that research into the anaerobic degradation of
ardous organic chemicals has been retarded by the belief that anaerobic degra-
dation, particularly the nethanogenic fermentation, requires a long lag period
before detectable loss of the study compound is observed.
The importance of the acclimation period may be overstated. In earlier
studies, Sleat and Robinson (1983) showed that the length of the acclimation
periods are the result of sample preparation. Increasing the sediment to water
ratio markedly reduced the acclimation period. When benzoate was added to a
10* suspension of sediment, methanogenesis was observed after acclimation
periods ranging from 50 to 80 days. In undiluted sediments amended with
[ring-U-C14] benzoate, 14C-labelled gas production was detected after as little
as 4 hours of anaerobic incubation. They also found that the jtemperature optimum
for benzoate mineralization dropped from 37°C to 28°C when undiluted sediments
were used instead of diluted sediments. The difference in temperature optima
apparently resulted from the a change in the rate limiting step in the conver-
sion of benzoate to methane and carbon dioxide. In undiluted samples ring
cleavage or the formation of nethanogenic precursors is the rate limiting
process whereas in diluted samples methanogenesis is the rate 'limiting step.
Sleat and Robinson (1984) suggested that undiluted samples mayj be more approp-
riate for examining environmental effects on anaerobic degradation processes.
Other investigators have also advocated the use of undiluted sediment or
undisturbed sedimeiif^ores in examining the anaerobic degradation or transfor-
mation of organic and inorganic compounds. Jorgensen (197fi) found that disturb-
ing sediment cores by mixing resulted in a small loss of activity whereas
dilution of the sediment caused a marked decrease in the rate of sulfate reduc-
tion calculated per unit volume of undiluted sediment. Undiluted mixed sediment
had two—fold lower activity sediment cores^ A 1»10 dilution of sediment resulted
in a 25-fold decrease in activity when compared to sediment cores. In a second
study Jorgenson (1978) found no difference in sulfate reduction for sediments
that were directly injected with labelled sulfate; that were pooled in a vessel,
carefully mixed, cored again, and then injected; or that were pooled, carefully
mixed with labelled sulfate, and cored again. When repeated at 14"C, the mixed
incubations were somewhat lower than the injected samples, although the differ-
ence was significantly less than the two-fold difference found in the initial
experiment. Jones and Simon (1984) observed similar results when they examined
the mineralization of acetate if*-emall sediment cores. They cautioned, however
that the effects of sorption and diffusion within the sediment cores should be
closely examined.
CONCLUSIONS
The anaerobic degradation of potentially hazardous chemicals released to
the environment has received increased attention over the last 10 to 20 years.
This increased interest has led to significant advances in identifying the
principal microorganisms involved in the anaerobic degradation process,
unravelling the complex degradative pathways used by these organisms, and
demonstrating the potential for anaerobic degradation of a large number of
compounds. As these studies have developed, a number of key processes have
been recognized. Several common degradative pathways appear to be shared by"
31
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all bacteria and bacterial communities capable of anaerobic degradative
activity. Ring reduction precedes ring cleavage in the degradation of aromatic
compounds. Substitutents of aromatic compounds are generally removed to produce
benzoate or phenol before ring reduction and ring cleavage occurs. In most
substrates, including sewage sludge, sediments, and aquifer material, degrada-
tion results from the activity of microbial consortia having methanogenic and
sulfidogenic organisms acting as the final electron acceptors of the community.
Die removal of ring substituents, ring reduction and ring cleavage is generally
performed by fermentative members of the consortia.
Despite these advances there is a lack ofjinformation on the turnover
rates of hazardous organic chemicals in anaerobic environments. The lack of
attention apparently results from the difficul|ties in working with bacteria and
bacterial communities under strict anaerobic conditions, the long adaptation
periods before degradation is observed for many compounds and the statistical
problems inherent in working with undisturbed sediments (Jones and Simon 1984).
Work in this area may, however, be facilitated by the increasing number of
aerobic studies directed toward prediction of the persistence and concentration
of toxic chemicals in natural environments. A! number of kinetic models have
been proposed to characterize degradation in natural environments (Paris et al.
