FEMS MicrobiologyEcology38 (1986) 161-169
Published by Elsevier
161
FEC 00068
Anaerobic production and transformation of aromatic
hydrocarbons and substituted phenols by ferulic acid-degrading
BESA-inhibited methanogenic consortia
(Anaerobic pathways; fermentation; toluene; p-cresol; 2-ethylphenol)
Dunja Grbi6-Gali6
Department of Civil Engineering, Stanford Universi(v, Stanford, CA 94305. U.S.A.
Received 5 February 1986
Accepted after revision 16 April 1986
1. SUMMARY
Sewage sludge-derived methanogenic enrichments degrading ferulic acid as sole carbon and
energy source were partially inhibited with 2bromoethanesulfonic acid. The various intermediates and products formed under inhibition of
methanogenesis were studied using gas chromatography/mass spectrometry ( G C / M S ) . In addition to aromatic, alicyclic, and aliphatic acids
previously shown to be intermediates of ferulate
degradation to C02 and CH4, the following compounds were detected: toluene, ethylbenzene, phenol, p-cresol, 2-ethylphenol, catechol, and 3-hydroxy-4-ethylphenol. The character and the sequence of appearance of the compounds indicate
that fermentative bacteria which initiate the
anaerobic transformation of ferulic acid, in case of
disruption of interspecies hydrogen transfer, dispose of electrons by converting part of the substrate to reduced derivatives. Aromatic hydrocarbons are further partially oxidized through hydroxylation of the ring (and, to a lesser extent, the
side-chain), and partially reduced to saturated
alicyclic rings. Some of these compounds seem to
be gradually degraded to branched or straightchain aliphatic acids. Some compounds, like
catechol and ethylphenol, accumulate transiently
or persistently in high concentrations (up to 16
mM carbon out of the initial concentration of 30
mM substrate carbon), indicating that hydroxylation of the aromatic ring might be an important
metabolic reaction in these systems.
2. I N T R O D U C T I O N
It has been shown that lignin-derived monoaromatic alcohols and acids can be completely
degraded under methanogenic conditions [1-5].
The populations capable of such transformations
are complex communities consisting of strictly
anaerobic [6] and facultatively anaerobic fermentative bacteria [7,8], methanogens [5], and possibly
also hydrogen-producing acetogens as an intermediary link between the two groups. Fermentative bacteria initiate the degradation process and
are capable of carrying out the fermentation to a
certain degree even when methanogenesis is inhibited. In that case, the fermentation pattern
changes and more reduced products are formed
instead of molecular hydrogen. It has been reported that ferulic acid-degrading methanogenic
consortia affected by 2-bromoethanesulfonic acid
0168-6496/86/$03.50 ~ 1986 Federation of European MicrobiologicalSocieties
162
(BESA) which inhibits methanogenesis excrete reduced ring compounds like toluene, benzene,
cyclohexane and methylcyclohexane into the culture fluid [5]. The compounds appear transiently
and are subsequently transformed to unknown
products. The details of this phenomenon, however, have not been investigated.
The present study, based on detailed G C / M S
analyses of the culture fluid from inhibited
methanogenic consortia, demonstrates the capability of ferulic acid-transforming mixed fermentative cultures to produce various unusual compounds including aromatic hydrocarbons, cyclohexane derivatives, and substituted phenols. Most
of these compounds are also slowly transformed,
ultimately to branched and straight-chain aliphatic
acids and CO 2. Tentative pathways for production
and transformation of aromatic hydrocarbons,
based on the appearance and disappearance of
various intermediary products with time, are proposed.
