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Biodegradation of 4-aminobenzenesulfonate by Ralstonia sp. PBA and Hydrogenophaga sp. PBC isolated from textile wastewater treatment plant
Chemosphere 82 (2011) 507–513
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Biodegradation of 4-aminobenzenesulfonate by Ralstonia sp. PBA and Hydrogenophaga sp. PBC isolated from textile wastewater treatment plant Han Ming Gan a, Shafinaz Shahir b, Zaharah Ibrahim b, Adibah Yahya a,⇑ a b
Department of Industrial Biotechnology, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Department of Biological Sciences, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
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Article history: Received 10 July 2010 Received in revised form 27 October 2010 Accepted 28 October 2010 Available online 20 November 2010 Keywords: Hydrogenophaga Ralstonia 4-Aminobenzenesulfonate Biodegradation 16S rDNA
a b s t r a c t A co-culture consisting of Hydrogenophaga sp. PBC and Ralstonia sp. PBA, isolated from textile wastewater treatment plant could tolerate up to 100 mM 4-aminobenzenesulfonate (4-ABS) and utilize it as sole carbon, nitrogen and sulfur source under aerobic condition. The biodegradation of 4-ABS resulted in the release of nitrogen and sulfur in the form of ammonium and sulfate respectively. Ninety-eight percent removal of chemical oxygen demand attributed to 20 mM of 4-ABS in cell-free supernatant could be achieved after 118 h. Effective biodegradation of 4-ABS occurred at pH ranging from 6 to 8. During batch culture with 4-ABS as sole carbon and nitrogen source, the ratio of strain PBA to PBC was dynamic and a critical concentration of strain PBA has to be reached in order to enable effective biodegradation of 4-ABS. Haldane inhibition model was used to fit the degradation rate at different initial concentrations and the parameters lmax, Ks and Ki were determined to be 0.13 h1, 1.3 mM and 42 mM respectively. HPLC analyses revealed traced accumulation of 4-sulfocatechol and at least four unidentified metabolites during biodegradation. This is the first study to report on the characterization of 4-ABS-degrading bacterial consortium that was isolated from textile wastewater treatment plant. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction 4-Aminobenzenesulfonate (4-ABS) is one of the most commonly found sulfonated aromatic amines. It is widely used as an intermediate in the production of textile dyes, sulfonamide drugs, optical brighteners and pesticides. In nature, the biodegradation of 4-ABS is problematic. Unless there is a specific transport system for 4-ABS, the negatively charged sulfonate group would prevent uptake of the substrate through the bacteria membrane (Hwang et al., 1989). Even if bacteria develop an efficient uptake mechanism for 4-ABS, the thermodynamic energy barriers exerted by both the resonance-stabilized aromatic ring and C–S bond of 4-ABS have to be overcome in order to harness energy from this compound (Wagner and Reid, 1931). Furthermore, 4-ABS could exhibit bacteriostatic activity by inhibiting the folate synthesis pathway which is crucial for the maintenance of deoxynucleotide precursors pool and DNA synthesis (Brown, 1962; Dallas et al., 1992). To date, the biodegradation of 4-ABS is still a rare occurrence in the microbial community found in natural soil, polluted ⇑ Corresponding author. Address: Research Laboratory 2, Level 2, Building C08, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. Tel.: +60 75534157; fax: +60 75531112. E-mail addresses: [email protected] (H.M. Gan), shafi[email protected] (S. Shahir), [email protected] (Z. Ibrahim), [email protected] (A. Yahya). 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.10.094
harbor sediment and even activated sludge from some wastewater treatment plants (Alexander and Lustigman, 1966; Tan et al., 2005; Yemashova and Kalyuzhnyi, 2006). The consumption of most sulfonated aromatic amines commonly will result in the rapid excretion from organism (Greim et al., 1994). However, study in rats showed that 4-ABS has the longest retention time following ingestion as compared to its other counterparts (Honohan et al., 1979) thus making it the most significant compound to study. Under constant exposure to this compound, some negative effects of 4-ABS have been reported including hyperactivity in rats (Goldenring et al., 1982) and significant decrease in the nitrogen transformation processes in soil (Topac et al., 2009). One of the most important sources of 4-ABS is sulfonated azo dyes. In textile industries, sulfonated azo dyes are commonly used due to its stronger binding to the fiber and lower toxicity as compared to its non-sulfonated analog. Biological treatment of textile wastewater is highly favored due to its cost-effectiveness and higher sustainability as compared to physicochemical treatment (Pearce et al., 2003). In many of the studies found in the literature, the biological treatment of textile waste involves a two-stage process: decolorization and mineralization. In the decolorization stage, an anaerobic condition is provided to stimulate reductive cleavage of azo bond by redox active compounds of bacterial origin
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to release the parent aromatic amines. In the mineralization stage, air or specifically, oxygen, is introduced to enable further biodegradation of the aromatic amines. Through the actions of aromatic ring hydroxylation dioxygenase enzyme and beta-ketoadipate pathway (Harwood and Parales, 1996; Parales and Resnick, 2006), it is possible to convert sulfonated aromatic amines into simple compounds such as carbon dioxide, ammonium and sulfate. Under constant exposure to industrial effluent containing 4ABS, some microbial degradation of 4-ABS has been reported in the last two decades starting with a defined co-culture consisting of Hydrogenophaga palleroni S1 and Agrobacterium radiobacter S2 (Feigel and Knackmuss, 1988). Strain S1 was later reclassified as Hydrogenophaga intermedia S1 (Contzen et al., 2000) after further taxonomic studies. Some pure bacterial strains capable of degrading 4-ABS such as Pseudomonas paucimobilis (Perei et al., 2001), A. radiobacter PNS-1 (Singh et al., 2004) and Pannonibacter sp. W1 (Wang et al., 2009) have also been reported. A pure culture SAD 4i has been isolated from textile wastewater which can utilize 4-ABS as its carbon source but there is no report on its identification (Coughlin et al., 2002). In this study, the biodegradation of 4-ABS using two-membered bacterial consortium isolated from a pilot scale of textile wastewater treatment plant is investigated (Ibrahim et al., 2009). The strains have been identified as Hydrogenophaga sp. PBC and Ralstonia sp. PBA. This consortium can utilize 4-ABS as the sole carbon, nitrogen and sulfur source with ammonium and sulfate as part of the end products. In addition, there is no complete inhibition in growth even at 100 mM 4-ABS which underscores the potential of this consortium in treatment of severely 4-ABScontaminated wastewater. Results obtained in this work provide the much-needed fundamental studies for the biodegradation of aromatic amines generated from textile wastewater treatment. 2. Materials and methods 2.1. Culture condition Full strength (1) phosphate-buffered mineral (PB) medium consisted of 0.09 mM MgSO4, 0.042 mM KCl, 7.5 mM NaHPO4, 7.5 mM KHPO4, 15 mM KH2PO4, 0.0068 mM FeCl3 and 0.1 mM CaCl2. Required amount of neutralized 4-ABS from stock solution of 86.6 g L1 (500 mM) was added to the medium as sole carbon and nitrogen source. For the study of sulfate release, MgSO4 was replaced with MgCl2. In some cases, nitrogen source in the form of (NH4)2SO4 was added to a final concentration of 2.5 mM to give PBN medium. To assess the influence of vitamin supplement on population dynamics, p-aminobenzoate and biotin were added to a final concentration of 20 mg L1 and 2 lg L1 respectively. 