1981, Vashon and Schwab 1982, Lewis et al. 1984) and in pure cultures of micro-
organisms (Simkins and Alexander 1984, Robi(nson 1985). Many of these models
are based on the assumption that substrate disappearance can be modeled with
information only on substrate concentration and population density together
urith parameters of classical Monod kinetics (Simkins and Alexander -1984).
Recently, models were-described for the kinetics of biodegradation of organic
pompounds of bacteria growing on an alternative carbon source (Schmidt et al.
1985). Statistical means have been proposed £6r evaluating the fits of these
nodels to data obtained from laboratory studies.
32
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Table 1. Comprehensive List of Compounds Microbially Degraded or Transformed under Anaerobic Conditions.
Compound
acetyl salicyclic acid
acrylic acid
aUcyl pyridines
2-aminobenzoate
4-aminobenzoate
4-amino-3-chlorobenzoate
4-amino-3-5-
dichjLorobenzoic acid
ar.oranth
m-anisic acid
Reference
Shelton £ Tiedje, 84a
Shelton £ Tiedje, 84a
Rogers et al., 85
Shelton & Tiedje, 84a
Braun £ Gibson, 84
Shelton £ Tiedje, 84a
Suflita et al., ,82
Suflita et al. , 83
Suflita et al., 82
Dubin £ Wright, 75
Taylor, 83
Shelton £ Tiedje, 84a
Microorganism(s)
dilute sewage sludge
. _dilute sewage sludge
subsurface waters-£-soils
dilute sewage sludge
Pseudomonas sp.
dilute sewage sludge
dilute sewage sludge
dilute sediment
dilute sediment
3-ehlorobenzoate
enrich, meth. cult,
dilute sewage sludge
dilute sediment
Proteus vulgaris
Pseudomonas sp. Strain PN-1
dilute sewage sludge
Degradation/Transformation
deg. to CH4 £ CC>2
deg. to CH4 £ CO2
transformation*
deg. to CH4 £ CO2
mineralization13
deg. to CH4 £ CO2
dechlorination
dechlorination
dechlorination
reduction
demethylation
deg. to CH4 £ CO2
aIn the context of this table transformation indicates that the investigators monitored the disappearance
of the compound and no indication was given to indicate partial or complete degradation of the compound.
^Mineralization is used to indicate that the investigators followed formation of CO,. The
compound was
completely degraded with a constant fraction converted to CO2 and the remainder converted to biomass.
-------
Compound
anisol
benefin
benzidine-based azo dyes
benzene
benzo(j&)pyrene
benzoate
Reference
Bache & Pfennig, 81
Golab et al., 70
Manning et al., 85
Grbic-Galic £ Vogel,
Delaune et al., 81
Harvood & Gibson, 86
Fina £ Fiskin, 60
Proctor £ Scher, 60
Guyer £ Hegeman, 69
Dutton £ Evans, 69
Dutton £ Evans, 69
Nottingham £
Hungate, 69
Williams £ Evans, 75
Ferry £ Wolfe, 76
Balba £ Evans, 77
Evans, 77
BaXker, 77
Fina et al., 78
Keith et al., 78
Shlomei et al., 78
Healy £ Young, 79
Balba £ Evans, 80
Mountfort £ Bryant, 82
Microorganism(s)
Degradation/Transformation
Acetobacterium voodii
flooded soil
dilute human feces
i ferulic acid enriched
methanogenic consortium
dilute sediment
Rhodopseudomonas palustris
bovine rumen fluid
dilute sewage sludge
Rhodopseudomonas sp
Rhodopseudomonas palustris
Rhodops eudomonas palustris
Rhodopseudomonas palustris
enriched methanogenic cultures
Moraxella sp.
