FEMS Microbiology Ecology 86 (1992) 229-235
0 1992 Federation of European Microbiological Societies 0168-6496/92/$05.00
Published by Elsevier
229
FEMSEC 00368
Total degradation of 6-aminonaphthalene-2-sulphonic
acid
by a mixed culture consisting of different bacterial genera
'
Ruiica Rozgaj and Margareta Glancer-Soljan
'
' Institute for Medical Research and Occupational Health, University of Zagreb, and
Faculty of Food Technology and Biotechnology,
University of Zagreb, Zagreb, Yugoslavia
Received 11 February 1991
Revision received 8 October 1991
Accepted 8 October 1991
Key words: Biodegradation; 6-Aminonaphthalene-2-sulphonicacid; Adaptation to xenobiotic compound;
Mutual interactions
1. SUMMARY
2. INTRODUCTION
A mixed bacterial culture consisting of eleven
different strains was investigated in view of its
ability to degrade 6-aminonaphthalene-2-sulphonic acid (6"s).
Taxonomic characterization of the microorganisms showed that they belonged to three genera: Flauobacteriurn, Bacillus
and Pseudornonas. None of the single strains
could degrade 6A2NS. Some of 4-5-member cocultures degraded it, but lost the ability in future
subcultures. Only the mixed culture consisting of
all eleven strains were stable and efficacious in
degradation through numerous subcultures. The
well-adapted mixed culture degraded the compound fast and without accumulation of intermediates, with a low increase in cell biomass and a
high degree of mineralization.
Controlled microbial degradation of naphthalenesulphonates and their substituted analogues has been subject of numerous studies because of their accumulation in the environment.
Their xenobiotic character makes them resistant
to natural degradation. However, some microbial
populations, previously chronically exposed to
naphthalene and its derivatives, could, by selection of microorganisms and their progressive
adaptation to a certain compound, yield a culture
capable of metabolizing the compound [ll. Particularly convenient sources of such microbial populations are activated sludge from industrial sewage
plants [2,3], or soil and river or marine water
sampled near chemical industries [4-61.
A naphthalenesulphonate-degrading culture
derived by enrichment in a medium containing
naphthalenesulphonate as sole source of an essential nutrient may be composed of one or more
different strains. Sakota et al. [7] isolated Flauobacterium capsulatum capable of degrading 2-
Correspondence to: R. Rozgaj, Institute for Medical Research
and Occupational Health, University of Zagreb, Ksaverska
cesta 2, 410W Zagreb, Yugoslavia.
230
aminonaphthalene-1-sulphonic acid from soil.
Ohe and Watanabe [8] described a degradation of
the same compound and its degradation pathway
by Pseudumonas sp. TA-1. Degradation of naphthalenedisulphonic acids by Pseudornonas sp. in
monoculture was also reported [3,5].Mixed cultures are very efficacious in naphthalenesulphonate degradation [6,9]. It is believed that selected mixed cultures, capable of degrading certain groups of xenobiotic compounds, will become indispensable for biological treatment of
industrial wastewaters.
The present study deals with the biodegradation of 6-aminonaphthalene-2-sulphonicacid
(6A2NS). Emphasis is placed on the development
of a mixed bacterial culture with a stable number
and proportion of community members, which is
capable of degrading the compound quickly and
without accumulation of intermediates.
3. MATERIALS AND METHODS
3.1. Microorganisms
A mixed culture capable of metabolizing
6A2NS was isolated from activated sludge from
an industrial sewage plant. The first step in adaptation to 6A2NS was performed in continuous
culture in a 6-1 laboratory reactor by aerating it in
the presence of 6A2NS as sole source of carbon
and energy. During the adaptation period
(NH,)2HP04 was added as the nitrogen source
at a concentration of 0.5 g/l. After approximately
three months the culture, consisting of about
thirty different bacterial strains, degraded 6A2N2
present at a concentration of 0.250 g/l. Further
enrichment was carried out in batch cultures in
1000 ml Erlenmeyer flasks with baffles containing
mineral medium supplied with increasing concentrations of 6A2NS (0.125-1.00 g/O. (NH4)2HP0,
was ommited from the medium because the mixed
culture showed the capability of utilizing the
amino group of 6A2NS as the nitrogen source.
Cultures were incubated at 30°C on a rotary
shaker at 180 rpm. In that phase, which took
another three months, a number of strains disappeared from the mixed culture. The final culture,
capable of degrading 1 g/l of 6A2NS consisted of
Table 1
List of bacterial strains found to be present in the stable
mixed culture capable of degrading 6A2NS
Flatlobacterium decorans
F. deuorans
F. indoltheticum
B a c i l h cereus
B. cereus
B. circulans
Pseudomonas aeruginosa
P. aeruginosa
P. desmolytica
P. desmolytica
P. cepacia
LOV866
LOV922
L o v 9 12
LOV832
LOW86
LOV904
LOV83.5
LOV837
LOV848
LOV853
LOV877
eleven different bacterial strains. That mixed culture appeared to be stable in the number a n d
proportion of community members under laboratory conditions during a period of 2 years.
