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BF00248485
BF00248485
Micrnbiolegy
9 Springer-Verlag 1993
Abstract. A new mesophilic sulfate-reducing bacterium, Desulfotomaculum sapomandens, the thermophilic Desul-
strain Groll, was isolated from a benzoate enrichment .)Cotomaculum thermobenzoicum and a few strains of Desul-
culture inoculated with black m u d f r o m a freshwater fococcus multivorans are able to grow in fresh water
ditch. The isolate was a spore-forming, rod-shaped, media (Szewzyk and Pfennig 1987; De Weerd et al. 1990;
motile, gram-positive bacterium. This isolate was able of Tasaki et al. 1991).
complete oxidation of several aromatic compounds in- A m o n g the sulfate-reducing bacteria belonging to the
cluding phenol, catechol, benzoate, p- and m-cresol, genus Desulfotomaculum only the two species mentioned
benzyl alcohol and vanillate. With hydrogen and carbon above have been reported to metabolize aromatic com-
dioxide, formate or O-methylated aromatic compounds, pounds. While Desulfotomacuhtm thermobenzoicum was
autotrophic growth during sulfate reduction or homoace- able to grow only with benzoate, Desulfotomaculum
togenesis was demonstrated. Lactate was not used as a sapomandens could also use phenylpropionate, phenyl-
substrate. SO 2-, SO~-, and $2032- were utilized as acetate and 4-hydroxybenzoate (Cord-Ruwisch and Gar-
electron acceptors. Although strain Groll originated from cia 1985; Tasaki et al. 1991). With respect to the
a freshwater habitat, salt concentrations of up to 30 g 9 1- degradation of aromatic compounds strain Groll showed
were tolerated. The o p t i m u m temperature for growth was a similar versatility as the marine strains, Desulfobac-
35-37 ~ The G + C content of D N A was 42.1 tool%. terium phenolicum and Desulfobacterium anilini (Bak und
This isolate is described as a new species of the genus Widdel 1986; Schnell et al. 1989).
Desulfo t omaculum. In the present paper we describe an isolate that utilizes
aromatic compounds, fatty acids and alcohols as the only
Key words: Sulfate reduction - Desulfotomaculum - electron donor and carbon source. In addition, strain
Anaerobic catechol oxidation - Degradation and trans- Groll is able to O-demethylate and decarboxylate aro-
formation of aromatic compounds matic compounds. To our knowledge this is the first
report on a sulfate-reducing bacterium growing by O-
demethylation of an aromatic c o m p o u n d and subsequent
complete decomposition of the benzene nucleus.
During anaerobic microbial transformation and degrada-
tion of lignin-related aromatic compounds only a few
intermediary products, like benzoate or phenolic com- Materials and methods
pounds are formed (Healy and Young 1980; Bache and
Pfennig 1981; Kaiser and H a n s e l m a n n 1982). Anaerobic Sources of microorganisms
degradation o f phenolic compounds including catechol
Strain Groll was isolated from an enrichment culture inoculated
has been reported for methanogenic mixed cultures and with a black mud sample from a small freshwater ditch. Benzoate
pure cultures of phototrophic, nitrate-reducing and sulfa- was used as only carbon source and electron donor. Desulfoto-
te-reducing bacteria (for review see Evans and Fuchs macuhtm orientis (DSM 675) was kindly provided by H Cypionka,
1988). Sulfate-reducing bacteria capable to use several University of Konstanz, FRG.