benzoate enriched
methanogenic culture
enriched methanogenic culture
Rhodopseudomonas palustris
Pseudomonas strain (NPI)
enriched methanogenic cultures
enriched nitrifying cultures
enriched methanogenic culture
benzoate enriched
methanogenic cultures
benzoate enriched
methanogenic cultures
enriched meth. cultures
mixed culturest P. aeruginosa £
_D. vulgaris
eocultures of an anaerobic
bacterium with H2 utilizing
organism
demethylation
transformation
azo reduction
deg. to CH4 £ C02
deg. to CH4 £ C02
mineralization
deg. to CH4 £ CO2
mineralization
mineralization
mineralization
transformation
deg. to CH4 £ CO2
mineralization
deg. to CH4 £ CO2
deg. to CH4 £ C02
deg. to CH4 £ C02
transformation
deg.'to CH4 £ C02
transformation
transformation
deg. to CH4 and C02
deg. to CH4 £ C02
-------
Compound
Reference
benzoate (cont)
ben2yl alcohol
brominated
trihalonethanes
bromodichloro methane
bromoforn
2-brooobenzoate
3 -br omobe nz oa t e
4-bromobenzoate
2,3-butanediol
tutylbenzyl phthalate
caffeic acid
carbon tetrachloride
Sleat £ Robinson, 84
Shelton £ Tiedje, 84a
Harwood & Gibson, 66
Tarvin & Buswell, 34
. <
Sheltlon & Tiedje, 84a
Bouwer et al., 81
Bouwer £ McCarty, 83b
Bouwer fi McCarty, 83b
i
Bouwer £ McCarty, 83b
Suflita et al. , 82
Horowitz et al., 83
Suflita et al., 82
Suflita et al. , 82
Shelton £ Tiedje, 84a
Shelton £ Tiedje, 84a
Perez-Silva et al., 66
Bouwer £ McCarty, 83a
Bouwer £ McCarty, 83b
Microorganism(s)
freshwater sediment
dilute sewage sludge
Rhodopseudomonas palustrls
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
enriched nitrifying culture
Degradation/Transformation
deg. to CH4 £ C$2
deg. to CH4 £ CO2
mineralization
deg. to CH4 £ CO2
deg. to CH4 £ CO2
debromination
transformation
enriched nitrifying culture
enriched nitrifying culture
dilute sewage slydge
dilute sediment
dilute freshwater sediments
dilute sewage sludge
dilute sediment
dilute sewage sludge
dilute sediment
dTlute s ewage' s ludge
dilute sewage sludge
rat feces
enriched methanogenic culture
enriched nitrifying culture
transformation
transformation
deg. to CH4 £ CO2
debromination
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
dehydroxylation
transformation
transformation
-------
Compound
catechol
4-chloroacetanilide
3-chlorobenzoic acid
Chloroethene
Chloroform
2-chloropheno1
3-chlorophenol
4-chlorophenol
4-chlorophthalic acid
cholesterol
cinnamic acid
coniferyl alcohol
Reference
Healy £ young, 78
Shelton & Tied.j.e,_84a.-
Suflita et al. , 83
Suflita et al., 82
Shelton £ Tiedje, 84b
Parsons et al., 85
Bouwer £ McCarty, 83b
Boyd et al., 83 4
Shelton £ Tiedje, *84
Suflita £ Miller, 85
Boyd et al., 83
Suflita et al., 82
Suflita £ Killer, 85
Suflita £ Killer, 85
Taylor £ Ribbons, 83
Taylor et al., 81
Healy £ Young, 79
Tarvin £ Buswell, 34
Grbic-Galic, 83
Klcroorganism(s)
dilute sewage sludge
dilute sewage sludge
dilute sediment
3-chlorobenzoate enriched
roethanogenic community
dilute sewage sludge
dilute sediment
dilute sewage sludge
groundwater materials
enriched nitrifying culture
Degradation/Transformation
deg. to CH4 £ CO2
deg. to CH4 £ CO2
dechlorination
dechlorination
deg. to CH4 £ CC>2
transformation
transformation
dilute sewage sludge
deg. to CH4
£
CO 2
dilute sewage sludge
deg. to CH4
£
co2
aquifer materials
deg. to CH4
£
co2
dilute sewage sludge
deg. to CH4
£
CO2
dilute sewage sludge
deg. to CH4
£
co2
dilute sediment
aquifer materials
deg. to CH4
£
co2
groundwater materials
deg. to CH4
£
co2
a marine mixed culture
decarboxylation
$
Pjfeeudomonas sp. strain PN-1
demethylation
(...