3. M A T E R I A L S A N D M E T H O D S
The ferulic acid-degrading consortia used for
these experiments were enriched from anaerobic
sewage sludge [5] and maintained with this
aromatic acid as the sole carbon and energy source
for 5 yr. A serum bottle variation of the Hungate
technique [9] was used for strictly anaerobic
cultivation. The anaerobic culture procedures,
batch culture monitoring techniques, and anaerobic media composition have been described previously [10]. The cultures were fed 3 m M ferulic acid
(30 mM total carbon) monthly. 6 mth before the
onset of these experiments, 1 m M of BESA was
added to inhibit methanogenesis. After 5 mth, the
cultures were fed ferulic acid once more, and
substrate concentration in inhibited cultures was
monitored with UV-spectrophotometry while gas
production was measured with a glass syringe and
analyzed with a gas partitioner [2]. The initial
substrate disappeared completely in one month,
but only an average of 4.5 mmol gas (CO 2 exclusively) was produced from 30 mmol substrate
carbon. After this time period, 10 ml out of 3
parallel cultures were each transferred to 100 ml
fresh media, fed ferulic acid, and amended anew
with 1 m M BESA. These transferred cultures were
incubated 60 days at 35°C in the dark, and the
transformation products were followed with
GC/MS.
A Finnigan M A T 4000/4500 gas chromatog r a p h / m a s s spectrometer with an INCOS data
system was used for G C / M S analysis. Samples of
culture fluid were extracted with diethyl ether
under acidic conditions. 1 /~1 of the extract was
injected splitlessly (for 30 s) onto a 60-m DB-5
fused silica capillary column (0.32 m m internal
diameter, 1.0 ~ m film thickness). Helium was used
as carrier gas. The initial column temperature was
60°C, and was increased to 250°C at a rate of
4 ° C / m i n . The injector temperature was 250°C,
and the ionizer temperature was 140°C. The forepressure of the column was adjusted to 0.75 bar.
The scanning rate was approx. 1 s/scan, with an
ionization voltage of 1500 V. Identification of
unknowns was based on comparison with a library
of known spectra. An internal standard procedure
with 2-fluorophenol was used for quantitation.
The detection limit for aromatic and alicyclic
compounds was 1/~M.
4. RESULTS
The G C / M S procedure employed permitted
detection and identification of aliphatic acids,
aromatic hydrocarbons, substituted phenols,
alicyclic rings, aromatic alcohols, and aromatic
acids no more complex and with a boiling point
no higher than that of p-hydroxyphenylpropionic
acid. The compounds detected and their concentration on four chosen sampling days are listed
in Table 1. It is apparent that, in addition to
aromatic acids which are the intermediates in the
degradation pathway for ferulic acid [5], there are
two new major metabolites: 2-ethylphenol, and
catechol. It is also evident that in the early stages
of incubation, the majority of organic carbon (27.6
mmol) is missing. This means that carbon must be
stored in the substrate and more complex degradation intermediates, from caffeic to cinnamic
acid. These compounds were not detectable with
this procedure, but had been detected previously
163
Toluene, ~ - - C H 3
--0--
p-Cresol, H O - K ~ C H
3
--.Z~.-- Ethylbenzene,~ C H z C H 3
0.6
t
0.5
o
E
E
/
/
,,o,
\
\
\
I
\
I
0.4
t
o
i
\
t
~
~
0.5
t
/
0.2
i
#
o.I
I
I
'~
~
/
I
0
I
0
I0
__
20
50
40
50
60
Time (day)
Fig. 1. Time course of toluene, ethylbenzene, and p-cresol formation and degradation in BESA-inhibited ferulic acid-degrading
methanogenic consortia. Note that the product concentrations are in micromolar range.
Phenylpropionic ocld, ~CHzCHzCOOH
IO.O
Phenylacetic ood, <~--CHzCOOH
8.0
~
---0'~"- 2-Ethylphenol,<~CH2CH3
OH
~
P~'\
4.0
/.,.:;,-",,
.o
\
,,
o ~ ~ ~
O
-°'"
lO
,
20
,
50
Time (day)
,
40
.%
",, ,""
50
"W,
60
Fig. 2. Time course of 2-ethylphenol, phenylacetic acid, and phenylpropionic acid formation. The concentrations are in millimolar
range.
