University of Groningen
Growth of bacteria at low oxygen concentrations
Gerritse, Jan
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Chapter 7
Mineralization of the herbicide 2,3,6-trichlorobenzoic acid by a
co-culture of anaerobic and aerobic bacteria
Jan Gerritse and Jan C. Gottschal
In: FEMS Microbiology Ecology (1992) 101:89-98
Chapter 7
Mineralization of the herbicide 2,3,6-trichlorobenzoic acid
by a co-culture of anaerobic and aerobic bacteria
Jan Gerritse and Jan C. Gottschal
Departme", of Microbiology, U"iL'ersüy of Groningen, Haren, Netherlands
Received 3 December 1991
Revision received 16 MaTch 1992
Acceplcd 17 March 1992
Key words: Co-culture; Anaerobic plus ae robic bacteria; 2,3,6-Trichlorobenzoic acid ; Red uctive
dechlorin ation; 2,5-0ichl orobenzoic acid ; Microaerobic minera liza tion
1. SUMMARY
Bacteri a from an anaerobic enrichment reductively removed chlorine from th e ortho- position
of 2,3,6-trichiorobenzoic acid (2,3,6-TBA) producing 2,5 -dichlorobenzoate (2,5-0BA). Th e
strictly aerobic bacterium Pseudomonas aeruginosa JB2 subsequently used 2,5-0BA as a growth
substrate in the presence of oxygen . The anaerobic dechlorinating microbial popu lation was
grown with P. aeruginosa JB2 in continuous culture. Inside the liquid culture, a nylon netting, on
a sta inless-steel support, contained vermiculite
particles to provide a strictly anaerobic environment within the aerated culture. Complete mineralization of 2,3,6-TBA depended on the extent
of oxygen input into the reactor. Unde r strictly
anaerobic conditions 2,5-0BA and CI- we re produced stoichiometrically through the reductive
Correspondenee
10: J. Gerritse, Depa rtme nt of Microbiology,
U niversi ty of Gron ingen, Ke rkl aan 30, 975 1 NN Haren , The
Nethe rlands.
70
dechlorin ation of 2,3 ,6-TBA. T his process of reductive dechl orination was not inhibited by (moderate) aeration resulting in an 0 z-conce ntration
of 0.3-0.5 /LM in the culture liqui d.
2. INTROOUCTION
Halogenated aromatic compounds are serious
pollutants of the e nvi ronme nt world-wide. Many
of these man-made chemica is, such as I, I, l -tri chl oro -2,2-b is( p -chl oro ph e nyOe th a ne ( 0 OT),
polychlorinated biphe nyls (PC B), chlo rin ated
dibe nzo-p-d ioxi ns, chlo rina ted benze nes and
chlorinated phenols, persist in nature. Some of
these xe nobiotics can be degraded by (micro)
orga nisms. The me tabolism of ha logenated aromatic compounds has been stud ied extensive ly
during recent years and inform ation on their
degradation has been reviewed by seve ral autho rs
[1 -5]. Oegradation depends on a va rie ty of factors. Th e chemical structure of the compound,
the presence of organisms with appropriate
catabolic ca pacities and the prevail ing environ-
Mineralization of trichlorobenzoate
mental conditions are of critical importance. The
presence of mol ecu lar oxygen is of remarkable
influence on the degradation. Under aerobic conditions, the persistence of aromatic compounds
generally increases with increasing halogen substi tution. In particular the activity of dioxygenases, requi red for the oxidation of the aromatic
ring, is reduced progressively (or indeed completely blocked) by the presence of halogen substituents [6]. Quite the opposite is true for reductive dehalogenation reactions, catalyzed by some
(facultative) anaerobes. Increased halogen substitution may indeed enhance degradation [5 ,7].
