Chemosphere 141 (2015) 13–18
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The fate of a nitrobenzene-degrading bacterium in pharmaceutical wastewater treatment sludge Yuan Ren a,b,c,⇑, Juan Yang a, Shaoyi Chen a a
School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Panyu District, Guangzhou 510006, PR China The Key Laboratory of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, PR China c The Key Laboratory of Environmental Protection and Eco-Remediation of Guangdong Regular Higher Education Institutions, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
NBA-1 can grow and biodegrade
nitrobenzene under anaerobic and aerobic conditions. There was no obvious difference of NB degradation when glucose was added. NB biodegradation rate decreased when was added under aerobic condition. NBA-1 and some microbes are weakened when p-CNB was added under aerobic condition. The bacterial community was stable when p-CNB was added under anaerobic condition.
a r t i c l e
i n f o
Article history: Received 29 December 2014 Received in revised form 9 May 2015 Accepted 29 May 2015
Keywords: Nitrobenzene-degrading bacterium Fate Pharmaceutical sludge PCR-DGGE Bacterial community
a b s t r a c t This paper describes the fate of a nitrobenzene-degrading bacterium, Klebsiella oxytoca NBA-1, which was isolated from a pharmaceutical wastewater treatment facility. The 90-day survivability of strain NBA-1 after exposure to sludge under anaerobic and aerobic conditions was investigated. The bacterium was inoculated into sludge amended with glucose and p-chloronitrobenzene (p-CNB) to compare the bacterial community variations between the modified sludge and nitrobenzene amendment. The results showed that glucose had no obvious effect on nitrobenzene biodegradation in the co-metabolism process, regardless of the presence/absence of oxygen. When p-CNB was added under anaerobic conditions, the biodegradation rate of nitrobenzene remained unchanged although p-CNB inhibited the production of aniline. The diversity of the microbial community increased and NBA-1 continued to be one of the dominant strains. Under aerobic conditions, the degradation rate of both nitrobenzene and p-CNB was only 20% of that under anaerobic conditions. p-CNB had a toxic effect on the microorganisms in the sludge so that most of the DGGE (denaturing gradient gel electrophoresis) bands, including that of NBA-1, began to disappear under aerobic conditions after 90 days of exposure. These data show that the bacterial community was stable under anaerobic conditions and the microorganisms, including NBA-1, were more resistant to the adverse environment. Ó 2015 Elsevier Ltd. All rights reserved.
⇑ Corresponding author at: School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Panyu District, Guangzhou 510006, PR China. E-mail address: [email protected] (Y. Ren). http://dx.doi.org/10.1016/j.chemosphere.2015.05.098 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
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1. Introduction Nitrobenzene and its chlorinated compounds are important organic intermediates for the production of organic pigments, azo dyes, pesticides, and medicines. Wastewater from aniline-producing factories, waste gas from chemical factories, and transport accidents are the main sources of nitrobenzene pollution. For treatment of moderate and high concentrations of nitrobenzene-containing wastewater, extraction, absorption, advanced oxidation, and airlift processes can be used to separate water and the pollutant with high efficiency. Biological treatment for nitrobenzene-containing wastewater is practical for lower nitrobenzene concentrations or for effluent from physical and chemical pretreatment. Many studies have screened and isolated nitrobenzene-degrading microorganisms from different samples, while the adaptability of bacterial pure culture to salinity, co-substrates and heavy metals still needs more consideration (Chao et al., 2009; Zheng et al., 2009; Lin et al., 2011; Li et al., 2012; Wang et al., 2012). The fate of a given bacterial species is a crucial