164
Table 1
C o m p o u n d s detected with G C / M S in the culture fluid of BESA-inhibited, ferulic acid-fed methanogenic consortia
In the control cultures fed no substrate, neither aromatic nor aliphatic intermediates were detected. In ferulate-fed uninhibited
cultures (no BESA), no aromatic hydrocarbons, phenols, ethylcyclohexane, benzyl alcohol, or cyclohexanepropionic acid were
detected.
Compound
Concentration (mM carbon) "
Day 13
Day 32
Day 47
Day 55
HO-~CH2CH2COOH
p-Hydroxyphenylpropionic acid b
~
0.06
0.4
0.0
0.0
0.002
3.7
0.2
1.6
0.8
6.8
8.5
1.0
0.3
16.5
0.7
16.0
CH2CH2COOH
Phenylpropionic acid
(~CH2COOH
Phenylacetic acid
--COOH
Benzoic acid
~
CH2CN2COOH
Cyclohexanepropionic acid b
0.0
0.004
0.02
0.0
0.08
0.6
0.0
0.0
Ethylbenzene b
0.02
0.04
0.003
0.0
Toluene
0.05
0.1
0.0
0.0
Ethylcyclohexane b
0.01
0.0
0.0
0.0
HO-~CH2CH3
OH
3-Hydroxy-4- ethylphenol b
0.0
0.2
0.0
0.0
0.4
2.4
5.4
7.3
0.005
0.6
0.02
0.2
~)--COOH
Cyclohexanecarboxylic acid
~
CH2CH3
<~CH3
~
~
CH2CH3
CH2CH3
OH
2-Ethylphenol b
HO--~CH3
p-Cresol b
165
Table 1 (continued)
Compound
Concentration (mM carbon) a
Day 13
Day 32
Day 47
Day 55
OH
Catechol b
0.6
2.0
16.0
5.0
0.1
0.8
0.4
0.8
0.005
0.0
0.0
0.0
0.0
0.03
0.0
0.01
0.0
0.0
0.02
0.0
0.0
0.2
0.01
0.002
0.0
0.05
0.1
0.0
0.0
0.004
0.1
0.0
Formic acid
0.0
0.0
0.0
0.2
Total carbon
2.4
34.4
31.4
32.1
@-o.
Phenol b
Q
CH2ON
Benzyl alcohol b
CH3 (CH2}4COOH
Hexanoic acid
CH3(CH2)2 COOH
Butanoic acid
CH3CH2CHCOOH
CH3
2-Methylbutanoic acid
CH3CH2COOH
Propionic acid
CH3COOH
Acetic acid
HCOOH
" The results are the means of 3 parallel cultures.
b Compounds found for the first time in ferulic acid-fed BESA-inhibited cultures (not previously detected by HPLC analyses in
similar cultures [51).
with high pressure liquid chromatography (HPLC)
in similar cultures [2,5]. In later stages of incubation, complex aromatic acids are converted mostly
to simpler intermediates (phenylpropionic, phenylacetic, benzoic acids) and substituted phenols
which temporarily accumulate and then slowly
disappear, or remain as dead-end metabolites (see
Figs. 1-3). The most complex phenol found was
3-hydroxy-4-ethylphenol, but it appeared only
transiently (see Table 1). Other compounds were
found in much lower concentrations. Shorter
aliphatic acids typically increased late during incubation.
Formation and disappearance with time of some
of the more interesting products are shown in
Figs. 1-3. The results presented are the average of
data obtained with three replicate cultures, p-
Cresol, catechol, phenylacetic acid, and phenylpropionic acid accumulated temporarily and were
slowly degraded after 30-40 days of incubation.
Benzoic acid and 2-ethylphenol persisted even on
day 60; benzoic acid, which had been lowered
considerably on day 47, rose rapidly to reach the
concentration of 12 mM carbon on day 50 of
incubation. After day 50, the pattern of products
changed again; phenylpropionic acid, phenol, pcresol, and catechol showed a secondary increase
while phenylacetic acid continued to decrease, and
benzoic acid continued to rise.