dilute sewage sludge
deg. to CH4
£
CO 2
del'lute sewage sludge
deg. to CH4
£
C02
ferulic acid enriched
deg. to CH4
£
co2
enriched methanogenic culture
-------
Compound Reference
m-cresol
Bakker,
77
Boyd et
al., 83
Fedorak
£ Hrudey,
84
Shelton
£ Tiedje,
84a
£-cresol
Bakker,
77
Fedorak
£ Hrudey,
84
£-creBol
Bakker,
77
Boyd et
al., 83
Shelton
£ Tiedje,
84 a
DDT
Miskus et al. , 65
Glass, 72
Sethunathan, 73
Zoro et
Al., 74
diazinon
Sethunatha-ii7 3—
2, 3-dibromobutane
Castro £
Belser, 68
dibrooochloromethane
Bouwer £
KeCarty,
83b
1,2-dibromo-3-chloropropane Castro £ Belser, 68
di-ja-butylphthalate Benckiser & Ottow, 8 2
Shelton & Tiedje, 84a
2,5-dichlorobenzoate Suflita et al., 82
i
3,4-dichlorobenzoate Suflita et al., 82
3,5-dichlorobenzoate
Horowitz et al. , 83
Suflita et al., 82
Microorganism(s)
Degradation/Transformation
enriched nitrifying culture
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
enriched nitrifying culture
^ilute sewage sludge
enriched nitrifying-culture
dilute sewage sludge
dilute sewage sludge
rumen fluid
flooded soil
flooded soil
dilute sewage sludge
flooded soil
flooded soil
enriched methanogenic culture
flooded soil
Pseudomonas pseudoalcaligenes
dilute sewage sludge
transformation
deg. to CH4 £ C02
deg. to CH4 s CO2
deg. to CH4 £ CO2
transformation
deg. to CH4 £ CO2
trans formation
deg. to CH4 £ CO2
deg. to CH4 £ CO2
dechlorination
dechlorination
transformation
dechlorination
transformation
debrocd nation
transformation
dehalogenation
transformation
transformation
dilute sewage sludge dechlorination
dilute sediment
dilute sewage sludge dechlorination
dilute sediment
dilute freshwater sediment
dilute sewage sludge
dilute sediment
dechlorination
deg. to CH4 £ C02
-------
6
Compound
1,1-dichloroethene
cis-dichloroethene
trans-di chloroe thene
2.3-dichlorophenol
I
2.4-dichlorophenol
2.5-di chloropheno1
2.6-dichlorophenol
diethylphthalate
dihydrodiranillin
3-hydroxybenzoate
3,4-dihydroxybenzoic acid
2,4-dihydroxyphthalic acid
3, 5-dimethoxybenzoic acid
2,6-dimethylphenol
3,4-dimethylphenol
Reference
Barrio-Lage et al., 86
Barrio-Lage et al., 86
Parsons et al., 85
Parsons et al., 84
Barrio-Lage et al., 86
Parsons et al., 85
Parsons et al., 84
Boyd £ Shelton, 84
Boyd £ Shelton, 84
Suflita £ Miller, 85
Boyd £ Shelton, 84
Suflita £ Miller, 85
Boyd £ Shelton, 84
Shelton £ Tiedje, 84a
Chen et al., 85
Taylor et al., 70
Bakker, 77
Taylor £ Ribbons, 83
Taylor, 83
Bache £ Pfennig, 81
Fedorak £ Hrudey, 84
Fedorak' £ Hrudey, 84 -
Microorganism(s)
dilute sediment
' dilute sediment
groundwater materials
aquifer material
dilute sediment
groundwater materials
aquifer material
fresh £ acclimated sludge
fresh £ acclimated sludge
aquifer materials
fresh £ acclimated sludge
aquifer materials
fresh £ acclimated sludge
dilute sewage sludge
runen fluid
Pseudomonas PN-1
enriched nitrifying culture
a marine mixed culture
Pseudomonas sp. Strain PN-1
Acetobacterium strains
dilute sewage sludge
dilute sewage sludge
Degradation/Transformation