2.2. Isolation of 4-ABS-degrading bacteria Sample was withdrawn from the aerobic reactor of a textile wastewater treatment plant (Ramatex Textile Industrial, Malaysia). The seed inoculum was enriched in repeated batch cultures containing PB medium and 3 mM 4-ABS as sole carbon and nitrogen source. The enrichment was performed at 30 °C and 150 rpm in an orbital shaking incubator. Growth was observed indirectly from the turbidity readings and once sufficient turbidity was achieved, 1% (v/v) cultivated inoculum was transferred into new medium. After 10 successive transfers, serial dilution was performed and the inoculum was plated on solid PB medium with 4-ABS as sole carbon and nitrogen source for the purpose of bacteria isolation. Single colonies were then transferred individually using a sterile toothpick onto the same solid medium to confirm their 4-ABS degrading ability. Then, subsequent streaking was performed on
nutrient agar to obtain single colonies. Two strains designated PBA and PBC were isolated from the agar plates. 2.3. Characterization and identification of bacteria The isolates were first characterized using Gram-staining. Then identification was performed by 16S rDNA sequence analysis. Genomic DNA was extracted using Qiagen DNAeasy Blood and Tissue Kit according to the manufacturer’s instruction. The sequence of 16S rDNA was amplified by the polymerase chain reaction (PCR) method using primer fD1 and rD1 without linker sequences (Weisburg et al., 1991). The PCR condition consisted of an initial denaturation step at 94 °C for 5 min which was followed by 30 cycles of 94 °C (1 min), 50 °C (30 s), 72 °C (1.5 min), plus a final 10 min-chain elongation cycle at 72 °C. The amplicons were purified and sequenced. Then, the sequences were subjected to the BLAST search in NCBI to obtain closely related sequences. The relevant sequences were then aligned under the default ClustalW algorithm as set in MEGA4.1. Phylogeny analysis was performed using neighbor-hood joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and the positions containing alignment gaps and missing data were eliminated only in pair-wise sequence comparisons (pair-wise deletion option). A bootstrap value of 2000 was used for the phylogeny analyses. 2.4. Analytical methods Microbial growth was monitored by measuring the absorbance at 600 nm. An optical density of 1.0 represented 420 mg cell dry weight per liter. 4-ABS was detected and quantified through reaction with Ehrlich’s reagent (Meyer et al., 2005; Adegoke and Nwoke, 2008). Ehrlich’s reagent was prepared by dissolving 2 g of p-dimethylaminobenzaldehyde (Sigma) in 95 mL of 95% ethanol which was then acidified with 20 mL of concentrated hydrochloric acid. Upon the addition of 20 lL of Ehrlich’s reagent to 1 mL of cellfree supernatant, the formation of yellow reaction product was inspected visually and/or by measuring the absorbance at 440 nm. All experiments were done aerobically in 250 mL flasks at 30 °C with constant shaking (150 rpm). The microbial oxidation of 4-ABS was monitored by measuring the Chemical Oxygen Demand (COD) in the medium. COD was measured using a Hach spectrophotometer model DR4000 following Hach method 8000 (Jirka 2 and Carter, 1975). Release of NHþ ion were quantified 4 and SO4 using Nessler’s and barium chloride method respectively. Double distilled water was used as the diluent for these assays when appropriate. HPLC analysis was performed using Waters 600 equipped with a 4.6 mm 250 mm reverse-phase C18 column (Agilent, USA). Cell-free supernatant was filtered through 0.22 lm nylon membrane prior to analysis. The mobile phase consisted of 98% water, 1% methanol and 1% phosphoric acid (85%). The flow rate was maintained at 1.0 mL min1. Detection was carried out at 230 and 245 nm. Under this condition, the retention time of 4-ABS and 4-sulfocatechol were 2.15 and 2.7 min respectively. 2.5. Substrate inhibition kinetics In order to study the effect of 4-ABS concentration on degradation kinetics and biomass growth, growth was monitored at concentration range of 2.5–100 mM after 10% (v/v) inoculation into the culture medium. Absorbance at 600 nm and concentration of remaining 4-ABS was measured at various time points. The specific growth rate during exponential growth phase was then determined. Haldane substrate inhibition model (Haldane, 1930) was used to fit the data points.
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2.6. Biodegradation of other aromatic compounds The ability of the bacterial strains to utilize aromatic compound was studied by inoculating 4-ABS-grown cells into separate PBN medium containing 3-aminobenzenesulfonate, protocatechuate, phenylacetate, 4-sulfocatechol, o-phthalate, p-aminobenzoate, chloroaniline or aniline as sole carbon source at initial concentration of 3 mM. 4-ABS-grown cells were washed twice with PBN medium and inoculated at initial optical density at 600 nm of approximately 0.002 into the respective medium. Growth on each substrate was determined by measuring the absorbance at 600 nm every 10–12 h. Prior to measurement, cells were spun down and resuspended in PBN medium to eliminate any influence of colored oxidized substrate such as protocatechuate and 4-sulfocatechol on measurement.
3. Results and discussion 3.1. Isolation and characterization of 4-ABS-degrading bacterial strains Upon inoculation of partially treated textile wastewater into PB medium with 3 mM 4-ABS, significant turbidity was observed after 72 h. Growth was consistent with the depletion of 4-ABS. After 10 rounds of subculture, serially diluted culture was plated on solidified PB medium with 3 mM 4-ABS. After 5 d of incubation, yellow-pigmented colonies could be observed on the plate. After subsequent subculture onto similar medium, less than 10% of the colonies retained the ability to grow. The isolates on this subculture plate were further streaked on nutrient agar to obtain single pure bacterial. Surprisingly, after 2 d of incubation, white-pigmented colonies appeared on the plate. However, when the incubation was prolonged to 4 d, yellowish and smaller colonies resembling the originally observed colonies on solidified PB medium appeared. The white- and yellow-pigmented isolates were designated as PBA and PBC respectively. When these isolates were individually inoculated into 4-ABS supplemented liquid PB medium, isolate PBC exhibited minor growth but subsequent subculture in the similar medium failed which indicated metabolic interdependence among isolate PBA and PBC. As expected, when both isolates were inoculated together, growth was observed without loss of 4-ABS degrading ability even after several transfers into fresh medium. Both isolates were Gram negative, catalase positive, oxidase positive and grew well on nutrient agar separately. The optimum growth temperature was 30 °C. Growth was severely impaired at 37 °C while no growth was observed at 50 °C and above.
Fig. 1. Phylogenetic position of strain PBA and PBC among 11 taxa of the family Comamonadaceae and 11 taxa of the family Burkholderiaceae based on 16S rDNA sequence. Achromobacter xylosoxidan was used as outgroup. The evolutionary history was inferred using the neighbor-joining method with bootstrap consensus tree inferred from 2500 replicates.