Thus, under anaerobic conditions these bacteria
may dehalogenate compounds that resist aerobic
degradation. In many cases this dehalogenation is
not complete but yields less-halogenated compounds, which subsequently may be degraded by
aerobic bacteria. For example, Alcaligenes eutrophus H850 metabolized most of the PCB congeners aerobically, resulting from anaerobic reductive dechlorination of more highly chlorinated
congeners of Arochlor 1242 [8]. Recently, Beunink and Rehm demonstrated the coupling of a
reductive and an oxidative reaction during the
degradation of DDT and 4-chloro-2-nitrophenol
using cultures composed of a facultatively anaerobic and a strictly aerobic bacterium immobilized
in Ca-alginate beads [9,10]. A similar scheme of
complementary catabolism of anaerobic and aerobic bactcria may be envisaged for complete mineralization of chlorobenzenes [11 - I3], DDT [14],
chloroanilines [15 ,16] and possibly of polyhalogenated aroma tic compounds in genera!.
Recently, we have demonstrated that strictly
anaerobic and aerobic bacteria can co-exist in
mixed cultures grown under 0 2-limited condition s [17,18]. This result suggests that co-cultures
of anaerobes and aerobes may be used to mineralize poly-halogenated aromatic compounds in a
one-step process. In the present study this hypothesis has been tested. A mixed culture of
anaerobic and aerobic bacteria ab Ie to mineralize
the herbicide 2,3,6-trichlorobenzoic acid (2,3,6TBA) was used as a model system for such a
process. This mixed culture was composed of a n
anaerobic enrichment culture, capable of 2,5-dichlorobenzoic acid (2,5-DBA) format ion by re-
ductive dechlorination of 2,3,6-TBA [18], and the
aerobic Pseudomol1as aeruginosa J B2, able to
grow on 2,5-DBA as the sole carbon and energy
source [19].
3. MATERlAL AND METHOOS
3.1. Souree and growlh oJ organisms
p. aeruginosa JB2 was kindly supplied by Or.
F.K. Higson and Or. D.D. Focht. The organism
grows on several mono-, di-, and trihalogenated
benzoic acids [19]. We routinely grew P. aerugi110sa JB2 at 30°C in a low-chloride mineral salts
medium (LMM) containing the following components (per liter): MgSO.· 7H 20 (0.1 g),
Ca(N0 3 )2.4H 20 (0.05 g), EDTA (J mg), FeSO•.
7H 20 (2 mg), ZnSO.· 7H 20 (0.1 mg), MnCl 2 '
4H p (0.03 mg), H 3 B0 3 (0.3 mg), CoCl 2 . 6H 20
(0.2 mg), CuCI 2 ' 2HP (0.01 mg), NiCI 2 ' 6H 20
(0.02 mg), Na 2MoO.· 2H 20 (0.03 mg), Na 2Se0 3
. 5H 20 (0.026 mg) and Na 2WO.· 2HP (0.033
mg). Nter autoclaving, the medium was completed by the addition of a K(NH . )PO.-buffer
(25 mM, pH 7.0) and (chloro)benzoate (2.5 mM)
from separately autoclaved stock solutions.
The anaerobic reductively dechlorinating enrichment used in this study was described earl ier
in a preliminary report [18]. Successful 2,3,6-TBA
dehalogenating enrichments could be obtained
repeatedly from 3 different sources. For further
growth of these enrichments the following components were added to 1 I of LMM-medium:
yeast extract (J g), peptone (I g), resazurin (0.1
mg), sodium benzoate (2 mmol) and a volatile
fatty acid mixture (10 mi). The latter mixture
contained acetate (120 mM), propionate (40 mM),
butyrate (20 mM), 2-methylbutyrate (5 mM),
isobutyrate (5 mM), valerate (5 mM) and isovalerate (5 mM). After autoclaving, vitamins (1 mI)
[20], 1,4-naphthoquinone (0.2 mg) and 2,3,6-TBA
(0.68 mmol) were added from filter-sterilized
stock solutions. The medium was prepared under
an N2-atmosphere and reduced with Na 2S (35
mg). Anaerobic batch cultures we re incubated
statically at 30°C in the dark, and transferred
(5-10% inocula) every 3-5 months in freshly
prepared media.
