change in bacterial community during biodegradation of aniline

Journal of Applied Microbiology 1998, 84, 859–864

Change in bacterial community during biodegradation ofaniline

K. Tani, M. Masuhara, N. Welikala, N. Yamaguchi and M. NasuFaculty of Pharmaceutical Sciences, Osaka University, Osaka, Japan

6053/01/97: received 24 September 1997 and accepted 30 September 1997

K. TANI, M. MASUHARA, N. WELIKALA, N. YAMAGUCHI AND M. NASU. 1998. The response ofriver water microbial communities to chemical compounds was monitored underlaboratory conditions using aniline as a model. Bacteria were collected from unpollutedand polluted sites. Bacterial abundance (plate and total direct counting) and its relation toaniline biodegradation was examined. Colony hybridization with 16S rRNAoligonucleotide probes was used to study the changes in microbial community structureduring biodegradation of aniline. The changes in bacterial abundance and communitystructure were related to biodegradation of aniline. Burkholderia–Pseudomonas(rRNA group III), an authentic Alcaligenes group became dominant despite the initialdifferences in the microbial communities, suggesting that these genera are themain aniline degraders in the aquatic environment.

INTRODUCTION

With rapid industrial development, many industries haveproduced a wide range of chemicals and some of these com-pounds have caused environmental pollution. The improperdischarge of wastes, accidental spills or deliberate release ofthese chemicals have all contaminated soils, undergroundwaters and aquatic environments, and pollution will continueinto the future. Microbial degradation of these pollutants isthe major natural process that leads to detoxification andelimination from the environment.

The microbial community structure changes in responseto the flow of pollutants and studies of such changes couldoffer valuable information on the effect of these pollutants.Microbial community changes are difficult to monitor asnumerous factors are involved but, generally, if the pollutantis toxic, a gradual decline in the number of surviving species isexpected. Alternatively, enrichment of particularly successfulpopulations may result. This study examined how microbialcommunities from river water responded to the stress ofaniline under laboratory conditions.

MATERIALS AND METHODS

Sampling sites

River water samples were collected at three sites in Osaka,Japan. The Minoh river originates in the Minoh Mountains

Correspondence to: Katsuji Tani, Faculty of Pharmaceutical Sciences, OsakaUniversity, 1-6, Yamada-oka, Suita, Osaka, 565, Japan.

© 1998 The Society for Applied Microbiology

and joins the Ina river about 1·5 km before Kuwazu bridge.Takiue (lat. 34°51?9ýN, long. 135°28?49ýE), considered to bean unpolluted site, is located in the Minoh National Park. Atthis point, the river is narrow, shallow and fast flowing. Thestream bed is very rocky and the water is exposed to neitherdomestic nor industrial effluents, Kuwazu (lat. 34°46?47ýN,long. 135°25?42ýE) on the Ina river is an industrial area andconsidered to be fairly polluted. At this point, the river iscomparatively wide. Kitahashi (lat. 34°41?27ýN, long.135°32?3ýE) is located in a commercial area (Osaka BusinessPark) and considered to be highly polluted. Domestic waterflows into the upstream of this river.

Test system

River water micro-organisms were trapped by 0·40 mmNuclepore polycarbonate membrane filters (Corning Costar)and these filters were transferred into test tubes (25×200mm) containing 25 ml of artificial river water (mineral solu-tion used for BOD measurement ; 21·75 mg K2HPO4, 8·5 mgKH2PO4, 17·7 mg Na2HPO4, 1·7 mg NH4Cl, 22·5 mg MgSO4,27·5 mg CaCl2, 250 mg FeCl3 per litre H2O ; pH 7·2) and 20ppm of aniline as the sole source of carbon. Test tubes wereincubated at 25 °C and shaken in the dark for a period of 14days. A sample containing micro-organisms without anilineand a sample containing only aniline were also incubated asa blank and a control respectively. Aliquots were withdrawnperiodically, and bacterial number and Total Organic Carbon(TOC) concentrations were determined.

860 K. TANI ET AL.

Measurement of bacterial number and total organiccarbon

Colony forming units (cfu) of samples were determined byspreading on 1/10 diluted PYG agar plate containing 0·5 gpolypeptone (Nihon Seiyaku Co.) ; 0·25 g yeast extract (DifcoLaboratories) ; 0·1 g glucose (Nacalai Tesque, Inc.) ; and 15 gagar (Nacalai Tesque, Inc.) l−1 H2O (Yoshikura et al. 1981).Plates were incubated at 25 °C for 1 week before counting.