Hydrogenophaga strains have been repeatedly isolated from activated sludge (Kampfer et al., 2005; Chung et al., 2007) and there is also a recent report on the isolation of Hydrogenophaga bisanensis from textile wastewater without elucidation on its role within the microbial community (Yoon et al., 2008). Some Hydrogenophaga strains are also able to degrade xenobiotic compounds such as polychlorinated biphenyl and methyl tert-butyl ether (Hatzinger et al., 2001; Lambo and Patel, 2006). On the contrary, the studies on biodegradation of aromatic compounds by the genus Ralstonia are more established (Schlomann, 1990; Sudip et al., 2000; Trefault et al., 2004).
3.2. Identification of strains PBA and PBC
3.3. Biotransformation products of 4-ABS
The 16S rDNA amplified were 1500 nucleotides and 1494 nucleotides in length for strains PBA and PBC respectively. The 16S rDNA nucleotide sequence of both strains PBA and PBC have been deposited in Genbank and assigned accession number HM194607 and HM194608 respectively. Comparison of 16S rDNA sequences with sequences deposited in NCBI showed that isolate PBA was most closely related to an uncultured bacterium clone 300A-F12 with sequence similarity of 99.1% followed by Ralstonia solanacearum with sequence similarity of 97.6%. Isolate PBC was most closely related to H. intermedia S1 with sequence similarities of 99.7%. Phylogenetic analyses based on the 16S rDNA sequence indicated that strain PBA was nested within the genus Ralstonia while strain PBC belonged to the genus Hydrogenophaga (Fig. 1). The topology of phylogenetic tree constructed using maximum parsimony also showed the similar pattern (data not shown).
After complete biotransformation of 4-ABS, approximately 0.18 mg of biomass yield was obtained per 1 mg of 4-ABS. During the course of 4-ABS biodegradation, nitrogen and sulfur in the form of ammonium and sulfate could be detected in the medium (Fig. 2). Only 50% of the theoretical stoichiometric value of ammonium was detected in the medium which implied that part of it was utilized by the biomass for protein production and other nitrogen-dependent biological processes. Using the empirical formula for bacterial biomass (C5H7O2N), the theoretical requirement of nitrogen was 0.12 mg per 1.0 mg biomass. However, based on the difference between initial 4-ABS concentration and final ammonium concentration, the nitrogen incorporated into biomass was determined to be 0.23 mg per 1.0 mg biomass which was approximately twofold higher than the typical theoretical requirement. Higher-than-average nitrogen content in bacterial biomass has been reported in
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Fig. 2. Biotransformation of 4-ABS and release of ammonium and sulfate during growth of bacterial consortium in PB medium containing 4-ABS as sole carbon and nitrogen source.
some strains of Alcaligenes and Pseudomonas from the marine environment (Kogure and Koike, 1987). In addition, since 4-ABS is the sole nitrogen source, additional enzymes may have to be synthesized to effectively harness the nitrogen from 4-ABS thus increasing the nitrogen content in biomass. Sulfate was also a major product of the biodegradation of 4-ABS and its release into the medium was about 88% of the theoretical value. Sulfate was utilized rather sparingly in the synthesis of some amino acids. 3.4. Population dynamics analysis and COD removal During the biodegradation of 4-ABS, the ratio of strain PBC to strain PBA was not constant. Prior to the sharp increase in 4-ABS depletion rate at 48 h, the cell number of strain PBA was almost equal to strain PBC with the ratio of 1:1.375 (Fig. 3a). It appeared that a concentration threshold of strain PBA has to be reached to enable effective biotransformation of 4-ABS. This was in contrast with report on the interaction between H. palleronii S1 and A. radiobacter S2 whereby the ratio of strain S1–S2 was about 3:1 throughout the growth (Dangmann et al., 1996). Strain PBC could degrade 4-ABS in axenic culture when p-aminobenzoate and biotin were added into the medium. Therefore, it was of interest to study the effect of such supplementation on the population dynamics. In the presence of vitamins, the cell number of strain PBA in the consortium during stationary phase was about one magnitude lower compared to those grown in the absence of vitamins (data not shown). The lack of growth promoting factors which could not be completely substituted by p-aminobenzoate and biotin might have induced secretion of growth substrate albeit in lesser amount into the medium to support growth of strain PBA so that optimum 4-ABS degradation could be achieved. In the study of the interaction between H. palleronii S1 and A. radiobacter S2 (Dangmann et al., 1996), similar trend was observed whereby the supplementation of vitamins essential for the growth of Hydrogenophaga strain did not cause complete inhibition in the growth of strain S2 but merely shifted the relative number of CFU of S1–S2 from 3:1 to 40:1. We initially proposed that the syntropic interaction between strain PBA and PBC was similar to that reported by Dangmann et al. (1996) since minor amount of 4-sulfocatechol was detected in the medium during growth (Fig. 6) which could putatively be used by strain PBA to maintain its viability and role as growth factor provider. We tested the ability of strain PBA to utilize 4-sulfocatechol as the sole carbon source. Surprisingly, strain PBA lacked the ability to grow on 4-sulfocatechol thus eliminating the possi-
Fig. 3. Utilization of 4-ABS as the sole carbon and nitrogen source by the co-culture. (A) Viable cell count of strain PBA and PBC at different time points during the biodegradation of 4-ABS in minimal medium. (B) Reduction of COD attributed to 4ABS and increase in cell biomass during biodegradation.
bility of 4-sulfocatechol being the energy source supplied by strain PBC. In contrast, strain PBA could grow on other aromatic compounds such as protocatechuate, p-hydroxybenzoate and phenylacetate as the sole carbon source. The conversion of 4-ABS to these non-sulfonated aromatic compounds is unlikely. Further work is necessary to identify the metabolites generated during biodegradation of 4-ABS so that syntropic interaction between strains PBA and PBC could be elucidated. It is worth noting that even though this work shows the first report on the interaction between the genus Ralstonia and Hydrogenophaga, there is a recent report on the interaction between the genus Ralstonia and Klebsiella in the biodegradation of thiocyanate (Chaudhari and Kodam, 2010). The elimination of COD by the co-culture was also studied as COD is one of the important parameters in the assessment of water quality. The theoretical COD for 4-ABS is 1.39 mg COD per mg 4ABS. When the co-culture was grown at 3464 mg L1 of 4-ABS, our experimental COD value at 0 h was approximately 4400 mg COD L1 (Fig. 3b) which was lower than theoretical value of 4814 mg COD L1. At the end of exponential growth phase, 78% of the initial COD in cell-free supernatant was eliminated. During the stationary phase, the COD reduction still continued albeit at a lower rate towards near complete elimination. In 118 h, 98% of the COD removal in cell-free supernatant could be observed. The COD reduction of suspended cells did not progress to completion (approx 69% removal) even after prolonged incubation. Higher value of COD reported for the suspended cells coupled with the disappearance of 4-ABS suggested that the component of 4-ABS was incorporated into biomass. 3.5. Influence of pH on 4-ABS biodegradation When the co-culture was grown in mineral medium containing 20 mM 4-ABS at initial pH of 5.5, 6, 7 and 8, complete degradation
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was only observed at pH 7 and 8. The pH of the medium started to decrease during log phase and remained somewhat constant during stationary phase. Since biodegradation was performed using batch culture, the drop in pH was most probably due to the accumulation of acidic metabolite by the biomass. Similar observation has been reported previously in the biotransformation of 4-ABS by P. paucimobilis SA1 (Perei et al., 2001). At initial pH of 6, only 68% of the 4-ABS was degraded (Fig. 4b). It is worth noting that biotransformation was severely inhibited only when the pH dropped to 5.5 and below (Fig. 4a and b). Neither detectable degradation nor growth could be observed when the initial pH was 5.5 (data not shown). Interestingly, although only 75% of the 4-ABS was biotransformed when the co-culture was grown at initial pH of 6, the highest biomass was obtained under this condition (Fig. 4c). There are two possible hypotheses to account for the continual increase in biomass when 4-ABS biotransformation was inhibited. First, the energy and NADH generated for synthesis and activation of the putative dioxygenase for oxidation of 4-ABS (Parales and Resnick, 2006) was channeled into biomass production in strain PBC. Second, in a state of excess energy, partially oxidized intermediates were secreted by strain PBC, a phenomenon coined ‘‘metabolism overflow’’, to promote growth of strain PBA (Russell and Cook, 1995). It will be interesting to study the changes in bacterial population and accumulation of metabolites when the 4-ABS-biotransformation activity is inhibited during growth.