Taxonomic identification of organisms, performed by routine methods [10,11], showed that
the isolated microorganisms belonged to three
genera: Flaciobacterium, Bacillus and Pseudomonas (Table 1). Stock cultures of isolates were
stored on nutrient agar plates at 4°C and subcultured monthly.
3.2. Media and chemicals
For the cultivation of bacteria, a liquid mineral
medium was used containing 4 g KH2P04, 0.2
MgSO, . 7 H 2 0 , 0.05 g CaCI, * 2 H 2 0 and 0.01
FeCl, per litre. The medium was supplemented
with 1 g/l 6A2NS as carbon source (and with 0.5
g/l (NH4)2HP0, as nitrogen source during t h e
adaptation to 6A2NS). In adaptation and enrichment procedures 6A2NS concentrations Varied
from 0.125-1.00 g/l.
To isolate monocultures and to propagate
biomass, a solid medium (Nutrient agar) was used,
containing 2.5 g proteose peptone, 2.5 g bacto
peptone, 5.0 g meat extract, 2.5 g brain-heart
bouillon, 3.0 g yeast extract, 3.0 g glucose, 3.0
sodium chloride, and 25 g agar per litre. The PH
of both mineral and nutrient media was adjusted
to 7.2-7.4 by 20% NaOH. The media were sterilized by autoclaving at 121°C for 30 min.
6A2NS was obtained from Bayer AG, Leverkusen, Germany. All mineral salts were of aria-
231
lytical grade and were product of Merck AG,
Darmstadt, Germany. Proteose, meat extract and
brain-heart bouillon were also supplied by Merck.
Bacto peptone, yeast extract and agar were purchased from Difco, Detroit, MI.
3.3. Mixed culture deueloping from bacterial monocultures
Bacterial monocultures from stock cultures
were transferred to nutrient agar plates and incubated at 30°C for 48 h. After growth, bacterial
mass was separately transferred to culture tubes
with a mineral medium containing 0.125 g/1
6A2NS and incubated at the same temperature
without shaking for 96 h. Bacterial suspensions
were then transferred to Erlenmeyer flasks on a
shaker. After 96 h bacterial cells were harvested
by centrifugation and joined in homoIogous
medium. A newly formed mixed culture incubated on a rotary shaker at 30°C degraded the
added amount of 6A2NS after 4-6 days.
Further transfers were performed after centrifugation of cell suspension following exhaustion of substrate using the entire biomass as an
inoculum for the next subculture.
3.4. Analytical methods
Supernatants of previously centrifuged cell
suspensions (6700 X g for 10 min) were analysed
t o estimate changes in the concentration and
structure of 6A2NS during degradation. Routine
control of samples during the adaptation and
degradation processes was performed with a Unicam SP 1805 UV Spectrophotometer. Changes in
optical densities at wavelengths 200 and 244 nm
were compared with those from the calibration
curve prepared from 6A2NS solutions in the concentration range 0.05-0.75 g/l.
High-performance liquid chromatography
(HPLC) analysis was done at intervals during
degradation at room temperature. A LKB Bromma, Sweden HPLC instrument equipped with a
LKB HPLC pump 2150-012, a Rheodyne 7125
sample injector with a sample loop of 20 pl, a
LKB variable wavelength monitor 2151-002 and
a LKE! computing integrator were used. The column (4.250) was prepacked with 5 l m LiChrosorb NH, (Merck). The eluent was a mixture of
H,O : MeOH :50% (NHJ2SO, (80 :20: 0.1) with
a flow rate of 0.7 ml per min. The UV detector
was set at 226 nm.
Total organic carbon (TOC) was determined
by standard methods [12] with a Shimadzu TOC500 analyzer equipped with an ASI-502 automatic
sample injector.
The sulphate concentration in supernatants
was analysed by the colorimetric method based
on the reaction of barium chloranilate with sulphate ion at pH 4 [13]. Photometric measurement
was performed at 530 nm.
6A2NS and its metabolites were analysed by
infrared (IR) spectrophotometry on previously
lyophilized samples of supernatants with a Perkin
Elmer 257 Gratin Infrared Spectrophotometer.