71
Chapter 7
A reactor was set up to grow mixed cultures of
anaerobic and aerobic bacteria (Fig. I). A chemostat similar to that described before was used
[17]. ft was equipped with probes allowing continuous monitoring and regulation of temperature,
dissolved meygen concentration, redox potential
and pH of the culture liquid. The pH was maintained by automatic titration with H ) P0 4 • A Servomex 1100 oxygen analyzer was used in combination with a gas flow meter to quantify the
oxygen consumption by the bacteria in the reactor. The total volume of the Iiquid phase of the
reactor was 900 mI. Particles of vermiculite, a clay
mineral abundant in soils [21], enclosed in a
porous nylon bag (pore size I mm) we re present
as asolid phase in the reactor liquid. The bag
contained 100 g vermiculite and occupied a volume of 570 mI. The reactor-liquid was stirred by
means of a magnetic stirrer (l000 rpm) immediately below the nylon bag. Oxygen was supplied
by mixing air with N 2-gas supplied via a needIe
positioned just below the vermiculite bag. The
reactor was operated at 30°e.
3.2. Analytical procedures
Chloride was measured colorimetrically according to the method of Bergman and Sanic [22]
with NaCl as a standard. Methane concentrations
in the head-space gas we re measured gas chromatographically [17]. Chlorinated benzoates were
analyzed, after methylation and extract ion in
CHCI ) , through capillary gas chromatography
(Packard, GC model 438 A) using a flame ionization detector (275°C) and split injection (250°C,
vent ratio 1 : 40). AChrompack CP-sil-8-CB column (25 m X 0.25 mm) was used to separate
methyl esters of chlorobenzoates with a linear
temperature gradient (J O°C per min from 70 up
to 250°C). Standard solutions of commercially
obtained chlorobenzoates were used for quantification. Using 4-chlorobenzoic acid as an internal
standard, integration of the peak areas recorded
with a Shimadzu C-3A integrator yielded a linear
response over a concentration rang ... from 10 J.LM
to 10 mMo Coefficients of variation [(standard
deviation j mean) x 100] of the concentrations
ranged from 2 to 5% . Protein was measured
72
according to Lowry et al. [23] with bovine serum
albumin as the standard.
Substrate oxidation kinetics of P. aeruginosa
JB2 we re determined in a (Yellow Springs-type)
biological oxygen monitor equipped with a polarographic oxygen electrode. Cells were washed
and resuspended in an isotonic K(NH 4 )P0 4 -buffer
with chloramphenicol (30 mg 1- I) to block protein synthesis. Oxygen tracings were performed at
30°C, and kinetic parameters we re obtained from
direct linear plots.
For fluorescent microscopy a Carl Zeiss G42llO-e Axioscope was used. The microscope was
equipped with an Osram high-pressure mercury
lamp, and a Zeiss BP 400-44, FT 460, LP 470
filter-combination, and allowed detection of fluorescence of methanogenic bacteria.
3.3. Chemicals
All chemicals were of analytical grade, except
2,3,6-trichlorobenzoic acid (Pfaltz and Bauer,
Flushing, NY), which was demonstrated (by capillary GC) to contain 71.5 % 2,3 ,6-TBA, 4.6%
2,3,5-trichlorobenzoic acid, 3.5% 2,4,6-trichlorobenzoic acid, 0.2 % 2,4-dichlorobenzoic acid, 0.4 %
2,6-dichlorobenzoic acid, 1.1 % 2,5-dichlorobenzoic acid, 0.2% 2,3-dichlorobenzoic acid , and
18.5% unidentified compounds.
4. RESULTS
4.1. Degradation of chlorobenzoates by P. aeruginosa 182
As reported by Hickey and Focht [19], P.
aeruginosa JB2 was capable of growth with benzoate or 2,5-DBA as the sole energy and carbon
sources in mineral medium. No growth occurred
in a batch culture supplied with 2,3,6-TBA (2.5
mM) within 100 days of incubation. In batch
cultures containing a mixture of benzoate (2.5
mM) and 2,3,6-TBA (2.5 mM) growth occurred
on benzoate while no 2,3,6-TBA was metabolized. Thus P. aeruginosa J B2 is not able to
co-metabolize 2,3 ,6-TBA with benzoate as a primary substrate.