loss of substrate
loss of substrate
transformation
dechlorination
transformation
transformation
dechlorination
transformation
transformation
deg. to CH4 £ C02
transformation
deg. to CH4 £ CO2
transformation
deg. to CH4 £ C02
transformation
mineralization
transformation
decarboxylation
demethylation
demethylation
deg. to CH4 s C02
-deg.. to CH4 £ CO2
-------
Compound Reference Microorganism(s) Degradation/Transformation
3,5-dimethylphenol
FedoraX & Ha^adey* 84
dilute sewage sludge
deg. to CH4 £ CO2
dimethylphthalate
Shelton & TlMje, 84a
-dllnt* sewage sludge
deg. to CH4 £ CO2
di-n-nonylphthalate
o'Grady et al*, 85
activated sludge
tr ans f 0 rma ti 0 n
t
ethylacetate
Shelton & Tittff 84a
dilute sewage sludge
deg. to CH4 £ CO2
ethylene dibromide"
Castro £ Bftlser, 68
flooded soil
debromination
Bouwer £ McCarty, 85
enriched methanogenic culture
debromination
ethylene glycol
Dwyer & Tij&dje, 83
enriched methanogenic culture
deg. to CH4 £ CO2
ferulic acid
Taylor, 83
Pseudomonas sp. Strain PN-1
demethylation
Bache & Pfennig, 81
Acetobacterium strains
demethylation
Healy et al., 80
mixed anaerobic cultures
deg. to CH4 £ CO2
Healy £ Young, 79
acclimated anaerobic nixed
deg. to CH4 £ CO2
culture
Grbic-Galic, 85
facultative anaerobic bacterium
transformation
Grbic-Galic £ Young, 85 methanogenic consortium
deg. to CH4 £ CO2
Frazer £ Young, 85
Gram-negative anaerobic
demethylation
i
bacterium
2-f luor obe nr oa te
Schenr.en et al., 85
benzoate-degrading denitrifying
mineralization
bacteria
3-fluotophthalic acids
Taylor £ Ribbons, 83
a marine mixed culture
decarboxylation
gallic acid
Kaiser £ Hanselmann, 82.
_enriched_anaerobic culture
deg. to CH4 £ CO2
Schink £ Pfennig, 82
Pelobacter acidigallici
transformation
geraniol
Shelton £ Tiedje, 84a
dilute sewage sludge
deg. to CH4 £ CO2
guaiacol
Kaiser £ Hanselmann, 82
enriched anaerobic culture
deg. to CH4 £ CO2
Taylor, 83
Pseudomonas sp. strain PN-1
demethylation
hexadecene
Schink, 85
dilute sediment £ sludge
deg. to CH4 £ C02
-------
Compound
4-hydroxyacetanilide
hydrocinnamic
2-hydroxybenzoic acid
3-hydroxybenzoic acid
4-hydroxybenzoic acid
4-hydroxybenzyl alcohol
3-hydroxybutanone
4-hydroxyphthalic acid
indole
2-iodobenzoate
3-iodobenzoic acid
4-iodobenzoate
Reference
Shelton £ Tiedje, 84a
Tarvin £ Buswell, 34
Shelton £ Tiedje, 84a
Bakker, 77
Dutton £ Evans, 69
Bakker, 77
Healy £ Young, 79
Shelton £ Tiedje, 84a
Shelton £ Tiedje, 84a
Shelton £ Tiedje, 84.a
Tiylor £ Ribbons, £3
Kang et al., 84
Suflita et al., 82
Horowitz et al., 83 '
Suflita et al., 82
Suflita et al., 82
lignin
Benner £ Hodson, 85
Microorganism(s)
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
enriched nitrifying culture
Rhodopseudomonas palustris
enriched nitrifying culture
acclimated anaerobic mixed
culture
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
a marine mixed culture
enriched nethanogenic cultures
dilute sewage sludge
dilute sediment
dilute freshwater sediment
dilute sewage sludge
dilute sediment
dilute sewage sludge