aspect for its application in wastewater treatment because competition among microorganisms is very common in the environment. Generally, a single organic compound is often used as the sole carbon source and energy source to screen for the most effective microorganisms to use in the biodegradation of waste (Arora et al., 2012). When two or more species of bacteria exist in the same system, the competition for carbon sources, oxygen, temperature and other nutritional factors can affect the survival of the dominant strain (Polymenakou and Stephanou, 2005; Giorni et al., 2009; Chamkha et al., 2011). Some studies have been conducted to examine the variations of some specific bacterial communities with different types of molecular biological technologies that do not rely on the traditional culturing methods (Fang et al., 2002; Gilbride et al., 2006; O’Reilly et al., 2009; Meynet et al., 2012). Compared with the papers published in the field of effective microorganism isolation, identification and determination of other characteristics (Song et al., 2005; Batista et al., 2006; Hassanshahian et al., 2012; Li et al., 2012; Wang et al., 2012), less work has been conducted to investigate the fate of the microbes in full or laboratory scale wastewater treatment facilities (Sahlstrom et al., 2008; Harrison, 2013). Although some composite microbial preparations, such as effective microorganisms and growth-promoting microorganisms, are commercialized in food additives and wastewater treatment (Glick et al., 1999), many of these new bacterial agents have to be added regularly during the process to compensate for loss or inactivation of the amended strains. The dominant strain or clones that are responsible for the degradation have different fates due to their responses to carbon sources, oxygen availability and many other environmental factors. Therefore, the results of adaptability studies using pure culture strains have important applications in both production and environmental situations. As a case in point, to understand the fate of a pure strain in pharmaceutical wastewater, a nitrobenzene-degrading stain was isolated and added to lab scale nitrobenzene-containing wastewater treatment sludge amended with nitrobenzene and/or p-CNB as carbon sources. The concentrations of nitrobenzene, p-chloronitrobenzene (p-CNB) and aniline (a prominent degradation product of nitrobenzene) were monitored under anaerobic or aerobic conditions while the bacterial community structure was studied using the PCR-DGGE/cloning method. The diversity changes associated with the different treatments were assessed to examine the survivability of the pure cultures and gather additional data that will be useful in judging the utility of the microorganism in the treatment of pharmaceutical wastewater. Moreover, we hope to obtain more
scientific data regarding the characteristics and factors that influence the bioactivity of the pure cultures. 2. Materials and methods 2.1. Chemicals and cultures Nitrobenzene (>99.0%) and p-CNB (99.0%) were purchased from Fuchen Chemical Reagents Co. Ltd. (Tianjin, China). All other chemicals used in this study for culture medium were obtained from Kermel Chemical Reagents Co. Ltd. (Tianjin, China). The reagents for HPLC flow phase were all HPLC grade pure. The inorganic culture medium contained (w/v): 0.5% NaCl; 0.05% MgSO4; 0.5% K2HPO4: 0.3% KH2PO4; and a mixture of 1.0% trace elements which contained (g L1): 0.19 CoCl26H2O, 0.024 NiCl26H2O, 0.03 CuCl22H2O, 0.05 ZnCl2, 0.05 H3BO3, 0.5 MnCl24H2O and 0.09 (NH4)6Mo7O242H2O. The isolation medium contained (w/v): inorganic culture medium, 1.5% agar with nitrobenzene as the sole source of carbon, nitrogen and energy. Na2S9H2O 0.2 mL L1 (stock solution 500 g L1) was added as an oxygen scavenger when anaerobic medium was being prepared. Enrichment cultures were established using a medium containing (w/v) inorganic culture medium, 1.0% trace elements mix (see content above), 1.0% yeast extract and 1.0% peptone. Nitrobenzene was added after sterilization. 