Oxygen consumption kinetics of P. aeruginosa
JB2 we re analyzed with cells grown on 2,5-DBA
Mineralization of tricblorobenzoatr
in a statically insubated (Ol-limited) and a weil
aerated (O l-saturated) batch culture. With 2 mM
2,5-DBA as a substrate an apparent Km for
oxygen of 25 p.M was obtained with eells grown
under both conditions. The maximum specific
oxygen consumption rate (Qü~X) of the eells grown
0l-saturated was 7.2 p.mol -mg protein - I h - I .
The Qü;x of the eells grown O l-limited was
somewhat higher, 12.0 p.mol mg protein - ' h - ' .
4.2. Reductiue dechlorination of 2,3,6- TBA in an
anaerobic reactor
An anaerobic reactor was set up to establish a
population of anaerobic bacteria capable of reductive dechlorination of 2,3,6-TBA (Fig. 1). Preliminary results of enriehments from freshwater
sediments, reductively dechlorinating 2,3,6-TBA
and producing 2.5-DBA, have been published
elsewhere [18]. Because initial experiments indicated a very low growth rate for such cultures
(doubling time 25-50 days) a nylon bag filled with
particies of the ciay-mineral vermiculite was
placed inside the reactor to prevent wash-out of
the dechlorinating bacteria. The reactor was inoculated with an actively dechlorinating batch culture (l50 mI) grown in the presence of vermiculite. Reductive dechlorination continued without a lag and the retention time of liquid phase of
the reactor was set at 830 h. When 2,3,6-TBA was
degraded to a concentration < 0.01 mM, the
retention time of the culture liquid was decreased
stepwise to 200 h, 100 h, and 50 h over a period
of 30 days. Under these conditions the 2,3,6-TBA
concentration remained below 0.2 mM o A further
dec rea se in the retention time to 40 h resulted in
accumulation of the substrate, 2,3,6-TBA, in the
reactor liquid, and a concomitant decreasing concentration of the dechlorination products, 2,5DBA and chloride.
An analysis of substrates consumed and products formed was made after the reactor was operated anaerobically for two months at aretention
time of 50 h (Tabie 1). Under these growth conditions the redox potential measured in the liquid
phase of the reactor remained between - 200
and - 215 mV. About 35% of the total carbon
supplied to the reactor in the form of benzoate,
yeast extract and peptone was recovered as
Medium
supp1y
Gassupp1y
(Nilrogen t Air)
Waste
Uquid phase
o
o
o
Support
Magnetic stirrer
Fig. l. Reactor used for continuou s growth of a co-culture of
an anaerobic enrichment culture, reductively dechlorinating
2,3,6·TBA 10 2,5-DBA and Ihe aerobic P. aernginosa !B2,
metabolizing 2,5-DBA. The reactor contained a porous nylon
bag filled with vermiculite. Probes were inserted in the liquid
phase above the vermiculite 10 continuously monitor (and
regulate) pH, temperature. redox potential and oxygen con -
centration . Gasses (N l , or air) were added via a needie with
its outlet just below the vermiculite .
methane. With fluorescent microscopy no fluorescent bacteria we re detected in samples of 1-10
p.1 taken from the liquid phase, indicating the
presence of a very low number of methanogens,
probably < 10 3 _10 5 per mI. A thick biofilm on
the vermiculite, and a relatively low protein concentration in the culture liquid « 45 mg I- I)
indicated that most of the bacteria in the reactor
were present on the vermicu lite particles. Ben-
73
Chapter 7
2 .0 , . . - - - - - - - - - - - - - - ,
zoate was metabolized to a concentration < I
ILM . The residual concentration of 2,3,6-TBA in
the reactor liquid (0.045 mM) showed that > 90%
of the 2,3,6-TBA entering the reactor (0.68 mM)
was consumed. In the reactor liquid 0.99 mol of
chloride and 0.93 mol of 2,5-DBA were recovered
per mol 2,3,6-TBA consumed. In addition to 2,5DBA, minor concentrations ( < 0.03 mM) of some
other dichlorobenzoates (2,4-DBA and 3,5-DBA)
were produced.