Total Direct Counts (TDC) of samples were measured bythe Hobbie method (Hobbie et al. 1977) with slight modi-fication. Samples were fixed in 2% paraformaldehyde, andstained with DAPI (4?,6-diamidino-2-phenylindole ; Sigma)(Porter and Feig 1980) for 15 min (final concentration : 1ppm). The cells were held on 0.20 mm Nuclepore poly-carbonate membrane filters (Corning Costar) with a slightvacuum. Subsequently, TDC was measured using BH-2 epi-fluorescence microscope (Olympus) equipped with a dichroicmirror (DM400), and absorption filter (L420) and an exci-tation filter (UG1). Total carbon and total inorganic carbonwas determined by TOC-500 analyser (Shimadzu). The dif-ference between total carbon and inorganic carbon was takenas total organic carbon.

Isolation of bacterial strains and examinations oftheir aniline degradability

About 100 bacterial colonies were isolated from each 1/10diluted PYG agar plate at random after the cfu count wastaken and transferred onto new 1/10 diluted PYG agar plates.Isolates were cultured at 25 °C for testing for aniline degrad-ability using a spectrophotometer and identification by colonyhybridization.

Table 1 Oligonucleotide probes designed by multiple sequence alignment—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Probe name Oligonucleotide sequence Location—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Pseudomonas (rRNA group I) 5?-ATTTC AGCCT ACCAC CTTAA-3? 1467–1486*2

Vibrio–Aeromonas 5?-ACGAC GCACT TTTTG GGATTCGCTC ACTAT CGCAA G-3? 1262–1297*1

Flavobacterium–Cytophaga 5?-AGGTA CCCCC AGCTT CCATG GCT-3? 1408–1434*1

Burkholderia–Pseudomonas (rRNA group III), authentic 5?-GTGTG CCGGT TCTCT TTCGA GCAC-3? 1022–1044*1

Alcaligenes

Acinetobacter 5?-GCGCC ACTAA AGCCT CAAAG GCC-3? 836–858*1

EUB338 5?-GCTGC CTCCC GTAGG AGT-3? 338–355*1

—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

*1 � Homologous position on Echerichia coli 23S rRNA ; *2 � Homologous position on Echerichia coli 16S rRNA.

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 859–864

Aniline degradability of isolates was examined by the fol-lowing method ; all isolates were inoculated into 1/10 dilutedPYG liquid medium (0·5 g polypeptone ; 0·25 g yeast extract ;0·1 g glucose per litre H2O) and precultivated for 2 days at25 °C. Harvested bacterial cells were collected by cen-trifugation and resuspended in saline to reach a final opticaldensity of 5. Of aliquots, 100 ml, were withdrawn and trans-ferred into a carbon-limited medium (2·0 g (NH4)2SO4, 2·0 gKH2PO4, 3·0 g Na2HPO4 · 12 H2O, 0·01 g MgSO4 · 10 H2Oin 1000 ml H2O) containing 40 ppm of aniline as sole sourceof carbon. The samples were incubated for 3 days at 25 °C inthe dark with shaking and the cleavage of the benzene ringof aniline was used to measure degradation of aniline usingspectrophotometer model U-2000 (HITACHI). Absorbanceof culture was measured at 280 nm.

Oligonucleotide probes

Oligonucleotide probes for identification of bacterial groupsare listed in Table 1. Five probes were used, i.e. EUB338(Amann et al. 1990), PI, BPA, FC and VA that are specific todomain Bacteria, Pseudomonas rRNA group I, Burkholderia–Pseudomonas rRNA group III, authentic Alcaligenes, Flavo-bacterium–Cytophaga and Vibrio–Aeromonas groups ofbacteria, respectively. The specificity of these probes havebeen confirmed by colony hybridization (Yamaguchi et al.1996).

Colony hybridization

Autoclaved nitrocellulose filters (BA85 ; pore size, 0.45 mm ;Schleicher & Schuell Co.) were placed on 1/10 diluted PYG

CHANGE IN BACTERIAL COMMUNITY 861

agar plates and moistened by 1/10 diluted PYG liquidmedium. All isolates were inoculated onto these moisturizedfilters and cultivated overnight at 25 °C. Filters were treatedwith 10% sodium dodecyl sulfate (SDS) for 15 min at roomtemperature. Subsequently, filters were immersed in 3×SSC(1×SSC is 0·15 mol l−1 sodium chloride, 0·015 mol l−1

sodium citrate (pH 7·0)), at 65 °C for 15 min. The membranefilters were allowed to air dry, and baked at 80 °C for 2 h.Baked filters were washed vigorously twice in 3×SSC, 0·1%SDS at 65 °C for 90 min. Then these filters were transferredto 10 ml of hybridization buffer (1 mol l−1 sodium chloride,0·1% SDS, 100 mg salmon sperm DNA (Sigma) per ml) inplastic bags. Membranes were prehybridized overnight at50 °C, followed by the addition of 32P-labelled oligonucleotide(total radioactivity of 1×107–3×107 cpm) ; and then hybrid-ized for 24 h at 42 °C (EUB338 probe) or 50 °C (BPA, FC,and PI probes) or 60 °C (VA probe). The filters were washedtwice at 70 °C for 15 min in 0·25 mol l−1 sodium chloride :0·1% SDS (FC probe) ; in 1 mol l−1 sodium chloride : 0·1%SDS (PI probe) ; in 0·5 mol l−1 sodium chloride : 0·1% SDS(VA probe) ; or washed twice for 15 min at 65 °C in 1 moll−1 sodium chloride : 0·1% SDS (EUB338) and BPA probes).The filters were air-dried and exposed to X-ray film.