dilute sediment
thermophilic anaerobic
enrichment cultures
Degradation/Transformation
deg. to CH4 £ CO2
deg. to CH4 £ COj
deg. to CH4 £ CO2
transformation
mineralization
transformation
deg. to CH4 £ CO2
deg. to CH4 £ C02
deg. to CH4 £ CO2
deg. to CH4 £ CO2
decarboxylation
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deiodination
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
-------
Compound
lindane
3-methoxycinnamic acid
2-methoxyphenol
3-nethoxypheno1
4-ne thoxypheno1
3-nethylbutanol
3-o-nethylgallic acid
methyl parathion
nicotinic acid
Reference
Sethunathan, 73
Beland et al., 76
Yule et al., 67
MacRae et al. , 69
Jagnow et al., 77
Heritage £ MacRae,
77a, b
Tu, 76
Mathur £ Saha, 75
Raghu £ MacRae, 66
Bache £ Pfennig, 81
Boyd et al., 83
Shelton £ Tiedje, 84
Boyd et al., 83
Shelton & Tiedje, 84>
Boyd et al., 83
Shelton £ Tiedje, 84
Shelton £ Tiedje, 84
Taylor, 83
Ou, 85
Harary, 75
Imhoff-Stuckle
£ Pfennig, 83
Stadtman et al., 72
nitrilotriacetic acid
Ward, 85
Microorganlsm(s)
Degradation/Transformation
flooded soil
dilute sewage sludge
flooded soil
Clostridium sp.
anaerobic bacteria
Clostridium splenoides
artaer obi e-ba ete r i a
flooded soil
flooded 8oil
Acetobacterium strains
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
dilute sewage sludge
Pseudomonas sp. Strain PN-1
flooded soil
anaerobic bacterium
Desulfococcus niacini
transformation
dechlorination
dechlorination
dechlorination
dechlorination
dechlorination
dechlorination
dechlorination
dechlorination
demthoxylation
deg. to CH4 £ CC>2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
demethoxylation
mineralization
fermentation
mineralization
Clostridium barkeri
mi ne r a liz a ti on
subsurface soils
)
mineralization
-------
Compound
Reference
2-nitrophenol
3-nitrophenol
I
4-nitrophenol
octadecane
1-octanol
2-octanol
orange 22
oxalate
Ponceau 3R
Ponceau SX
parathion
pentachlorophenol
phenol
Boyd et al., 83
She1ton £ Tiedje, 84a
Boyd et kl. , 83
Boyd et al. , 83
Shelton £ Had j e, 84a
Hambrick,III, et al.,
80
Shelton £ Tiedje, 84a
Shelton £ Tiedje, 84a
Dubin £ Wright, 75
Smith £ Orecland, 83
Dubin £ Wright, 75
Dubin £ Wright, 75
Rajaram £
Sethunathan, 75
Sethunathan, 75
Guthrie et al., 84
Bakker, 77
Evans, 77
Healy £ Young, 78
Healy £ Young, 79
Boyd et al., 83
Fedorak £ Hrudey, 84
Microorganism(s)
Degradation/Transformation
dilute
sewage
sludge
dilate
sewage
sludge
dilute
s ewage
sludge
dilute
sewage
sludge
dilute
s ewage
sludge
dilute
sediments
dilute sewage sludge
dilute sewage sludge
Proteus vulgaris
dilute aquatic sediments
Proteus vulgaris
Proteus vulgaris
flooded soil
flooded soil
anaerobic sewage sludge
enriched nitrifying culture
Rhodopseudomonas palustris
Pseudomonas strain NP-1
methanogenic enriched cultures
dilute sewage sludge
acclimated anaerobic mixed
cultures
dilute sewage sludge
dilute sewage sludge
deg.
to
CH4
£
C02
deg.
to
ch4
£
CO
deg.
to
ch4
£
CO 2
deg.
to
ch4
£
co2
deg.
to
ch4
£
co2
deg.
to
CH4
£
co2
deg.
to
ch4
£
co2
deg.