A control experiment was performed to investigate whether 2,3,6-TBA was dechlorinated in
vermiculite containing non-steri le medi a not inoculated with the dechlorinating enrichment culture, but containing a sufficient number of nondehalogenating bacteria to ensure reducing conditions over long periods of time. No reduction of
th e concentration of 2,3,6-TBA was observed
within 200 days indicating that the reductive
dechlorination was not an aspecific reaction
catalized by vermiculite or many bacteria not
selected for the ability to dechlorinate.
4.3. Min eralization of 2,3,6-TBA by the combined
activity of the anaerobic reductively dechlorinating
enrichment and P. aeruginosa JB2
When a sample of the anaerobic reactor Iiquid
(100 mI) was incubated aerobically, no further
degradation of 2,5-DBA occurred, which indicates that 2,5-DBA degrading aerobic bacteria
were not present in the dechlorin ating enrichment culture. However, when such a sample was
inoculated with P. aeruginosa lB2, 2,5-DBA was
metabolized immediately (Fig. 2). Chlorine release correlated weil with 2,5-DBA consumption.
In an experiment designed to investigate
whether reductive dechlorination of 2,3,6-TBA
i'
.§.
co
"oc:
"0.
E
o
.."
.:
(;
:ë
: f~
0 .5 "". _ _ _ _ _ _ _ _ _ _ _
o
00
o
10
Time (days)
Fig. 2. Degradalion of 2.5-DBA resulting from reductive
dechlorination of 2,3,6-TBA in an anaerobic reactor fo llowing
aerobic incuba tion of (he reactor liquid with (c1osed symbols)
and wi thout P. aerugillosa J82 (open symbols) . .. , 2,5- DBA:
- , C1 - .
and oxidative degradation of 2,5-DBA cou ld occur simulta neously, P. aeruginosa 1 B2, grown on
2,5 -DBA in an 0 r limited batch cu lture, was concentrated by centrifugation and inocul ated into
the dechlorinating reactor. This procedure resulted in an initi al density of approximately 70 mg
P. aeruginosa lB2 prote in pe r liter reactor liquid.
It was observed that individual cells of P. aeruginosa lB2 could be detected in the reactor microscopically, using the sa me filter combina tion to
detect flu orescence of me thanogen ic bacte ri a (see
MATERlALS AND METHODS). Immediately af ter inoculation the fl ow of N 2-gas was replaced by air,
which was bubbled through the reactor liquid at a
Table I
Mass·ba la nce of the reactor with a co-culture of a n anaerob ic dechlorinating enrichment and Ihe aerobic bacterium PseudomonDS
aeruginosa J82
Reactor
condi tions
Anaerobic
Microaerobic
0,
reading
(/L M)
Redox
reading
(mV)
0.0
0.4
-2 14
+299
Co nsumptio"
(/Lmol h -
Produclion
(/Lmol h - I )
I)
0,
BA
2.3.6-TBA
eH,
2.5-DBA
Cl
0
3840
38.8
38.8
13.6
13.7
530
0
12.6
0.0
13.4
39.0
The reactor was ope rated under a naerobic a nd microaerobic condi tions at a retention time of 50 h. Substrates in th e resevo ir
med ium: 2.3,6·TBA (0.68 mM), and BA ( 1.94 mM).
74
Mineralization of trichlorohenzoate
2 .0
~
.s
1.5
lil
c:
::l
0
0.
1.0
0
0
Q,
.~=
c:
É
.s
07,,-\\ .''
~
.<:
0
E
°'0/
0,
° ,
"C
E
~
U
~óI6~
6 ..,p
~
0.5
c:
0
\
E
::l
Ul
;):~;;:~
c:
0
~
.<:
Ü
cS
0 .0 4
-20
_ =___
· '0
~-""""""':":::""-..;n._-.J
10
20
30
40
50
60
Time (days)
Fig. 3. Time course of 2.3,6-TBA. 2,5-DBA and CI - ca ncen-
day 15) and a further increase of the redox reading from + 134 to + 299 mV. Although under
these conditions the 0 2-conceniration in the culture liquid still remained < 1 ILM , CH. could no
longer be detected in the head-space gas. After 7
volume changes the concentration of 2,5-DBA in
th e reactor liquid had decreased to < 1 IL Mand
that of 2,3,6-TBA to 17 ILM (Fig . 3; day 34).