3.5. Measurement of growth
The growth of bacterial culture was followed
by measuring the optical density (OD) and dry
weight of mixed cultures. OD was determined
spectrophotometrically at 546 nm with a Unicam
SP 1805 UV Spectrophotometer. 10 ml samples
of culture liquid were then centrifuged at 5500 X g
for 25 min. The cell pellet was washed once with
distilled water, dried at 105°C for 24 h and then
weighed. Measurements were performed in triplicate. Results are expressed as mean values.
4. RESULTS
4.1. Estimation of the minimal number of members
of the 6A2NS degrading culture
The eleven bacterial monocultures were incubated in the liquid mineral medium containing
0.125 g/1 6A2NS on a rotary shaker at 30°C to
test their ability to degrade the compound. Not
one monoculture degraded it individually. Comparable results were obtained with mixed cultures
consisting of two or three strains. Some mixed
cultures with four or more members which included representatives from every genus degraded 6A2NS at a concentration of 0.125 g/l.
(Fig. 1). However, degradation was incomplete
and resulted in accumulation of intermediates
causing loss of degrading ability after several subcultures. The intermediates were identified by
232
ion paired-HPLC as inorganic nitrates, mostly
ammonium nitrate, with maximum absorbance at
200 nm of UV spectrum. The degradation process in such cultures was also disturbed by an
increase of the 6A2NS concentration. Only the
mixed culture with eleven members showed a
degradation ability, stable under previously mentioned conditions of culturing through numerous
subcultures (Fig. 1).
4.2. Adaptation of mixed culture to increasing
6A2NS concentrations
Single strains were introduced into the community in equal biomass quantities. The newly
formed community was submitted to a progressive adaptation in batch cultures to develop a
stable mixed culture. The first subculture in the
mineral medium containing 0.125 g/16A2NS took
4-6 days before the compound completely disappeared from the medium. The concentration of
6A2NS was gradually increased in subsequent
subcultures. The best results in culture adaptation were achieved by three consecutive cultivations in a medium with the same concentrations
of 6A2NS before the concentration was increased
(Fig. 2).
O ’ * ’ b
Time(hrs1
Fig. 2. Adaptation of a mixed culture to growth in a mineral
medium containing increasing concentrations of 6A2NS from
0.125-1.00 g/l during sixteen consecutive subcultures. cuttures were incubated on a rotary shaker at 30°C.
During the adaptation period mutual interactions between community members had to be
restored. Fifteen subcultures were sufficient to
achieve an optimal proportion of strains, yielding
a stable mixed culture capable of 6A2NS degradation (Fig. 3). The relative participation of
Fig. 1. UV spectra of 6A2NS degradation products obtained after degradation by four different mixed cultures. 1, F l a ~ ~ o b a c t e ~ u ~
devorans LOV866, Bacillus circulans LOV904, Pseudomonas aeruginosa LOV837 and P. desmolytrca LOV853; 2. Flal>obacterium
der,orans LOV922, Bacillus cereus LOV886, Pseudomonas aerugrnosa LOV835, P. desmolytica LOV848 and P. cepacia LOV877; 3.
Flai,obacterium indoltheticum LOV912, Bacillus cereus LOV832, Pseudomonas aeruginosa LOV835, P. aeruginosa LOV837 and p.
desmolytica LOV853; 4, complete mixed culture with eleven members. A: UV spectrum of 6A2NS; B, C, D: UV spectra of
unidentified products after incomplete degradation of 6A2NS; E. UV spectrum of mineral medium after complete degradation of
6A2NS.
233
The organic sulphur which originated from the
compound degradation was converted to sulphate. No intermediate was detected by UV of
HPLC analyses. The results were confirmed by
IR spectrophotometry showing a complete disappearance of the aromatic compound (1500 cm-'1
and hydroxy and amino groups (3400 cm-') with
sulphate (1150 an-'), nitrate (860 cm-'1 and
nitrite (810 cm-') as final products of degradation.
strains in mixed culture was followed by plating
of culture samples on nutrient agar. The strains
could be distinguished on the basis of their morphology and the colour of their colonies. About
85% of the entire biomass resulted from growth
of Flavobacterium strains, Bacillus circulans and
Pseudomonas desmolytica LOV853. The other
strains remained present in small proportions and
were not removed from the cultures even after
extensive subculturing. Incomplete mixed cultures which lost some 'escorting' members appeared to be unstable with respect to degradation
potential which resulted in acccumulation of nitrates, as described earlier.
5 . DISCUSSION
Studies on biodegradation of aminonaphthalenesulphonates (ANS), are usually performed with
single strains isolated from various natural habitats exposed to that class of compounds [7,8,14].