2.2. Molecular biological analysis of strains in pharmaceutical sludge The sludge sample was taken from a pharmaceutical wastewater treatment plant (Guangzhou, Guangdong, China) and screened for nitrobenzene-degrading microorganisms using a gradient flow under anaerobic/aerobic conditions. The detailed screening, isolation and molecular identification process can be found in Supplementary Material 1. After enrichment, the screened strains were added to a sequencing batch reactor (SBR) with nitrobenzene and other substrates to investigate the variation of microbial community and the competitive relationships between the screened strains and the indigenous flora. 2.3. The addition method of pure culture A 2.5 L sealable plastic bottle was used as an anaerobic batch reactor. After enrichment, the supernatant solution obtained by centrifugation was discarded. The screened microbes were washed three times with sterile water and then placed into 2 L of nitrobenzene-containing wastewater, which was then mixed with 500 mL anaerobic sludge obtained from a pharmaceutical wastewater treatment plant. Nitrogen gas was used for 20–30 min to purge the dissolved oxygen to achieve anaerobic conditions. After screwing the cap, the Anaerobic Sequencing Batch Reactor (ASBR) was fastened on a horizontal shaker at room temperature. The liquid was sampled every seven days to determine the degradation of
Table 1 Type of substrates and additional carbon sources in the systems. Label
Respiration types
Substrates
Co-substrates
A1 A2 A3
Anaerobic
Nitrobenzene
None Glucose Glucose and p-CNB
O1 O2 O3
Aerobic
Nitrobenzene
None Glucose Glucose and p-CNB
Y. Ren et al. / Chemosphere 141 (2015) 13–18
nitrobenzene and the slurries were taken at the same time for subsequent molecular analysis. The liquid supernatant was discarded after the settling and the fresh culture media was added to the ASBR bottle. For the aerobic process, the screened strains were put in a 2.5 L SBR with 300 mL aerobic sludge. The substrates in the anaerobic and aerobic reactors are shown in Table 1. 2.4. Analytical methods Nitrobenzene, p-CNB and aniline were analyzed using an HPLC system (Shimadzu LC20A; SPD-M20A UV–Vis Detector; SIL-20A Auto sampler; Agilent HC-C18 column, 4.6 mm 250 mm, 5 lm). A 1.5–2.0 mL sample of the cultures was collected for analysis. The liquid was centrifuged at 4 °C for 15 min at 13,000 rpm. The supernatant was collected and filtered through a 0.45 lm membrane filter before HPLC analysis. Methanol:water = 70:30 (v/v) was the mobile phase at a flow rate of 1.0 mL min1. The UV detector was set at 245 nm. The retention times for aniline, nitrobenzene and p-CNB were 3.92, 5.38 and 7.12 min, respectively. The recoveries of NB, 4-CNB, and aniline are 88–105%, 91–115%, and 91–106%, respectively. All experiments and measurements were performed in triplicate, and the arithmetic averages were taken for additional calculations and data analysis. 2.5. DNA extraction from sludge and subsequent PCR Total DNA of 1.5 mL sludge sample was extracted with OMEGA DNA kits, following the manufacturer’s instructions. The extracted and purified DNA was used as the template with F34l (50 -ACTCCT ACGGGAGGCAGCAG-30 ) (GC clamp: 50 -CGCCCGCCGCGCCCCGCGC CCGTCCCGCCGCCCCCGCCCG-30 ) as the forward primer sequence and R518 (50 -ATTACCGCGGCTGCTGGC-30 ) as the reverse primer. The PCR reaction system and procedures were the same as that described in Supplementary Material 2. 2.6. DGGE, gel segmentation, cloning and sequencing Samples of the PCR products (20 mL) were mixed with 2 DGGE dye and loaded onto 8% polyacrylamide gels in 1 Tris–acetate EDTA (TAE) buffer using a DCode universal mutation detection system (Bio-Rad, USA). The polyacrylamide gels were made with a linear denaturing gradient from 35% at the top of the gel to 70% denaturant at the bottom. The electrophoresis was run for 14 h at 60 °C and 60 V, after which the gels were stained with ethidium bromide and photographed on a UV transilluminator. DGGE bands of interest were cut and immersed overnight in 50 mL of MilliQÒH2O at 4 °C. After centrifuging the prepared bands, DNA in the supernatant was amplified using