RESULTS

Quality of the water samples

The quality of the river water samples are shown in Table 2.Colony forming units, TDC, cfu/TDC and TOC valuesincreased in river water samples, beginning from the leastpolluted Takayama and fairly polluted Kuwazu to the highlypolluted Kitahashi. Significant differences were observed incfu and cfu/TDC while TDC was least affected by the levelof pollution.

Biodegradation of aniline

Aniline was degraded in the artificial river water by theKuwazu and Kitahashi bacterial samples much faster than by

Table 2 Quality of the river water samples—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Sampling Atmospheric Water cfu TDC cfu/TDC TOCsites temp. (°C) temp. (°C) pH (cells ml−1) (cells ml−1) (%) (ppm)—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Takiue 18·5 17·6 8·1 7·6×103 1·4×106 0·54 1·9Kuwazu 27·5 21·3 7·9 1·2×105 3·3×106 3·6 2·6Kitahashi 25·0 22·9 7·1 1·1×106 7·3×106 15 6·5—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Sampling date : 29 September 1993.


Fig. 1 Biodegradation of aniline. �, Takiue ; ž, Kuwazu ; �,Kitahashi

the Takiue sample (Fig. 1). By the third day, the bio-degradation of the aniline in the Kuwazu and Kitahashi sam-ples was almost completed, although biodegradation in theTakiue sample had not begun. In the Takiue sample, bio-degradation began on the third day and was almost completeon the 10th day. The maximum rate of biodegradation in theTakiue sample was reached by the fifth day.

Change in bacterial abundance and cfu/TDC ratio

The introduction of aniline into the test system broughtdifferent reactions from different communities of bacteria(Table 3). Thus, the cfu, TDC and cfu/TDC values of theTakiue sample continued to increase throughout incubation.The increase in the cfu/TDC sample was especially signifi-cant. A 100-fold increase in the cfu value was observed onthe third day in the Kuwazu sample, and increase in thecfu/TDC value was observed which corresponded to the cfuincrease. However, no drastic changes in the cfu and

862 K. TANI ET AL.

Table 3 Change in bacterialabundance in the test system

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Sampling Days after cfu TDC cfu/TDCsites incubation (cells ml−1) (cells ml−1) (%)—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Takiue 0 7·6×103 1·4×106 0.54

3 2·4×105 1·4×106 1010 4·1×106 1·6×107 25

Kuwazu 0 1·2×104 3·3×106 3·63 1·4×106 5·6×106 25

10 7·6×105 5·4×106 14

Kitahashi 0 1·1×106 7·3×106 153 1·4×106 4·3×106 33

10 4·1×105 4·2×106 9·8—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

cfu/TDC values of the Kitahashi sample were observed dur-ing the first 3 days.

Change in relative abundance of aniline degraders

The relative abundance of aniline degraders in all the com-munities at the inception was insignificant constituting lessthan 1% of the colonies studied (Table 4). This was com-pletely reversed in the Kuwazu and Kitahashi communitiesby the third day, with over 90% of bacteria being anilinedegraders. The amount of aniline degraders in these com-munities dropped to their original levels on the 10th day afterincubation. The Takiue community, on the other hand, didnot show any significant increase in aniline degraders untilday 10, when they constituted approximately 20% of thecommunity.

Change in microbial community structure duringincubation

Initial community structures as revealed by colony hybrid-ization indicate dominance of BPA in the Takiue and Kuwazucommunities, constituting approximately 30% of the popu-

Table 4 Abundance of aniline degraders—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Sampling Aniline degraders (%)—––––––––––––––––––––––––––––––––––––––––

sites 0 d 3 d 10 d—–––––––––––––––––––––––––––––––––––––––––––––––––––––Takiue ³1 (n � 94) ³5 (n � 94) 20 (n � 73)Kuwazu ³1 (n � 77) ×90 (n � 94) ³1 (n � 49)Kitahashi ³1 (n � 88) ×90 (n � 92) ³1 (n � 48)—–––––––––––––––––––––––––––––––––––––––––––––––––––––


lation (Fig. 2). The initial Kitahashi community, however,differed in that both BPA and FC groups contained approxi-mately 25% of the population each. Approximately 50–60%of bacteria were not accounted for by probes in all communi-ties.