Most of those sources contain communities of
numerous organisms. Continuous exposure to
naphthalene and its derivatives causes natural
selection of the organisms. Therefore isolated
microorganisms may have been adapted to ANS
4.3. 6A2NS degradation and product analysis
A well adapted mixed culture of eleven strains
was able to degrade 6A2NS efficiently and at a
constant rate. Degradation of 1 g/l of 6A2NS
started immediately and was complete after 6 h
(Fig. 4). The concentration of 6A2NS and total
organic carbon decreased linerarly. Increase in
biomass concentration was 0.240 g/g of 6A2NS.
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Fig. 3. Relative participation, based on biomass, of the individual bacterial strains in a mixed culture during adaptation to 6A2NS.
Flavobacterium devorans ( 0 ) ; F. indoltheticum ( m ); Bacillus cereus (0); B. circulans (0); Pseudomonas aeruginosa ( A h P.
desmolytica ( v h P. cepacia ( A ).
234
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Fig. 4. Growth of the mixed culture of all eleven strains in
mineral medium containing 1 g/l of 6A2NS as the sole carbon
and energy source. The incubation was carried out at 30°C on
a rotary shaker, pH 7.2. Cell growth was expressed as OD at
546 nm ( A ); concentration of 6A2NS measured by HPLC ( 0 ) ;
sulphate ( A 1; TOC ( 0 ) .
and acquired the ability to metabolize these compounds, although it is sometimes necessary to
support growth with additional nutrients such as
vitamins, glucose, certain intermediates of ANS
degradation, etc. [6,71.
Degradation by bacterial monocultures usually
takes a long time. Ohe and Watanabe [8] reported degradation of 0.1% 2AlNS by Pseudomonas sp. TA-1 which took 90 h. Another
problem with single-strain degradation is its possible inhibition by accumulation of non-degradable intermediates [14]. In some cases this may be
due to incomplete genetic information for complete degradation pathways, especially in the case
of complex compounds [151.
Results obtained with mixed cultures have b e e n
more promising. Nortemann et al. 161 used a
mixed culture with two different Pseudornonas
strains. Interaction between two community
members, in which one member’s product of
6A2NS transformation, 5-aminosalicylate, was a
substrate for the other, resulted in degradation of
4.5 m M of 6A2NS (approx. 1 g/l) within 30 h.
However, the interspecies transfer could be obstructed by autooxidation of 5-amino-salicylate
and its polymerization. To avoid a loss of degrading activity, the microorganisms had to be maintained on nutrient broth agar, containing ANs
(or some intermediates to permit growth of possible escorting strains). Diekmann et al. [9] isolated
another, previously unobserved strain from t h e
Same culture. This latter strain, which was a more
efficient aminosalicylate degrader, indicated that
the role of escorting strains should not be neglected. Tagger et al. [161 described a nine-member bacterial community capable of degrading
naphthalene. Two dominant members grew With
naphthalene as the sole carbon and energy Source,
while seven other strains used their metabolic
products.
The mixed culture used in this study was cornposed of eleven different strains belonging to
Seven species and three different genera (Table
1). The culture was composed of stable number
and proportion of community members. Although
none of the strains was able to degrade 6A2Ns
individually each single strain played a part in t h e
degradation process and exibited a high degree of
cooperation. Even after numerous subcultures t h e
culture composition remained unchanged. Once
its composition was defined, the culture could
simply be restored by combining the individual
strains over a relatively short period of time,
using a mineral medium containing 6A2NS as the
sole source of carbon, nitrogen and energy. T h e
process, which started with bacterial monocultures maintained on nutrient agar without 6A2Ns,
resulted in a stable mixed culture capable of
degrading 6A2NS, within a period no more than
three weeks (Fig. 2). Participation of individual
strains, initially introduced in equal proportions,
gradually changed during the adaptation period.
In a well-adapted culture 85% of the entire
235
biomass consisted of Flauobacterium strains,
Bacillus circulans and Pseudomonas desmolytica
LOW53 (Fig. 3). The culture degraded 1 g/l of
6A2NS in 6 h, which was much faster than observed by other authors [6,8]. The intermediates
described by Nortemann et al. [6] and Ohe et al.
[14] were not observed during the degradation
process by any of the analytical methods used in
the present study. This points to well-synchronized activity of community members with probably simultaneous consumption of both sulpho and
amino groups, ring cleavage and futher transformation of the compound to complete mineralization.
As industrial water treatment technologies call
for microbial cultures capable of fast degradation
of xenobiotic compounds without transformation
to other, more dangerous non-degradable intermediates with a low increase of biomass, further
investigations are to be focussed on stability of
the culture under conditions of varying pH, oxidation, temperature, concentration of 6A2NS and
Some other naphthalene derivatives, and its possible application to the treatment of industrial waters.
ACKNOWLEDGEMENT
We are grateful to Dr. B. GovorEin for the
indentification and characterization of strains
used in this study.
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