aromatic compounds as the only carbon source were
almost exclusively isolated from marine habitats. On- Media and conditions ./'or cultivation
ly DesuIfobacterium catecholicum, Desulfomonile tiedjei,
The freshwater basal medmm was prepared according to Pfennig
et al (1981). The bicarbonate-buffered (30raM) medmm was
* Present address: Department of Biochemistry, Biotechnology reduced with sulfide (1.5 raM), and contained trace element solution
Building, Cornell University, Ithaca, NY 14853 USA SL 10 (1 ml 1-1), and selenite-tungstate solution (2 ml-1 1), and
5 ml - 1-1 vitamin solution (Widdel and Pfennig 1984). Na-dithiomte
Correspondelwe to K.-H. Blotevogel (8 mg - 1-1) was supplemented. Resazurin (0.4 mg - 1-1} was added
283
Chemicals
Fig. 1. Phase contrast photomicrograph of Desulfotomaculumstrata
All chemicals were of analytical grade. GrolI. Bat"represents 10 gm
284
Growth conditions and nutrition (1-5 raM) if yeast extract was added. Crotonate and
pyruvate were completely fermented to acetate. Fatty
Strain Groll grew at 20 to 40 ~ the o p t i m u m tem-
acids, alcohols, aromatic compounds and other substrates
perature being at 35 37 ~ The p H range was between
were oxidized completely. Fastest growth occurred with
6.0 and 8.0 with an o p t i m u m at p H 7.0 7.4. The isolate
butyrate and butanol with a minimum doubling time of
required no organic supplements. Vitamins were not
about 12 to 14h (for benzoate 22-25 h). Increased
essential for growth. After transfer to fresh media the
substrate concentrations resulted in an excretion of
isolate required Na-dithionite (100 gM) as additional
acetate. Some aromatic compounds were transformed by
reductant to initiate growth. Without dithionite rather
O-demethylation or decarboxylation prior to complete
long lag-phases up to one week were observed.
oxidation (Fig. 2).
Strain Groll is a strictly anaerobic sulfate-reducing
Sodium chloride was not required for growth, alt-
bacterium, able to grow either chemolithoautotrophically
hough the isolate showed high salt tolerance. Even a
with H 2 as electron donor and C O 2 as only carbon source,
sodium chloride concentration of 35 g . 1-1 was not
or with formate, methoxylated aromatic and several other
inhibitory (data not shown). Variations of the initial
compounds (Table 1). The isolate was capable to grow
sulfide concentration (up to 10 raM) in the freshly in-
with H 2 plus CO2, formate and methoxylated aromatic
oculated media demonstrated that strain Groll was more
compounds in media lacking sulfate by homoacetogenesis
tolerant to sulfide than other members of the genus
as indicated by the production of small amounts of acetate
(Cord-Ruwisch und Garcia 1985). The m a x i m u m ODs78
was around 0.2 in the presence of 10 m M sulfide added.
Table 2. Results of stoichiometric measurements with Desulfotomacuhlm sp. strain Groll on benzoate, catechol, phenol, vanillate,
protocatechuate, butyrate and butanol as electron donor and carbon source, and 10 mM sulfate as electron acceptor
Substrate given Acetate Cell dry Substrate Substrate mol HS formed Growth yield:
and utilized excreted weight consumed oxidized per mol g dry weight
formed for cell material a by sulfate substrate per mol
reduction oxidized substrate oxidized
[mM] [raM] [mg- i - 1] [mM]
Benzoate
1.7 0 42.2 0.23 1.47 3.0 27.0
2.1 1.1 43.2 0.24 1.86 38 27.9
Catechol
1.8 0 32.1 0.20 1.60 3.19 23.2
2.4 0 39.4 0.25 2.15 2.74 18.3
Phenol
1.9 0 29.7 0.16 1.74 3.56 15.9
Vanillate
2.2 0 49.6 0.26 1.94 3.56 25.6
Protocatechuate
2.4 0 41.1 0.27 2.13 3.19 19.3
a Substrate consumed for cell material was calculated according to Schnell et al. (1989):
Benzoate:
17 CoHsCOO- + HCOj + 71 H20 -, 30 (C4H703) + 18 OH-; thus 0.0055 mmol benzoate are required for 1.0 mg cell dry weight.
Catechol:
17 Co602 + 2 HCOj + 40 H20 ~ 26 (C4H703) + 2 OH- ; thus 0.0063 mmol catechol are required for 1.0 mg cell dry weight
The AG ~ values (for p H 7.0) were calculated from the Degradation of catechol and phenol
data of Thauer et al. (1977) and Kaiser and Hanselmann
(1982). G r o w t h of strain Groll with catechol and phenol as only
electron donor was only possible in bicarbonate-buffered
media, whereas benzoate and butanol allowed also
O-demethylation of aromatic compounds growth in phosphate-buffered media without bicarbo-
nate. The complete oxidation of 2 m M phenol by strain
Strain Groll was able to O-demethylate several aromatic Groll was more than two times slower than of 2 m M
compounds. The specifity of O-demethylation was tested catechol, which was completely oxidized within 8 days.