C hlorine production (2.85 mol per mol 2,3,6-TBA
consumed) proved that under these conditions
95% of 2,3,6-TBA had been dechlorinated completely (Tab Ie 1).
When, at day 34, air was replaced by N 2 ,
anaerobic conditions were restored and the redox
potential dropped to -210 mV within seve ra l
hours. Gradually, 2,5-DBA and Cl - returned to
their initial anaerobic concentrations (Fig. 3).
tratÎons in an aeraled reactor containi ng a mixed cultu re of
anaerobic 2,3 ,6-TBA dechlorinating bacter ia and the aerobic
2,S-DBA degrading P. aeruginosa 182. The reactor was aperated at a liquid retention time of 50 h. The reservo ir med ium
contained 0.68 mM 2,3,6-TBA. The act ual oxygen co ncentralion in the liquid phase remained be low 1 p.M. D . 2,3,6-TBA;
6., 2,S-DBA; 0, Cl - ; and - - - - - ', fal e of oxygen con sumption.
ra Ie of 4 I per h. Analysis of the in- and out-going
gas showed that oxygen was consumed at a rate
of 1.5-1.8 mmol h - J (Fig. 3; day 0). The reading
of the oxygen probe indicated an oxygen concentration in lhe reactor liquid of 0.4 ILM. Redox
readings increased by more than 300 m V (from
- 200 to + 134 m V) after aeration had started.
The rate of CH 4 production dropped significantly, but stabilized at a leve l 4 times less than
during strict anaerobiosis. The concentration of
2,3,6-TBA was slightly higher (about 0.1 mM)
than during strict anaerobiosis, but apparently
dechlorination was not significantly inhibited by
the addition of air. The decreasi ng concentration
of 2,5 -DBA and the concomitant increasing concentration of CI - in the reactor liquid (Fig. 3)
indicated that P. aerugil10sa 1B2 metabolized 2,5DBA resulting from the anaerobic dechlorination
of 2,3,6-TBA (see a lso Fig. 2).
Further increase of the air-flow resulted in
concomitant increased 0 2-consumption (Fig. 3;
5. DISCUSSION
The degrada tion of 2,3,6-TBA by the combined activity of anaerobic and aerobic bacteria
was studied. To the best of our knowledge this is
the first report in which the compl ete mineralization of this persistent herbicide is demonstrated.
Chlorinated benzoic acids have often bee n used
as model compounds in biodegra dation studies o f
ha loge nated aromatics. Although information of
th e ir occurrence in nature is scarce, it is certain
they have bee n released into the e nvironment
due to extensive use as he rbicides [24]. Furthermore, chlorobenzoates are known to be produced
during the aerobic metabolism of PCBs a nd a lkyl
be nze nes [1,3]. 2,3,6-Trichlorobenzoic acid itself
has bee n used in large doses to con trol weeds,
often as a component of mixtures with other
herbicides. Although slow (partiaI) decomposition
of 2,3,6-TBA in soil may occur [25], th is compound was repeatedly found to be persiste nt and
effective for many months (up to five years) after
application [24,26,27].
The persistence of poly-chlorinated benzoic
acids unde r aerobic conditions has been noticed
by various investigators [26-28] . Especially
chlorobenzoates containing two ortho-substituted
chlorines, like 2,3,6-TBA or 2,6-dichlorobenzoic
acid (2,6-DBA), resist aerobic degrad ation . lt has
7S
Chapter 7
been suggested that an unsubstituted carbon on
the aroma tic ring next to the carboxyl group is
important for the oxidation of chlorobenzoa tes by
dioxygenases [19,29,30). The only aerobic bacterium 'known to metabolize 2,3,6-TBA is a Breuibacterium sp. [31]. When grown on benzoa te
th is organism co-oxidized 2,3,6-TBA. The product formed, 3,5-dichloroca techol, was toxic for
this bacte rium and was not metabolized further.