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3.6. Influence of initial 4-ABS concentration on specific growth rate When the consortium was grown in 4-ABS at a concentration range of 2.5–100 mM, growth was observed at all concentrations as indicated by increase in turbidity. Contrary to previous findings (Dangmann et al., 1996; Singh et al., 2004), no significant difference in lag phase was observed at all concentrations (Fig. 5a). Interestingly, when the initial 4-ABS concentration was 75 or 100 mM, the exponential phase deviated from the typical pattern with an extra transient lag phase beginning at 30 h. This may be due to the adaptation of the consortium to the higher concentration of toxic intermediate formed during biodegradation. Furthermore, the tolerance of consortium to high concentration of 4-ABS could further extend the potential of this consortium to the treatment of severely 4-ABS-contaminated wastewater such as those from 4-ABS-manufacturing plant (Perei et al., 2001). To determine the effect of initial 4-ABS concentration on degradation kinetics, Haldane inhibition model (Eq. (1)) was used to fit the specific growth rate (l) at different initial 4-ABS concentrations (S) using non-linear regression. Then, the maximum specific growth rate (lmax), saturation constant (Ks) and inhibition constant (Ki) could be determined from the graph constructed (Fig. 5b).
l¼
lmax S S þ ðS2 =K i Þ þ K s
ð1Þ
The parameters lmax, Ks and Ki were determined to be 0.13 h1, 1.3 mM and 42 mM respectively. The experimental values fit well with the calculated values with an R2 value of 0.9727. The Ki value was the highest so far obtained compared to previous studies which indicated that this consortium could tolerate high concentration of 4-ABS while maintaining satisfactory specific growth rate.
Fig. 4. Changes in (A) pH, (B) 4-ABS biotransformation and (C) biomass after 96 h of growth in PB medium supplemented with 20 mM 4-ABS adjusted to different initial pH values. Experiments were also conducted at pH 5.5 but no biotransformation and biomass increase could be observed.
Fig. 5. Effects of different initial concentration of 4-ABS on: (A) growth profile and (B) specific growth rate.
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bon and nitrogen source. The consortium could tolerate up to 100 mM of 4-ABS without significant inhibition in growth thus highlighting their potential in treating wastewater that contains high level of 4-ABS. The biodegradation of 4-ABS could only take place at a narrow pH range of 6–8. In order to ensure efficient mineralization during textile wastewater treatment, it would be crucial to monitor this parameter. HPLC analyses showed minor accumulation of intermediates with one of them being identified as 4-sulfocatechol during biodegradation. Results from this study provide insight into the characteristics and mechanisms of 4-ABS conversion by Ralstonia sp. PBA and Hydrogenophaga sp. PBC. These findings would be invaluable in designing an effective biological treatment of aromatic amines generated from textile wastewater. Acknowledgements
Fig. 6. HPLC profile of 4-ABS and 4SC standards (10 mM each) and cell-free supernatant obtained during biodegradation of 4-ABS by bacterial consortium.
3.7. HPLC analysis During growth on 20 mM 4-ABS as sole carbon and nitrogen source, the peak area of 4-ABS decreased through time and gave rise to two new peaks at 2.7 min and 3.3 min (Fig. 6). The peak at 2.7 min shared similar elution time with 4-sulfocatechol standard. 4-Sulfocatechol is a commonly secreted intermediate in the degradation pathway of 4-ABS by H. intermedia S1 (Feigel and Knackmuss, 1988; Dangmann et al., 1996). Towards the end of incubation at 84 h, 4-sulfocatechol and 4-ABS were degraded to near completion while the peak at 3.3 min still persisted and accounted for 55% of the total peak area. In addition, a few new peaks also appeared with the retention time of 2.3, 6.1 and 8.8 min. Work is underway for the identification of these unknown metabolites. 3.8. Substrate range of the consortium The ability of consortium to utilize other aromatic compounds as sole carbon source was studied. The consortium could grow on 4-ABS, 4-sulfocatechol, protocatechuate, p-hydroxybenzoate and phenylacetate as sole carbon source with the substrate preference phenylacetate > p-hydroxybenzoate > protocatechuate > 4ABS > 4-sulfocatechol according to the increase in optical density at 600 nm which was measured every 10–12 h. Bacterial growth on the utilizable substrates had an average lag phase of 10 h except for 4-sulfocatechol which required 48 h. Aniline, 4-chloroaniline, 3-aminobenzenesulfonate, p-aminobenzoate and phthalate were not utilized by the consortium presumably due to the lack of specific dioxygenase required for efficient degradation, narrow range of the native dioxygenase or the potential toxicity of some aromatic amines. 4. Conclusions The consistent exposure of microflora to aromatic amines commonly encountered in textile wastewater induced the selection of enzymatic pathway necessary to convert the recalcitrant compound such as 4-ABS into energy source. Ralstonia sp. PBA and Hydrogenophaga sp. PBC were isolated from textile wastewater after enrichment in minimal medium containing 4-ABS as sole car-
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H.M. Gan et al. / Chemosphere 82 (2011) 507–513 Kampfer, P., Schulze, R., Jackel, U., Malik, K.A., Amann, R., Srping, S., 2005. Hydrogenophaga defluvii sp. nov. and Hydrogenophaga atypica sp. nov., isolated from activated sludge. Int. J. Syst. Evol. Microbiol. 55, 341–344. Kogure, K., Koike, I., 1987. Particle counter determination of bacterial biomass in seawater. Appl. Environ. Microbiol. 53, 274–277. Lambo, A.J., Patel, T.R., 2006. Cometabolic degradation of polychlorinated biphenyls at low temperature by psychrotolerant bacterium Hydrogenophaga sp. IA3-A. Curr. Microbiol. 53, 48–52. Meyer, D., Witholt, B., Schmid, A., 2005. Suitability of recombinant Escherichia coli and Pseudomonas putida strains for selective biotransformation of m-nitrotoluene by xylene monooxygenase. Appl. Environ. Microbiol. 71, 6624–6632. Parales, R.E., Resnick, S.M., 2006. Aromatic ring hydroxylating dioxygenases. In: Ramos, J.-L., Levesque, R.C. (Eds.), Pseudomonas. Springer, Netherlands, pp. 287–340. Pearce, C.I., Lloyd, J.R., Guthrie, J.T., 2003. The removal of colour from textile wastewater using whole bacterial cells: a review. Dyes Pigments 58, 179–196. Perei, K., Rakhely, G., Kiss, I., Polyak, B., Kovacs, K.L., 2001. Biodegradation of sulfanilic acid by Pseudomonas paucimobilis. Appl. Microbiol. Biotechnol. 55, 101–107. Russell, J.B., Cook, G.M., 1995. Energetics of bacterial growth: balance of anabolic and catabolic reactions. Microbiol. Rev. 59, 48–62. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Schlomann, M., 1990. Enzymatic formation, stability, and spontaneous reactions of 4-fluoromuconolactone, a metabolite of the bacterial degradation of 4fluorobenzoate. J. Bacteriol. 172, 5119–5129. Singh, P., Mishra, L.C., Iyengar, L., 2004. Biodegradation of 4aminobenezesulphonate by a newly isolated bacterial strain PNS-1. World J. Microbiol. Biotechnol. 20, 845–849.