to
ch4
£
co2
reduction
deg. to CO2
reduction
reduction
transformation
transformation
transformation
transformation
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
-------
Compound
(phenol, cont)
phenol acetic acid
Reference
Shelton £ Tiedje, 84a
Barik et al., 85
Suflita fi Miller, 85
Barik et al., £5
phenolic mixtures
1-phenylazo-2-naphthal
0-phenylpropionate
Godsy et al., 83
Wuhrman et al., 80
Balba fi Evans, 79
Fhloroglucinol
Tsai s Jones, 75
Evans, 77
m-phthalic acid
o-phthalic acid
polyethylene .glycol
Dutton fi Evans, 78
Patel et al., 81
Schink fi Pfennig, 82
Walker fi Taylor, 83
Shelton fi Tiedje, 84a
Affring et al., 81
Affring et al., 81
Taylor fi Robbins, 83
Shelton fi Tiedje, 84a
l
Dwyer fi Tiedje, 83
Shelton fi Tiedje, 84a
propionaldehyde
Shelton fi Tiedje, 84a
Microorganism(s)
dilute sewage sludge
anaerohic bacterium plus
Wolinella succinogenes
groundwater materials
anaerobic bacterium plus
Wolinella succinogenes
sewage sludge
Bacillus cereus
benzoate enriched methanogenic
culture
Streptococcus bovis
fehodopseudomonas palustris
Fseudomonas strain NF-1
enriched methanogenic cultures
Rhodopseudomonas palustris
Caprococcus sp. Pe, 5
Pelobacter acidigallici
Fusarium solani
dilute sewage sludge
dentrifying mixed cultures
denitrifying mixed culture
a marine mixed culture
dilute sewage sludge
ejitiche_d_.me.thanogenic culture
dilute sewage sludge
Degradation/Transformation
deg. to CH4
fi
co2
deg. to CH4
fi
co2
deg. to CH4
fi
co2
deg. to CH4
fi
0
0
to
deg. to CH4 fi C02
transformation
deg. to CH4 fi C02
mineralization
deg. to CH4 fi CO2
mineralization
mineralization
fermentation
mineralization
deg. to CH4 fi CO2
mineralization
mineralization
decarboxylation
deg. to CH4 fi CO2
deg. to CH4 fi CO2
deg. to CH4 fi CO2
dilute sewage sludge
deg. to CH4 fi C02
-------
Compound
protocatechuic acid
pyrogallol
sinapic acia
Sunset Yellcw
squaline
syringic acid
Reference
Dutton & Evans, 69
Healy £ Young, 79
Shelton £ Tiedje, 84a
Kaiser £ HanselmaTmy "82
Sihink £ Pfennig, 82
Shelton £ Tiedje, 84a
Bache £ Pfennig, 82
Dubin £ Wright, 75
Schink, 85
Healy £ Young, 79
Bache £ Pfennig, 81
Kaiser £ Hanselmann, 82
Taylor, 83
Frazer £ Young, 85
Microorgani sm(s)
Degradation/Transformation
Rhodopseudomonas palustris
enriched methanogenic culture
dilute sewage sludge
enriched anaerobic culture
pQlohacter acldlgalllci
dilute sewage sludge
Acebacterium woodii
Proteus vulgaris
dilute sediment £ sludge
enriched methanogenic culture
Acetobacterlum woodii
dilute freshwater sediments
Pseudomonas sp. Strain PN-1
gram-neg, anaerobic rods
mineralization
deg. to CH4 £ CO2
deg. to CH4 £ CO2
deg. to CH4 £ CO2
fermentation
deg. to CH4 £ CO2
demethylation
reduction
deg. to CH4 £ CO2
deg. to CH4 £ CO2
demethylation
deg. to CH4 £ CO2
demethylation
demethylation
syringol
syringaldehyde
tartazine
tetracliloroquaiacol
1,1,2,2-tetrachloroethane
tetrachloroet^ylene
Kaiser £ Hanselmann, 82 enriched anaerobic consortium deg. to CH4 £ CO2
Kealy £ Young, 79
Bache £ Pfennig, 8.1
Dubin £ Wright, 75
Remberger et al., 86
t
Bouwe-'r £ McCarty, 8 3a
Bouwer et al., 81
Bouwer £ McCarty, 83a
Parsons et al., 84
Parsons et al., 85
Vogel £ McCarty, 85
enriched methanogenic culture
Acetobacterium woodii
Proteus vulgaris
sediment slurries
enriched methanogenic culture
dilute sewage sludge
enriched methanogenic culture
groundwater environment
groundwater materials
enriched methanogenic culture
deg. to CH4 6 CO2
demethylation
reduction
demethylation
transformation
dechlorination
transformation
dechlorination
trans forma ti on
transformation
-------
Compound
toluene
tetraehloroveratrol
te trachlcfroguai aco 1
2,3,6-trichlorobenzoa te
3,4,5-trichlorocatechol
1,1,1-trichloroethane
trichloroethylene
3,4,5-trichloroguaiacol
2,4,5-trichlorophen-
oxyacetic acid
3,4,5-trichloroveratrol
I
trifluralin
Reference.