5. DISCUSSION
The described methanogenic consortia were enriched from sewage sludge, but some of the
166
20 .,--0,--,-Benzoicacid,(~COOH
---o-- PhenoI,He@
/
[ -.,,-. oo,echo,.
E
/ ,
_
~../.il ~'/"
~ 211
o
0
I0
20
_.o
-
-
50
Time (day)
-
-
40
50
60
Fig. 3. Time course of phenol, catechol, and benzoic acid formation. Note that catechol and benzoic acid concentrations are in
moderately high millimolar range.
fermentative ferulate degraders from the consortia
[7] are ubiquitous organisms which can be found
in soil, water, sediments, and subsurface. In that
light, these findings gain in importance.
If deprived of a hydrogen scavenging mechanism (in this case, methanogens), fermentative
bacteria switch to products different from the
usual transformation pattern. Toluene and benzene were previously found in the culture fluid of
similar consortia [5], but this is the first time
microbial formation of ethylbenzene, p-cresol, and
2-ethylphenol is reported, except for the microbial
production of p-cresol from tyrosine in mammalian intestinal systems [11]. Toluene and ethylbenzene, which could theoretically be derived
through decarboxylation of phenylacetic and
phenylpropionic acids (Table 2), were found in
low concentrations (up to 105 #M carbon from
30 000 #M starting carbon in the substrate); therefore it is possible that they result from side-reactions which are not significant for the system as a
whole. However, our recent results (Grbie-Gali6
and Vogel, submitted) show that subcultures derived from these cultures have a remarkable capa-
bility to rapidly transform relatively high concentrations (15 mM) of aromatic hydrocarbons
(benzene, toluene) as sole carbon and energy
sources, to hydroxylated intermediates (phenol,
p-cresol, benzyl alcohol). Anaerobic degradation
of aromatic hydrocarbons (benzene, toluene,
xylenes) under denitrifying, not methanogenic,
conditions has already been reported [12,13]. Recently it has been found, by using asO-labeled
water, that the hydroxy-group from water is incorporated into toluene and benzene to yield phenols
under strictly anaerobic methanogenic conditions
(Vogel and Grbie-Gali6, submitted). For these reasons, it is suggested that toluene and ethylbenzene
are possibly also transformed to p-cresol and 2ethylphenol in these cultures (Fig. 4). This
suggestion is supported by the sequential appearance of toluene/p-cresol and ethylbenzene/
2-ethylphenol, which is presented in Figs. 1 and 2.
It is further supported by the ortho-position of the
hydroxy-group relative to the ethyl-group on 2ethylphenol, which could not be derived as a
product from any of the m- or p-hydroxylated
aromatic acids present in these cultures [2,5]. 2-
167
Table 2
S o m e of the r e a c t i o n s s u g g e s t e d to o c c u r in the B E S A - i n h i b i t e d
m e t h a n o g e n i c c o n s o r t i a d e g r a d i n g ferulic acid, a n d the c o r r e s p o n d i n g free e n e r g y c h a n g e s
N o t e t h a t the free e n e r g y c h a n g e s are c a l c u l a t e d p e r m o l e o f
o r g a n i c r e a c t a n t s , a n d t h a t t h e y o n l y s h o w if a given r e a c t i o n is
endergonic or exergonic. Actual concentrations of reactants
a n d p r o d u c t s a n d c o n s e q u e n t l y the s i g n i f i c a n c e of i n d i v i d u a l
r e a c t i o n s are p r e s e n t l y u n k n o w n .