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Biodegradation of 4-aminobenzenesulfonate by Ralstonia sp. PBA and Hydrogenophaga sp. PBC isolated from textile wastewater treatment plant Han Ming Gan a, Shafinaz Shahir b, Zaharah Ibrahim b, Adibah Yahya a,⇑ a b
Department of Industrial Biotechnology, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Department of Biological Sciences, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
a r t i c l e
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Article history: Received 10 July 2010 Received in revised form 27 October 2010 Accepted 28 October 2010 Available online 20 November 2010 Keywords: Hydrogenophaga Ralstonia 4-Aminobenzenesulfonate Biodegradation 16S rDNA
a b s t r a c t A co-culture consisting of Hydrogenophaga sp. PBC and Ralstonia sp. PBA, isolated from textile wastewater treatment plant could tolerate up to 100 mM 4-aminobenzenesulfonate (4-ABS) and utilize it as sole carbon, nitrogen and sulfur source under aerobic condition. The biodegradation of 4-ABS resulted in the release of nitrogen and sulfur in the form of ammonium and sulfate respectively. Ninety-eight percent removal of chemical oxygen demand attributed to 20 mM of 4-ABS in cell-free supernatant could be achieved after 118 h. Effective biodegradation of 4-ABS occurred at pH ranging from 6 to 8. During batch culture with 4-ABS as sole carbon and nitrogen source, the ratio of strain PBA to PBC was dynamic and a critical concentration of strain PBA has to be reached in order to enable effective biodegradation of 4-ABS. Haldane inhibition model was used to fit the degradation rate at different initial concentrations and the parameters lmax, Ks and Ki were determined to be 0.13 h1, 1.3 mM and 42 mM respectively. HPLC analyses revealed traced accumulation of 4-sulfocatechol and at least four unidentified metabolites during biodegradation. This is the first study to report on the characterization of 4-ABS-degrading bacterial consortium that was isolated from textile wastewater treatment plant. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction 4-Aminobenzenesulfonate (4-ABS) is one of the most commonly found sulfonated aromatic amines. It is widely used as an intermediate in the production of textile dyes, sulfonamide drugs, optical brighteners and pesticides. In nature, the biodegradation of 4-ABS is problematic. Unless there is a specific transport system for 4-ABS, the negatively charged sulfonate group would prevent uptake of the substrate through the bacteria membrane (Hwang et al., 1989). Even if bacteria develop an efficient uptake mechanism for 4-ABS, the thermodynamic energy barriers exerted by both the resonance-stabilized aromatic ring and C–S bond of 4-ABS have to be overcome in order to harness energy from this compound (Wagner and Reid, 1931). Furthermore, 4-ABS could exhibit bacteriostatic activity by inhibiting the folate synthesis pathway which is crucial for the maintenance of deoxynucleotide precursors pool and DNA synthesis (Brown, 1962; Dallas et al., 1992). To date, the biodegradation of 4-ABS is still a rare occurrence in the microbial community found in natural soil, polluted ⇑ Corresponding author. Address: Research Laboratory 2, Level 2, Building C08, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. Tel.: +60 75534157; fax: +60 75531112. E-mail addresses: [email protected] (H.M. Gan), shafi[email protected] (S. Shahir), [email protected] (Z. Ibrahim), [email protected] (A. Yahya). 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.10.094
harbor sediment and even activated sludge from some wastewater treatment plants (Alexander and Lustigman, 1966; Tan et al., 2005; Yemashova and Kalyuzhnyi, 2006). The consumption of most sulfonated aromatic amines commonly will result in the rapid excretion from organism (Greim et al., 1994). However, study in rats showed that 4-ABS has the longest retention time following ingestion as compared to its other counterparts (Honohan et al., 1979) thus making it the most significant compound to study. Under constant exposure to this compound, some negative effects of 4-ABS have been reported including hyperactivity in rats (Goldenring et al., 1982) and significant decrease in the nitrogen transformation processes in soil (Topac et al., 2009). One of the most important sources of 4-ABS is sulfonated azo dyes. In textile industries, sulfonated azo dyes are commonly used due to its stronger binding to the fiber and lower toxicity as compared to its non-sulfonated analog. Biological treatment of textile wastewater is highly favored due to its cost-effectiveness and higher sustainability as compared to physicochemical treatment (Pearce et al., 2003). In many of the studies found in the literature, the biological treatment of textile waste involves a two-stage process: decolorization and mineralization. In the decolorization stage, an anaerobic condition is provided to stimulate reductive cleavage of azo bond by redox active compounds of bacterial origin
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to release the parent aromatic amines. In the mineralization stage, air or specifically, oxygen, is introduced to enable further biodegradation of the aromatic amines. Through the actions of aromatic ring hydroxylation dioxygenase enzyme and beta-ketoadipate pathway (Harwood and Parales, 1996; Parales and Resnick, 2006), it is possible to convert sulfonated aromatic amines into simple compounds such as carbon dioxide, ammonium and sulfate. Under constant exposure to industrial effluent containing 4ABS, some microbial degradation of 4-ABS has been reported in the last two decades starting with a defined co-culture consisting of Hydrogenophaga palleroni S1 and Agrobacterium radiobacter S2 (Feigel and Knackmuss, 1988). Strain S1 was later reclassified as Hydrogenophaga intermedia S1 (Contzen et al., 2000) after further taxonomic studies. Some pure bacterial strains capable of degrading 4-ABS such as Pseudomonas paucimobilis (Perei et al., 2001), A. radiobacter PNS-1 (Singh et al., 2004) and Pannonibacter sp. W1 (Wang et al., 2009) have also been reported. A pure culture SAD 4i has been isolated from textile wastewater which can utilize 4-ABS as its carbon source but there is no report on its identification (Coughlin et al., 2002). In this study, the biodegradation of 4-ABS using two-membered bacterial consortium isolated from a pilot scale of textile wastewater treatment plant is investigated (Ibrahim et al., 2009). The strains have been identified as Hydrogenophaga sp. PBC and Ralstonia sp. PBA. This consortium can utilize 4-ABS as the sole carbon, nitrogen and sulfur source with ammonium and sulfate as part of the end products. In addition, there is no complete inhibition in growth even at 100 mM 4-ABS which underscores the potential of this consortium in treatment of severely 4-ABScontaminated wastewater. Results obtained in this work provide the much-needed fundamental studies for the biodegradation of aromatic amines generated from textile wastewater treatment. 