Grbic-Gallc £ Vogel, 86
Remberger et al., 86
Remberger et al., 86
Horowitz et al., 83
Suflita et al., 82
Microorganism(s)
ferulic acid enriched
methanoge'nic culture
sediment slurries
sediment slurries
dilute freshwater sediment
dilute sewage sludge
dilute sediment
Degradation/Transformation
deg. to CH4 £ C02
demethylation
demethylation
dechlorination
deg. to CH4 £ CO2
deg. to CH4 £ CO2
Remberger et al., 86 sediment slurries
Bouwer £ McCarty, 83a
Parsons et al., 85
Larsons £ Lage, 85
Parsons £ Lage, 85
Bouwer et al., 81
Parsons et al., 84
Parsons et al., 85
Kleopfer, 85
Remberger et al., 86
enriched nethanogenic culture
groundwater materials
groundwater material
aquifer materials
dilute sewage sludge
groundwater environment
groundwater materials
flooded soil
dilute sediment
demethylation
transformation
transformation
transformation
dechlorination
dechlorination
dechlorination
transformation
transformation
demethylation
Suflita et al. , 84
enriched nethanogenic culture dechlorination
Remberger et al., 86
dilute sediment
demethylation
Parr £ Smith, 73
Williams £ Feil, 71
Golab et al., 69
flooded soil
Rumen bacteria
flooded soil
transformation
transformation
transformation
2,4,-6-trihydroxybenzoate
Schink £ Pfennig, 82
Pelobacter acldigalllcl
fermentation
-------
Compound
3,4,5-trisiethoxybenzoate
I
3.4.5-trim«thoxyci nnami c
acid
2.4.6-trinitrotoluene
vanillate
vanillin
ve ratiate
m-xylene
o-xylene
£-xylene
Reference
I
t
Bache £ Pfennig, 81
Kaiser & Hanselm^nn,
Taylor, 83
Bache & Pfennig, 81
Taylor, 83
Carpenter et al., 78
Golab et al., 69
Healy £ Young, 79
Bache £ Pfennig, 81
Kaiser £ Hanselrann,
Taylor, 83
Prazer £ Young, 85
Grazer £ Young, 86
Bache £ Pfennig, 81
Taylor, 83
Mlcroorganism(s)
Acetobacterium voodii
82 dilute freshwater sediments
Pseudomonas sp. Strain PN-1
Acetobacterium voodii
Pseudomonas sp. Strain PN-1
Activated sludge system
flooded soil
Degradation/Transformation
demethylation
deg. to CH4 & CO2
demethylation
demethylation
demethylation
transformation
transformation
enriched methanogenic culture
Acetohacterium woodii
82 enriched anaerobic consortium
Pseudomonas sp. Strain PN-1
grao-neg anaerobic rods
anaerobic acetogen
Acetobacterium woodii
Pseudomonas sp. Strain PN-1
deg. to CH4 £ CO2
demethylation
deg. to CH4 £ CO2
demethylation
denethylation
deg. to COj £ acetate
demethylation
demethylation
Kuhn et al., 85
Grbic-Galic & Vogel, 86
Kuhn et al., 85
aquifer material nitrifying
conditions
ferulic acid enriched
methanogenic culture
aquifer material-nitrifying
conditions
transformation
deg. to CH4 & CO2
transformation
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