In the Takiue community, BPA increased to over 80% onthe third day, after which no significant decrease wasobserved. The amount of bacteria unaccounted for by probesdropped with the increase in PA and stayed at a constant20%.

In the Kuwazu and Kitahashi communities, the relativeabundance of BPA increased to approximately 60–80% onthe third day of incubation, after which a drop was observedon the 10th day. In all three communities, dominance of BPAcoincided with a period of rapid biodegradation of aniline.The bacteria unaccounted for by the probes increased as theabundance of BPA decreased. In the Takiue and Kuwazucommunities, VA and FC groups were not significant anddid not change during incubation. In the Kitahashicommunity, on the other hand, the FC group constituted amajor fraction in the beginning but diminished on the thirdday after incubation.

Composition of aniline degraders

The composition of aniline degraders indicated the pre-dominance of the BPA group during peak biodegradationthat coincided with the 10th day after incubation for theTakiue sample and the third day after incubation for theKuwazu and Kitahashi communities. It consisted of 50–90%of all aniline degraders (Table 5).

DISCUSSION

It has been reported that cfu/TDC can be used as an indicatorof the degree of water pollution (Nasu et al. 1992) ; a con-

CHANGE IN BACTERIAL COMMUNITY 863

Fig. 2 Change in bacterial community structure determined by 32P labelled 16S rRNA probes using colony hybridization. (a) Takiueinoculum (b) Kuwazu inoculum (c) Kitahashi inoculum. Ž, P(I) : Pseudomonas (rRNA I) ; , VA : Vibrio–Aeromonas ; , FC : Flavobacterium–Cytophaga ; ;, BPA : Burkolderia–Pseudomonas (rRNA III), authentic Alcaligenes ; , NI : bacteria unaccounted for by probes

tention which is further supported by this study. The Takiue,which is considered the least polluted river, had the lowestcfu/TDC ratio, while the Kuwazu and Kitahashi had higherratios in increasing order.

Aniline and aniline derivatives are used in pesticides, dyes,plastics, pharmaceuticals, etc., and are found in coal tar as anatural product (Anson and MacKinnon 1984 ; Konopka etal. 1989). These chemical compounds are eliminated fromthe environment by evaporation, photo-oxidation, chemicalbinding and biodegradation. Biodegradation by bacteria,however, has been found to be the most significant mech-anism of aniline removal from aquatic environments. Thus,Lyons et al. (1984) found higher aniline biodegradationactivity in polluted pond water than in unpolluted water.

The slow rate of biodegradation of aniline in unpolluted

Table 5 Bacterial composition ofaniline degraders

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Samplingstations 0 d 3 d 10 d—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––Takiue 0/94* 6/94 BPA 2/6 13/73 BPA 12/13

VA 1/6 NI 1/13NI 2/6

Kuwazu 0/77 94/94 BPA 67/94 0/49FC 5/94NI 15/94

Kitahashi 0/88 89/92 BPA 43/89 0/48VA 1/89NI 26/89

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

* � No. of aniline degraders/no. of isolates, BPA � Burkholderia–Pseudomonas (rRNAIII), authentic Alcaligenes ; FC � Flavobacterium–Cytophaga ; VA � Vibrio–Aeromonas ; andNI � Not identified (hybridized with EUB338 but not with other probes).


Takiue water and the higher rates of biodegradation in thepolluted Kuwazu and Kitahashi water are in agreement withthe findings of Nasu et al. (1993). The differing rates ofbiodegradation in environments with different degrees ofpollution could be used as an indicator for the degree ofpollution of a given aquatic environment.

The changes in cfu, TDC, cfu/TDC, and the relativeabundance of aniline degraders were in agreement with therate of aniline biodegradation in each environment. Thecfu/TDC of the Takiue sample had increased 50-fold fromthe initial value by the end of biodegradation on the 10th day.In contrast, it increased only sixfold in the Kuwazu sampleand twofold in the Kitahashi sample by the third day fol-lowing incubation, which also coincided with complete bio-degradation of aniline. This indicates that microflora in

864 K. TANI ET AL.

unpolluted water adapt dramatically to chemical organic com-pounds while those in polluted water undergo less changesince they have already adapted to these compounds.

The abundance of the BPA group increased as the rate ofbiodegradation increased and fell when the biodegradationwas completed. This clear relationship between aniline bio-degradation and relative abundance of the BPA group in allthree communities—in spite of their differences in abun-dance, composition and degree of pollution—suggests theimportance of this group for aniline degradation in the aquaticenvironment.

This work illustrates the very dynamic changes that takeplace in microbial populations during the biodegradation ofchemical compounds. Although these results were obtainedunder laboratory conditions, similar phenomena are likely tooccur in the natural environment when xenobiotic com-pounds are biodegraded.

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change in bacterial community during biodegradation of aniline

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