2. Materials and methods 2.1. Culture condition Full strength (1) phosphate-buffered mineral (PB) medium consisted of 0.09 mM MgSO4, 0.042 mM KCl, 7.5 mM NaHPO4, 7.5 mM KHPO4, 15 mM KH2PO4, 0.0068 mM FeCl3 and 0.1 mM CaCl2. Required amount of neutralized 4-ABS from stock solution of 86.6 g L1 (500 mM) was added to the medium as sole carbon and nitrogen source. For the study of sulfate release, MgSO4 was replaced with MgCl2. In some cases, nitrogen source in the form of (NH4)2SO4 was added to a final concentration of 2.5 mM to give PBN medium. To assess the influence of vitamin supplement on population dynamics, p-aminobenzoate and biotin were added to a final concentration of 20 mg L1 and 2 lg L1 respectively. 2.2. Isolation of 4-ABS-degrading bacteria Sample was withdrawn from the aerobic reactor of a textile wastewater treatment plant (Ramatex Textile Industrial, Malaysia). The seed inoculum was enriched in repeated batch cultures containing PB medium and 3 mM 4-ABS as sole carbon and nitrogen source. The enrichment was performed at 30 °C and 150 rpm in an orbital shaking incubator. Growth was observed indirectly from the turbidity readings and once sufficient turbidity was achieved, 1% (v/v) cultivated inoculum was transferred into new medium. After 10 successive transfers, serial dilution was performed and the inoculum was plated on solid PB medium with 4-ABS as sole carbon and nitrogen source for the purpose of bacteria isolation. Single colonies were then transferred individually using a sterile toothpick onto the same solid medium to confirm their 4-ABS degrading ability. Then, subsequent streaking was performed on
nutrient agar to obtain single colonies. Two strains designated PBA and PBC were isolated from the agar plates. 2.3. Characterization and identification of bacteria The isolates were first characterized using Gram-staining. Then identification was performed by 16S rDNA sequence analysis. Genomic DNA was extracted using Qiagen DNAeasy Blood and Tissue Kit according to the manufacturer’s instruction. The sequence of 16S rDNA was amplified by the polymerase chain reaction (PCR) method using primer fD1 and rD1 without linker sequences (Weisburg et al., 1991). The PCR condition consisted of an initial denaturation step at 94 °C for 5 min which was followed by 30 cycles of 94 °C (1 min), 50 °C (30 s), 72 °C (1.5 min), plus a final 10 min-chain elongation cycle at 72 °C. The amplicons were purified and sequenced. Then, the sequences were subjected to the BLAST search in NCBI to obtain closely related sequences. The relevant sequences were then aligned under the default ClustalW algorithm as set in MEGA4.1. Phylogeny analysis was performed using neighbor-hood joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and the positions containing alignment gaps and missing data were eliminated only in pair-wise sequence comparisons (pair-wise deletion option). A bootstrap value of 2000 was used for the phylogeny analyses. 2.4. Analytical methods Microbial growth was monitored by measuring the absorbance at 600 nm. An optical density of 1.0 represented 420 mg cell dry weight per liter. 4-ABS was detected and quantified through reaction with Ehrlich’s reagent (Meyer et al., 2005; Adegoke and Nwoke, 2008). Ehrlich’s reagent was prepared by dissolving 2 g of p-dimethylaminobenzaldehyde (Sigma) in 95 mL of 95% ethanol which was then acidified with 20 mL of concentrated hydrochloric acid. Upon the addition of 20 lL of Ehrlich’s reagent to 1 mL of cellfree supernatant, the formation of yellow reaction product was inspected visually and/or by measuring the absorbance at 440 nm. All experiments were done aerobically in 250 mL flasks at 30 °C with constant shaking (150 rpm). The microbial oxidation of 4-ABS was monitored by measuring the Chemical Oxygen Demand (COD) in the medium. COD was measured using a Hach spectrophotometer model DR4000 following Hach method 8000 (Jirka 2 and Carter, 1975). Release of NHþ ion were quantified 4 and SO4 using Nessler’s and barium chloride method respectively. Double distilled water was used as the diluent for these assays when appropriate. HPLC analysis was performed using Waters 600 equipped with a 4.6 mm 250 mm reverse-phase C18 column (Agilent, USA). Cell-free supernatant was filtered through 0.22 lm nylon membrane prior to analysis. The mobile phase consisted of 98% water, 1% methanol and 1% phosphoric acid (85%). The flow rate was maintained at 1.0 mL min1. Detection was carried out at 230 and 245 nm. Under this condition, the retention time of 4-ABS and 4-sulfocatechol were 2.15 and 2.7 min respectively. 2.5. Substrate inhibition kinetics In order to study the effect of 4-ABS concentration on degradation kinetics and biomass growth, growth was monitored at concentration range of 2.5–100 mM after 10% (v/v) inoculation into the culture medium. Absorbance at 600 nm and concentration of remaining 4-ABS was measured at various time points. The specific growth rate during exponential growth phase was then determined. Haldane substrate inhibition model (Haldane, 1930) was used to fit the data points.
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2.6. Biodegradation of other aromatic compounds The ability of the bacterial strains to utilize aromatic compound was studied by inoculating 4-ABS-grown cells into separate PBN medium containing 3-aminobenzenesulfonate, protocatechuate, phenylacetate, 4-sulfocatechol, o-phthalate, p-aminobenzoate, chloroaniline or aniline as sole carbon source at initial concentration of 3 mM. 4-ABS-grown cells were washed twice with PBN medium and inoculated at initial optical density at 600 nm of approximately 0.002 into the respective medium. Growth on each substrate was determined by measuring the absorbance at 600 nm every 10–12 h. Prior to measurement, cells were spun down and resuspended in PBN medium to eliminate any influence of colored oxidized substrate such as protocatechuate and 4-sulfocatechol on measurement.
3. Results and discussion 3.1. Isolation and characterization of 4-ABS-degrading bacterial strains Upon inoculation of partially treated textile wastewater into PB medium with 3 mM 4-ABS, significant turbidity was observed after 72 h. Growth was consistent with the depletion of 4-ABS. After 10 rounds of subculture, serially diluted culture was plated on solidified PB medium with 3 mM 4-ABS. After 5 d of incubation, yellow-pigmented colonies could be observed on the plate. After subsequent subculture onto similar medium, less than 10% of the colonies retained the ability to grow. The isolates on this subculture plate were further streaked on nutrient agar to obtain single pure bacterial. Surprisingly, after 2 d of incubation, white-pigmented colonies appeared on the plate. However, when the incubation was prolonged to 4 d, yellowish and smaller colonies resembling the originally observed colonies on solidified PB medium appeared. The white- and yellow-pigmented isolates were designated as PBA and PBC respectively. When these isolates were individually inoculated into 4-ABS supplemented liquid PB medium, isolate PBC exhibited minor growth but subsequent subculture in the similar medium failed which indicated metabolic interdependence among isolate PBA and PBC. As expected, when both isolates were inoculated together, growth was observed without loss of 4-ABS degrading ability even after several transfers into fresh medium. Both isolates were Gram negative, catalase positive, oxidase positive and grew well on nutrient agar separately. The optimum growth temperature was 30 °C. Growth was severely impaired at 37 °C while no growth was observed at 50 °C and above.