Reaction
F r e e e n e r g y c h a n g e at
physiological conditions,
AG6 (k J / r e a c t i o n ) a
P h e n y l p r o p i o n i c a c i d + 3 H 2 ---'
Cyclohexanepropionic acid
- 103.5
P h e n y l p r o p i o n i c acid
E t h y l b e n z e n e + CO:~
- 79.4
P h e n y l a c e t i c a c i d ---, T o l u e n e
+CO 2
-69.6
B e n z o i c a c i d ---, Benzene + C O 2
- 51.7
B e n z o i c acid + H zO '~" P h e n o l
+CO2+H 2
+16.6
E t h y l b e n z e n e + 3 H 2 -~
Ethylcyclohexane
- 82.8
E t h y l b e n z e n e + H 2 0 ---,
2-Ethylphenol + H 2
+ 70.6
Toluene + 3H 2
Methylcyclohexane
- 103.1
Toluene + H20
p-Cresol + H 2
+ 70.6
T o l u e n e + H 2 0 ~ Benzyl
alcohol + H 2
+ 102.1
Benzene+ 3H 2 ~ Cyclohexane
- 101.7
Benzene + H 20 ~ Phenol + H 2
+ 73.3
2 - E t h y l p h e n o l + H 2 0 ,--,
3-Hydroxy-4-ethylphenol + H 2
+ 70.6
p - C r e s o l + 3 H 2 --*
4-Methylcyclohexanol
- 71.6
Benzyl a l c o h o l
Benzaldehyde + H 2
+ 49.5
B e n z a l d e h y d e + H zO -~
Benzoic acid + H 2
+ 4.2
P h e n o l + H 2 0 ,--, C a t e c h o l
+H 2
+73.3
F u r t h e r r e a c t i o n s (alicyclic r i n g s --* a l i p h a t i c acids, l o n g e r - c h a i n
a l i p h a t i c a c i d s ~ s h o r t e r a l i p h a t i c acids, a l i p h a t i c a c i d s ---, C O 2
+ H z) are e n d e r g o n i c ; the reversed r e a c t i o n s a r e e x e r g o n i c .
a F r e e e n e r g y c h a n g e s c a l c u l a t e d a c c o r d i n g to the m e t h o d o f
P a r k s a n d H u f f m a n [18]. T h e values s h o w n c o r r e s p o n d to
f o r w a r d reactions.
Ethylphenol could be the source of 3-hydroxy-4ethylphenol, another phenolic compound transiently appearing in the culture fluid (see Table 1).
Since 2-ethylphenol accumulates towards the end
of incubation, it is conceivable that this transformation is reversible and that 2-ethylphenol can be
considered a dead-end metabolite.
In addition to p-cresol, 2-ethylphenol, and 3hydroxy-4-ethylphenol, 2 other phenolics, phenol
and catechol, were detected. The formation and
the relationship between these two compounds
and another intermediate, benzoic acid, are shown
in Fig. 3. Phenol, which might be formed from
benzoic acid (Fig. 3), does not increase significantly during the monitored incubation period,
which is consistent with its role as a possible
degradation intermediate of aromatic compounds
under anaerobic conditions [14]. Catechol, on the
other hand, is a known intermediate of aerobic
degradation of aromatics [16], but is not normally
found during anaerobic transformation of such
compounds. The abrupt increase in the concentration of catechol which follows the mild maximum
in the phenol curve (Fig. 3) might indicate that it
is produced from phenol through hydroxylation.
Since phenol is toxic to microorganisms [17], hydroxylation probably represents a detoxifying
mechanism. The transformation reactions between
benzoic acid and phenol, as well as phenol and
catechol, are proposed as reversible under the
inhibited culture conditions (see Fig. 4); this could
explain a secondary maximum in the benzoic acid
concentration (Fig. 3). Reversible exergonic reactions could also explain fluctuations in the concentration of other aromatic acidic intermediates
(phenylacetic and phenylpropionic acids) over time
(Fig. 2), and the decrease of aliphatic acids towards the end of incubation (Table 1).