with m o n o - m e t h o x y b e n z o a t e isomers and Desulfoto- Degradation of catechol was studied with dense cell
maculum orientis as reference strain. Strain Groll O- suspensions (50-80 gg protein/ml) of benzoate- and
demethylated only 2-methoxybenzoate, whereas Desul- catechol-grown cells of strain Groll. Benzoate-grown cells
fotomaculum orientis utilized only the para-isomer. Ho- of strain Groll degraded benzoate and 4-hydroxyben-
wever, both strains O-demethylated vanillin, vanillate, zoate without any lag-phase, whereas degradation of
vanillyl alcohol, anisol, ferulate, 2-methoxyphenol, 2,6- 3-hydroxybenzoate ( > 6 0 h ) , catechol ( > 1 0 5 h), proto-
dimethoxyphenol and syringate. Besides this transforma- catechuate (60 h), and phenol ( > 1 9 0 h) occurred after
tion also decarboxylation of protocatechuate to catechol different prolonged lag-phases. In the case of protoca-
occurred. The same high decarboxylation activity was techuate degradation, catechol was identified as an inter-
observed with catcchol-grown cells of strain Groll, but mediate by HPLC-analysis.
not with cells grown with benzoate, phenol, butyrate or Catechol-grown cells degraded the same substrates
butanol. 4- or 3-hydroxybenzoate or other aromatic acids without any lag-phases, except the degradation of phenol,
were not decarboxylated and cells grown with these which only occurred after a lag-pase of more than 190 h.
substrates showed no decarboxylation activity of proto- During protocatechuate degradation catechol was for-
catechuate. Aromatic aldehydes were rapidly oxidized to med as an intermediate. As shown in Fig. 2 A - E , catechol-
the corresponding acids by strain Groll. Vanillin was grown cells rapidly degraded catechol, benzoate, 3-
oxidized to vanillate which was O-demethylated and hydroxybenzoate, 4-hydroxybenzoate, and in addition
decarboxylated to catechol prior to complete oxidation transformed protocatechuate to catechol, whereas phenol
(Fig. 3). Benzyl alcohol was rapidly metabolized without was utilized only rather slowly (data not shown).
detection of intermediates in the culture fluid. Vanillyl
alcohol was first O-demethylated before oxidation of the
Discussion
alcohol group occurred. Concentrations up to 0.5 m M
were completely oxidized. At higher concentrations pro-
tocatechyl alcohol was excreted, which was not further
Physiological acpects
metabolized. Desulfotomacuhon orientis showed only O- To our knowledge the newly isolated strain Groll is the
demethylation of vanillyl alcohol (data not shown). first sulfate-reducing bacterium growing by O-deme-
280
AI,, i I i ,:F
CO
r-
0 , I . ] " - - ~ ,.~ I , I I
30 60 90 120 150 180
m 10
5- Time [h3
E
ua 08
0 0.6
N E 14 i i i i
C
s 0.4
1.2
02 1.o I
, I , I , I , I , I
0 30 60 90 120 150 180 0.8
:D
.c
Time [h3 U
| 0.6
'0
B 1.4 0
I I I I I
04
1-2 d
0.2
1.o I
0 I , I J I , I I
30 60 g0 120 150 180
E
u 0.8 Time [h3
-6
"~ 0.6 Fig. 2. Degradation of benzoate (A), catechol (B), 4-hydroxyben-
r~ zoate (C), 3-hydroxybenzoate(D) and decarboxylationof protoca-
L)
0.4 techuate (E) in dense cell suspensions of strain Groll pregrown with
catechol (~) and benzoate ([])
0.2
substrate added
I , I , I , I I
o 30 60 90 12o 15o 18o
Time Eh3
' I i ' I ' I '
C 1.4 I I I I
E
i.J 1.2
Strain Groll is a completely oxidizing sulfate-reducing Culture conditions. Strict anaerobe. The temperature
bacterium, as shown by the stoichiometry of substrate optimum is 35-37 ~ the p H optimum is between 6.9
oxidation (Table 2). However, with increased concentra- and 7.2. Addition of NaC1 or vitamins to the medium is
tions of butyrate, butanol and benzoate strain Groll not required for growth. Addition of 100 gM Na-di-
produced acetate. Strain Groll oxidized 1 tool substrate thionite favours initiation of growth.