Fig. 1. Phylogenetic position of strain PBA and PBC among 11 taxa of the family Comamonadaceae and 11 taxa of the family Burkholderiaceae based on 16S rDNA sequence. Achromobacter xylosoxidan was used as outgroup. The evolutionary history was inferred using the neighbor-joining method with bootstrap consensus tree inferred from 2500 replicates.
Hydrogenophaga strains have been repeatedly isolated from activated sludge (Kampfer et al., 2005; Chung et al., 2007) and there is also a recent report on the isolation of Hydrogenophaga bisanensis from textile wastewater without elucidation on its role within the microbial community (Yoon et al., 2008). Some Hydrogenophaga strains are also able to degrade xenobiotic compounds such as polychlorinated biphenyl and methyl tert-butyl ether (Hatzinger et al., 2001; Lambo and Patel, 2006). On the contrary, the studies on biodegradation of aromatic compounds by the genus Ralstonia are more established (Schlomann, 1990; Sudip et al., 2000; Trefault et al., 2004).
3.2. Identification of strains PBA and PBC
3.3. Biotransformation products of 4-ABS
The 16S rDNA amplified were 1500 nucleotides and 1494 nucleotides in length for strains PBA and PBC respectively. The 16S rDNA nucleotide sequence of both strains PBA and PBC have been deposited in Genbank and assigned accession number HM194607 and HM194608 respectively. Comparison of 16S rDNA sequences with sequences deposited in NCBI showed that isolate PBA was most closely related to an uncultured bacterium clone 300A-F12 with sequence similarity of 99.1% followed by Ralstonia solanacearum with sequence similarity of 97.6%. Isolate PBC was most closely related to H. intermedia S1 with sequence similarities of 99.7%. Phylogenetic analyses based on the 16S rDNA sequence indicated that strain PBA was nested within the genus Ralstonia while strain PBC belonged to the genus Hydrogenophaga (Fig. 1). The topology of phylogenetic tree constructed using maximum parsimony also showed the similar pattern (data not shown).
After complete biotransformation of 4-ABS, approximately 0.18 mg of biomass yield was obtained per 1 mg of 4-ABS. During the course of 4-ABS biodegradation, nitrogen and sulfur in the form of ammonium and sulfate could be detected in the medium (Fig. 2). Only 50% of the theoretical stoichiometric value of ammonium was detected in the medium which implied that part of it was utilized by the biomass for protein production and other nitrogen-dependent biological processes. Using the empirical formula for bacterial biomass (C5H7O2N), the theoretical requirement of nitrogen was 0.12 mg per 1.0 mg biomass. However, based on the difference between initial 4-ABS concentration and final ammonium concentration, the nitrogen incorporated into biomass was determined to be 0.23 mg per 1.0 mg biomass which was approximately twofold higher than the typical theoretical requirement. Higher-than-average nitrogen content in bacterial biomass has been reported in
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Fig. 2. Biotransformation of 4-ABS and release of ammonium and sulfate during growth of bacterial consortium in PB medium containing 4-ABS as sole carbon and nitrogen source.
some strains of Alcaligenes and Pseudomonas from the marine environment (Kogure and Koike, 1987). In addition, since 4-ABS is the sole nitrogen source, additional enzymes may have to be synthesized to effectively harness the nitrogen from 4-ABS thus increasing the nitrogen content in biomass. Sulfate was also a major product of the biodegradation of 4-ABS and its release into the medium was about 88% of the theoretical value. Sulfate was utilized rather sparingly in the synthesis of some amino acids. 3.4. Population dynamics analysis and COD removal During the biodegradation of 4-ABS, the ratio of strain PBC to strain PBA was not constant. Prior to the sharp increase in 4-ABS depletion rate at 48 h, the cell number of strain PBA was almost equal to strain PBC with the ratio of 1:1.375 (Fig. 3a). It appeared that a concentration threshold of strain PBA has to be reached to enable effective biotransformation of 4-ABS. This was in contrast with report on the interaction between H. palleronii S1 and A. radiobacter S2 whereby the ratio of strain S1–S2 was about 3:1 throughout the growth (Dangmann et al., 1996). Strain PBC could degrade 4-ABS in axenic culture when p-aminobenzoate and biotin were added into the medium. Therefore, it was of interest to study the effect of such supplementation on the population dynamics. In the presence of vitamins, the cell number of strain PBA in the consortium during stationary phase was about one magnitude lower compared to those grown in the absence of vitamins (data not shown). The lack of growth promoting factors which could not be completely substituted by p-aminobenzoate and biotin might have induced secretion of growth substrate albeit in lesser amount into the medium to support growth of strain PBA so that optimum 4-ABS degradation could be achieved. In the study of the interaction between H. palleronii S1 and A. radiobacter S2 (Dangmann et al., 1996), similar trend was observed whereby the supplementation of vitamins essential for the growth of Hydrogenophaga strain did not cause complete inhibition in the growth of strain S2 but merely shifted the relative number of CFU of S1–S2 from 3:1 to 40:1. We initially proposed that the syntropic interaction between strain PBA and PBC was similar to that reported by Dangmann et al. (1996) since minor amount of 4-sulfocatechol was detected in the medium during growth (Fig. 6) which could putatively be used by strain PBA to maintain its viability and role as growth factor provider. We tested the ability of strain PBA to utilize 4-sulfocatechol as the sole carbon source. Surprisingly, strain PBA lacked the ability to grow on 4-sulfocatechol thus eliminating the possi-
Fig. 3. Utilization of 4-ABS as the sole carbon and nitrogen source by the co-culture. (A) Viable cell count of strain PBA and PBC at different time points during the biodegradation of 4-ABS in minimal medium. (B) Reduction of COD attributed to 4ABS and increase in cell biomass during biodegradation.