Table 2 and Fig. 4 summarize the new reactions
proposed to occur in the BESA-inhibited methanogenic cultures fed lignin-derived aromatic acids
(the reactions already reported previously for similar cultures [2,5] are not included). All the compounds shown were detected either in these studies, or previously in experiments with similar cultures [5]. The prevailing reactions are reductions,
oxidations with oxygen derived from water (hydroxylations), and decarboxylations. Benzene,
toluene and ethylbenzene are most probably
formed from intermediary aromatic acids (benzoic, phenylacetic, and phenylpropionic) through
168
,~
6[HI
/- (~CHzCH2COOH ~
/-
/
~"
/"
Phenylproplo~c acnd
~O
~)-CH2COOH
~Cyclohex~epropionqc
®C
~
CH,C",
acid
~
COOH
®
6[HI J
t~
**
C.,CH,
OH
2- Elhylpinenol
, 4 f
3- Hydroxy- 4- Ethylphenol
~
@ "
OCt,
p Cresol
6[H]-*I
HOOCH
CH,OH ©
Benzytaleohot
~/
,
4- Methylcyck)hexonol
~
Benzece
""& ~
"04 C",
Methylcyclohexene
l
~ HO~ - C H , z C H3
Toluene
'~
Methyl:~tor~c oc~
Aliphahc acids
BenzoLc acid
/
Qc.,c.,
Dhylbenzene
Et hylcyc;ohexoce
> OCHzCH2COOH
Phenylece~ic acid
I
//
HzO
®
~
Cyclol]exane
*@-o.
Phenol
A
I
(~-OH
OH
Calechol
(~COOH
Benzoic c<:ld
~t '~
Ahph~ic oc~ds
Fig. 4. Suggested reactions showing production and transformation of aromatic hydrocarbonsand phenols in BESA-inhibitedferulic
acid-degrading methanogenic consortia. ® Compounds identified in the consortia; ** compounds previously detected in similar
consortia [5], or in toluene-degradingmixed methanogeniccultures (Grbid-Galidand Vogel, submitted); ---~,proposed reactions; -*,
previously confirmed reactions.
decarboxylation, and further transformed via
fermentative oxidation-reduction pathways, because they do not persist in the culture fluid. Since
phenol, substituted phenols, and benzyl alcohol
arise from their parent compounds through hydroxylation, it is proposed that hydroxylation reactions, although endergonic, have a special importance in the inhibited methanogenic consortia,
because they might form less inhibitory products
from potentially harmful compounds. Reduction
of aromatic hydrocarbons, which seems to occur
simultaneously with oxidation, results in production of cyclohexane derivatives whose fate is presently unknown. It is important to emphasize that
the pathways shown in Fig, 4 are only tentative,
but are supported by results of our previous work
with ferulate-, toluene-, or benzene-acclimatized
methanogenic consortia ([5], Grbid-Galid and
Vogel, submitted).
The results reported in this paper indicate that
fermentative bacteria adapted to anaerobic degradation of lignin-derived aromatic compounds
have a potential for production of compounds
which differ considerably from the usual microbial
metabolites. This potential seems to be expressed
under conditions of inhibited interspecies hydro-
gen transfer. Recent studies on one of the
fermentative bacteria isolated from the ferulic
acid-degrading mixed methanogenic cultures, the
facultative anaerobe Enterobacter cloacae DG-6
[7], suggest that most of the compounds described
above are produced by a pure culture as well
(Grbi6-Gali6, manuscript in preparation). These
peculiar capabilities of the fermentative microorganisms under conditions of disrupted hydrogen
removal can explain the appearance of methylated
and ethylated phenols in anaerobic subsurface
environments exposed to toxic leachates from
landfills [19] because it is known that gasoline
derivatives like toluene or benzene are inhibitory
for methanogens [20]. These results can also explain the extraordinary potential of mixed
methanogenic populations to degrade toluene,
benzene, ethylbenzene, and xylenes (B. Wilson,
personal colnmunication; Grbi6-Gali6 and Vogel,
submitted). The possibility of transformation (and
even production) of such a broad spectrum of
aromatic compounds by anaerobic communities is
significant for anaerobic environments in nature,
and especially for the environments which are
polluted by man-made aromatic contaminants.
169
ACKNOWLEDGEMENTS
This work was partially funded by EPA grant
CEE-811610.
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