p. aeruginosa JB2 can metabolize a wide range of
halogenated benzoic acids [1 9]. The following
chlorin ated benzoic acids are used as the sole
sources fo r ca rbon and energy by this bacterium:
2,3,5-TBA, 2,3- and 2,5- DBA , and 2- and 3-chlorobenzoate. In addition, whe n grown on benzoa te
P. aeruginosa JB2 co-metaboli zes (chlorine release) 2,4- , 2,6-, 3,4-, and 3,5-dichlorobe nzoa te.
In th e prese nt study it was substantiated th at P.
aeruginosa JB2 did neither grow on , nor cometabo li ze 2,3,6-TBA, thu s unde rlining the res istance of th is compound . to oxid ative degradation.
Unde r anaerobic conditions ma ny chlorinated
benzoic acids can be dechlorinated via red uctive
mecha nisms [5,7,32,33]. Anaerob ic e nrichm ents
transforming 2,3,6-T BA into 2,6-DBA, by substi tution of the meta - chlo rine fo r a proton we re
described by Horowitz et a l. [32]. No decrease of
the concentration of 2,6-DBA was observed after
a yea r of furth er anaerobic incubation of these
e nrichments. Recently, we obta ined anaerobic
enrichme nt cultures that reductive ly dechlo rin ate
2,3,6-T BA, and produce 2,5- DBA [1 8]. Altho ugh,
2,3,6-TBA deg radation in these batch cultures
was very slow, this enrichme nt was succesfull y
grown in a reactor by using vermiculite fo r the
attachment of the reductively dechl orin ating organisms.
The production of low concentrations of
dichlorobe nzoates othe r tha n 2,5-DBA in the reactor was probably ca used by reductive de halogenation of (tri)chlorobe nzoates present as impuri!Î es in the 2,3,6-TBA we used. Remova l of the
meta -substituted chlorine from 2,3,6-TBA did not
occur since 2,6- DBA was not p ro du ce d.
Dichlorobenzoates formed in the reactor (e .g.,
2,5- DBA, 2,4-DBA and 3,5- DBA) were not degraded under an aerobic conditions. Appa re ntly,
bacteri a like Desulfomonile tiedjei DCBI , able to
76
dechl orinate seve ral dichl orobenzoa tes [7], were
not selected in the enrichment used in th e present study.
Remova l of an ortho-substi tutcd chlorine fro m
2,3,6-TBA is crucia l for further mineralization of
th is compound since aerob ic chl orobcnzoa te catabolize rs like P. aeruginosa JB2 are capable of
growth on 2,5- DBA, but not on 2,3,6-TBA or
2,6- DBA [19,29,30]. Indeed , th is bacteri um was
shown to metabo li ze 2,5- D BA ae rob ica lly, whi ch
was form ed anae robica lly by reductivc dechlorinat ion of 2,3,6-TBA in th e e nrich'men t cul ture.
T he degradation of othe r chl orin ated compou nds
such as PCBs [1 ,8] and the pesticide Meth oxychl o r [1 ,1bist p- me th oxyph e nyIl-2,2,2- trichl oroeth ane] [34] ca n also be enh anced by a pc riod of
anaerobiosis preceding a pe riod of aerobiosis. In
ad dition, red uctive dechlorin ation of tri- and tetrachloroethylenes was shown to depend on a
tra nsiti on from ae robi c to anaerobic co nditions
[35]. These observa tions suggest th at alte rnat in g
anaerobic and ae rob ic conditions may improvc
degrada tio n of polychlori nated compo unds in
ge ne ra l. In th e e nvironm ent such conditions frequently occur in soils [36] and sed im ent s [37], a nd
it is te mpting to hypoth esize th at hab ita ts domi na ted by flu ctuating oxygen concent ra tions arc
best suited for the deve lopment of microb ia l communiti es ca pable of min eralizin g such xe nob iotics.