to 1 tool acetate. These findings are in agreement with
Growth substrates. Electron donors utilized with sulfate
butyrate oxidation by Desulfobacterium autotrophicum
as the electron acceptor: Hz, formate, propionate, buty-
which is also a complete oxidizer (Schauder et al. 1986).
rate, valerate, capronate, caprylate, iso-butyrate, 2-me-
Therefore, it is likely that strain Groll activates butyrate,
thylbutyrate, 3-methylbutyrate, crotonate, ethanol, pro-
and perhaps benzoate, by CoA transfer from acetyl-CoA
panol, butanol, hexanol, octanol, 2-methyl-l-propanol,
formed from butyryl-CoA by/~-oxidation. With increased
1,3-propanediol, pyruvate, malate, fumarate, succinate,
butanol concentrations ( > 2 raM) substrate oxidation
maleinate, benzoate, 3- and 4-hydroxybenzoate, 2-ami-
was incomplete (Table 2), and from 1 tool butanol oxidi-
nobenzoate, phenol, catechol, 3,4-dihydroxybenzoate,
zed two moles acetate were formed. At butanol concen-
phenylacetate, phenylpropionate, aromatic alcohols and
trations higher than 7 m M also butyrate was produced
aldehydes, methoxylated aromatic compounds. Substrate
from butanol. This formation of butyrate indicates that
oxidation is usually complete leading to CO2, but at high
the alcohol-oxidizing enzymes were not affected by high
substrate concentrations of butyrate, butanol and ben-
sulfide concentrations.
zoate acetate may accumulate.
Because of the decarboxylation of protocatechuate by
catechol grown cells and the COx-dependence ofcatechol Electron acceptors. Sulfate, sulfite, thiosulfate and CO 2
utilization, a carboxylation of catechol to protocate- serve as electron acceptors, whereas nitrate and sulfur
chuate as initial reaction of catechol metabolism by strain do not.
Groll is assumed. A reductive dehydroxylation of cate-
Biochemical characteristics. Cells contains cytochromes;
chol to phenol as postulated for methanogenic consortia
desulfoviridin is not present. The mol% G + C of D N A
by Balba and Evans (1980) is probably not involved in
is 42.1 + 1.6 (thermal denaturation).
catechol degradation by strain Groll. We propose a
carboxylation of catechol to protocatechuate similar to Source. Black sediment of a freshwater ditch. The
the carboxylation of phenol to 4-hydroxybenzoate as strain was deposited in the Deutsche Sammlung von
demonstrated for denitrifying bacteria (Tschech and Mikroorganismen (DSM), Braunschweig, F R G under
Fuchs 1989), because our results with strain Groll were No. 7213.
in good agreement with the postulated pathway for
anaerobic catechol oxidation since all catechol-oxidizing Acknowledgement. We thank F. Widdel (Bremen) for useful com-
strains are able to utilize protocatechuate, 3-hydroxyben- ments on the paper. The help of T. Gorontzy in preparing the
zoate, 4-hydroxybenzoate and benzoate (Szewzyk and manuscript is gratefully acknowledged.
Pfennig 1987; Schnell et al. 1989).
The results with strain Groll show that sulfate-
reducing bacteria are able to participate in the bioconver-
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