bility of 4-sulfocatechol being the energy source supplied by strain PBC. In contrast, strain PBA could grow on other aromatic compounds such as protocatechuate, p-hydroxybenzoate and phenylacetate as the sole carbon source. The conversion of 4-ABS to these non-sulfonated aromatic compounds is unlikely. Further work is necessary to identify the metabolites generated during biodegradation of 4-ABS so that syntropic interaction between strains PBA and PBC could be elucidated. It is worth noting that even though this work shows the first report on the interaction between the genus Ralstonia and Hydrogenophaga, there is a recent report on the interaction between the genus Ralstonia and Klebsiella in the biodegradation of thiocyanate (Chaudhari and Kodam, 2010). The elimination of COD by the co-culture was also studied as COD is one of the important parameters in the assessment of water quality. The theoretical COD for 4-ABS is 1.39 mg COD per mg 4ABS. When the co-culture was grown at 3464 mg L1 of 4-ABS, our experimental COD value at 0 h was approximately 4400 mg COD L1 (Fig. 3b) which was lower than theoretical value of 4814 mg COD L1. At the end of exponential growth phase, 78% of the initial COD in cell-free supernatant was eliminated. During the stationary phase, the COD reduction still continued albeit at a lower rate towards near complete elimination. In 118 h, 98% of the COD removal in cell-free supernatant could be observed. The COD reduction of suspended cells did not progress to completion (approx 69% removal) even after prolonged incubation. Higher value of COD reported for the suspended cells coupled with the disappearance of 4-ABS suggested that the component of 4-ABS was incorporated into biomass. 3.5. Influence of pH on 4-ABS biodegradation When the co-culture was grown in mineral medium containing 20 mM 4-ABS at initial pH of 5.5, 6, 7 and 8, complete degradation
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was only observed at pH 7 and 8. The pH of the medium started to decrease during log phase and remained somewhat constant during stationary phase. Since biodegradation was performed using batch culture, the drop in pH was most probably due to the accumulation of acidic metabolite by the biomass. Similar observation has been reported previously in the biotransformation of 4-ABS by P. paucimobilis SA1 (Perei et al., 2001). At initial pH of 6, only 68% of the 4-ABS was degraded (Fig. 4b). It is worth noting that biotransformation was severely inhibited only when the pH dropped to 5.5 and below (Fig. 4a and b). Neither detectable degradation nor growth could be observed when the initial pH was 5.5 (data not shown). Interestingly, although only 75% of the 4-ABS was biotransformed when the co-culture was grown at initial pH of 6, the highest biomass was obtained under this condition (Fig. 4c). There are two possible hypotheses to account for the continual increase in biomass when 4-ABS biotransformation was inhibited. First, the energy and NADH generated for synthesis and activation of the putative dioxygenase for oxidation of 4-ABS (Parales and Resnick, 2006) was channeled into biomass production in strain PBC. Second, in a state of excess energy, partially oxidized intermediates were secreted by strain PBC, a phenomenon coined ‘‘metabolism overflow’’, to promote growth of strain PBA (Russell and Cook, 1995). It will be interesting to study the changes in bacterial population and accumulation of metabolites when the 4-ABS-biotransformation activity is inhibited during growth.
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3.6. Influence of initial 4-ABS concentration on specific growth rate When the consortium was grown in 4-ABS at a concentration range of 2.5–100 mM, growth was observed at all concentrations as indicated by increase in turbidity. Contrary to previous findings (Dangmann et al., 1996; Singh et al., 2004), no significant difference in lag phase was observed at all concentrations (Fig. 5a). Interestingly, when the initial 4-ABS concentration was 75 or 100 mM, the exponential phase deviated from the typical pattern with an extra transient lag phase beginning at 30 h. This may be due to the adaptation of the consortium to the higher concentration of toxic intermediate formed during biodegradation. Furthermore, the tolerance of consortium to high concentration of 4-ABS could further extend the potential of this consortium to the treatment of severely 4-ABS-contaminated wastewater such as those from 4-ABS-manufacturing plant (Perei et al., 2001). To determine the effect of initial 4-ABS concentration on degradation kinetics, Haldane inhibition model (Eq. (1)) was used to fit the specific growth rate (l) at different initial 4-ABS concentrations (S) using non-linear regression. Then, the maximum specific growth rate (lmax), saturation constant (Ks) and inhibition constant (Ki) could be determined from the graph constructed (Fig. 5b).
l¼
lmax S S þ ðS2 =K i Þ þ K s
ð1Þ
The parameters lmax, Ks and Ki were determined to be 0.13 h1, 1.3 mM and 42 mM respectively. The experimental values fit well with the calculated values with an R2 value of 0.9727. The Ki value was the highest so far obtained compared to previous studies which indicated that this consortium could tolerate high concentration of 4-ABS while maintaining satisfactory specific growth rate.
Fig. 4. Changes in (A) pH, (B) 4-ABS biotransformation and (C) biomass after 96 h of growth in PB medium supplemented with 20 mM 4-ABS adjusted to different initial pH values. Experiments were also conducted at pH 5.5 but no biotransformation and biomass increase could be observed.
Fig. 5. Effects of different initial concentration of 4-ABS on: (A) growth profile and (B) specific growth rate.
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bon and nitrogen source. The consortium could tolerate up to 100 mM of 4-ABS without significant inhibition in growth thus highlighting their potential in treating wastewater that contains high level of 4-ABS. The biodegradation of 4-ABS could only take place at a narrow pH range of 6–8. In order to ensure efficient mineralization during textile wastewater treatment, it would be crucial to monitor this parameter. HPLC analyses showed minor accumulation of intermediates with one of them being identified as 4-sulfocatechol during biodegradation. Results from this study provide insight into the characteristics and mechanisms of 4-ABS conversion by Ralstonia sp. PBA and Hydrogenophaga sp. PBC. These findings would be invaluable in designing an effective biological treatment of aromatic amines generated from textile wastewater. Acknowledgements
Fig. 6. HPLC profile of 4-ABS and 4SC standards (10 mM each) and cell-free supernatant obtained during biodegradation of 4-ABS by bacterial consortium.
3.7. HPLC analysis During growth on 20 mM 4-ABS as sole carbon and nitrogen source, the peak area of 4-ABS decreased through time and gave rise to two new peaks at 2.7 min and 3.3 min (Fig. 6). The peak at 2.7 min shared similar elution time with 4-sulfocatechol standard. 4-Sulfocatechol is a commonly secreted intermediate in the degradation pathway of 4-ABS by H. intermedia S1 (Feigel and Knackmuss, 1988; Dangmann et al., 1996). Towards the end of incubation at 84 h, 4-sulfocatechol and 4-ABS were degraded to near completion while the peak at 3.3 min still persisted and accounted for 55% of the total peak area. In addition, a few new peaks also appeared with the retention time of 2.3, 6.1 and 8.8 min. Work is underway for the identification of these unknown metabolites. 3.8. Substrate range of the consortium The ability of consortium to utilize other aromatic compounds as sole carbon source was studied. The consortium could grow on 4-ABS, 4-sulfocatechol, protocatechuate, p-hydroxybenzoate and phenylacetate as sole carbon source with the substrate preference phenylacetate > p-hydroxybenzoate > protocatechuate > 4ABS > 4-sulfocatechol according to the increase in optical density at 600 nm which was measured every 10–12 h. Bacterial growth on the utilizable substrates had an average lag phase of 10 h except for 4-sulfocatechol which required 48 h. Aniline, 4-chloroaniline, 3-aminobenzenesulfonate, p-aminobenzoate and phthalate were not utilized by the consortium presumably due to the lack of specific dioxygenase required for efficient degradation, narrow range of the native dioxygenase or the potential toxicity of some aromatic amines. 4. Conclusions The consistent exposure of microflora to aromatic amines commonly encountered in textile wastewater induced the selection of enzymatic pathway necessary to convert the recalcitrant compound such as 4-ABS into energy source. Ralstonia sp. PBA and Hydrogenophaga sp. PBC were isolated from textile wastewater after enrichment in minimal medium containing 4-ABS as sole car-
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