A complicating fac tor occurring when employing alte rn ati ng anaerobic and aerobic conditions
to deg rade substanti al amount s of ch lorin atcd
compounds is that (tox ic) interm edi ates may accumulate du ring the anaerob ic or aerobic phase.
In addition, the anaerob ic bacteria involved may
not survive the pe riod of ae robiosis. T he fact th at
reductive dechlorin ation of 2,3,6-TBA a nd oxid ative min eralization of th c product 2,5- DBA occurred simultaneously in the ae rated reactor and
resulted in almost complete mine ra liza tion of
2,3,6-TBA shows th at anaerob ic and aerob ic bacte ri a ca n succesfully be combin ed at low oxyge n
te nsions (Fig. 4).
Because th e appa re nt Km fo r 0 2 of P. aeruginosa JB2 with 2,5-DBA as the substrate was 25
J.l.M , and the oxygen conce ntration in th e reactor
remained < 1 J.l. M, th e rate of 2,5- DBA oxid ation
Mineralization of trichlorobenzoate
IUquid ph..·1
a~a
~a
a
Óa
COOH
. ...J
"
2(H)
.'Ó::~:t5.
.......-H .CI
Fig. 4. Proposed scheme of chlorobenzoat e degradation in the
mixed culture reactor under aeraled conditions. 2,3.6·TBA
enters Ih e nylon bag with vermiculite wh ere reductive dechlo-
rination takes place. Thc anaerobic dechlorination product
2,5-0BA is degrad ed furth cr by P. aerugiflosa J82 ance it has
reached th e microaerobic liquid ph ase.
by P. aeruginosa J 82 must have been limited by
0 2' However, due to the availability of additional
organic substrates such as benzoate, yeast extract,
peptone and fermentation products of the anaerobic population P. aeruginosa could probably
maintain a sufficiently high cell-density to oxidize
all 2,5-D8A in spite of the low specific oxidation
ra te as a result of the low oxygen concentration.
Half-saturation constants for O 2 of aerobic
(halo)aromatic catabolizing bacteria are usually
relatively high [6,38). For example, for dioxygenases, which are essential enzymes in the
mctabolism of these bacteria, Km values for O 2
ranging from 9 J.L M to 33000 J.L M have been
reported. This would imply that relatively high
oxygen concentrations are necessary for an effi-
cient degradation of chlorinated aroma tics by
aerobic bacteria. This obviously complicates
growth of co-cultures composed of such organisms and anaerobes.
Aeration of the reactor reduced the activity of
the strictly anaerobic methanogenic bacteria
present in the mixed culture. However, reductive
dechlorination was not sign ificantly inhibited under these conditions. It is known th at strict anaerobes can be protected from O 2 by the respiratory
activity of (facultative) aerobes maintaining the
dissolved 0 2-concentration at a very low level
« 0.5 J.LM) [17]. Oxygen, present at such low
concentrations, surely does not always block reductive dechlorination [39-41]. Moreover, in this
particular reactor, O 2 concentrations (and redox
potentiaI) in the nylon bag with vermiculite were
probably much lower than in the liquid phase,
thus ensuring suitable growth conditions for strict
anaerobes.
The experiments described in this paper
demonstrate th at mixed cultures of strictly anaerobic and aerobic bacteria may be applied successfully to the degradation of poly-halogenated aromatic compounds. Use of biological purification
filters with habitats for both anaerobic and aerobic bacteria could have several advantages over
systems which are only (or sequentially) aerobic
or anaerobic for the following reasons: (J) both
aerobic and anaerobic species with different
catabolic capabilities can be active in close cooperation. This may reduce the problem of accumulation of (toxic) intermediates or dead-end products; (2) inactivation or death of aerobes or
anaerobes due to the periodic absence or presen ce of 0 2' respectively, is prevented; (3) only
one filter-system is needed, which can be operated con tinuously under 0 2-limitation.
ACKNOWLEDGEMENTS
This work was supported by the Netherlands
lntegrated Soil Research Programme. The authors thank Or. D.D. Focht, and Or. F.K. Higson
for their kind gift of Pseudomonas aeruginosa
J82.
77
Chapter 7
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