universal bacterial primers (F341 and R534). The PCR reaction system contained 5 lL 10 TE buffer, 4 lL dNTPs, 1 lL F341 and 1 lL R534 primers (10 lM), 3 lL MgCl2 (25 mM), 5 lL DNA template and 1.5 U Ex Taq (TaKaRa, Dalian, China). The mixture was then diluted to 50 lL with sterilized water. The PCR amplification was conducted using the following protocol: initial denaturation for 10 min at 95 °C and 40 cycles of denaturation for 1 min at 93 °C, 48 °C for 1 min, and 72 °C for 1 min 10 s, followed by a final extension for 3 min 50 s at 72 °C, after which the DNA was stored at 4 °C. A TaKaRa MiniBEST DNA Fragment Purification Kit was used to purify the PCR products. The DNA was purified following the instructions of the manufacturer. The PCR products were cloned into the pMD18-T vector using a pMD 18-T Simple Vector kit (TaKaRa Code: 103, TaKaRa, Dalian, China) according to the manufacturer’s instructions. The cloned PCR products were sequenced using a 16-capillary ABI PRISM 3100 Genetic Analyzer. Homology
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searches were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). MEGA 4 (Build# 4026) was used to align the sequences and compare their homologies to known bacterial 16S rRNA gene sequences. 2.7. Accession number of all the isolates After sequencing, all the sequences were submitted to Genbank with accession numbers of KC336083 and KF895813 for strains NBA-1 and NBO-1, respectively. The accession numbers of the sequences obtained from DGGE gel are KC315784–KC315797 for Band 1 to Band 14. 3. Results 3.1. Isolation and morphological identification After acclimation for several generations and repeated smear cultures, two bacterial strains were isolated based on their utilization of nitrobenzene as the sole carbon, nitrogen and energy source under either anaerobic or aerobic conditions. The strains were designated NBA-1 and NBO-1, respectively. The number of colonies that appeared under anaerobic conditions was more than that under aerobic conditions (see Supplementary Material 1), suggesting the preferential growth of nitrobenzene-degrading bacteria under anaerobic than that under aerobic conditions. 3.2. The molecular identification of pure strains NBA-1 and NBO-1 were purified by re-streaking single colonies three times (see Supplementary Material 2). Their 16S rRNA genes were amplified and sequenced and they were both identified as Klebsiella oxytoca strains by BLAST comparison, with >99% similarity to the type strain of the species. NBA-1 was used in the subsequent experiments because it was more efficient in degradation of nitrobenzene (see Supplementary Material 3). The sequence of NBA-1 was submitted to GenBank under accession number KC336083 and the strain is deposited in the Guangdong Culture Collection Centre of Microbiology (collection number: GIMCC1.724). 3.3. Reactor operation To investigate the fate of the screened strain, NBA-1 was introduced into nitrobenzene-containing wastewater as described above. Fig. 1 shows the concentrations of nitrobenzene, aniline, and p-CNB in the influent and effluent over different operating periods. As shown in Fig. 1(a) and (b), 0.7 mM NB could be degraded within 7 days by strain NBA-1 under anaerobic conditions. Similar results were obtained under aerobic conditions (Fig. 1(d) and (e)) which indicated that NBA-1 can degrade NB both with and without oxygen. So, we could detect the presence of aniline under anaerobic biodegradation conditions (in Fig. 1(a)–(c)). No aniline was detected under aerobic conditions (Fig. 1(d)–(f)) because it is readily degraded. The addition of glucose (Fig. 1(b)) had no impact on the performance of the system. The co-metabolic influence of glucose is prevalent in the degradation of PAHs and other aromatic compounds (Basheer et al., 2012; Li et al., 2012; Wang et al., 2012). Glucose provides energy and nutrients that are useful for the proliferation of microorganisms without selectivity, so there was no obvious variation of NB biodegradation when glucose was added to this system. When 20 mg L1 p-CNB was added (Fig. 1(c)) under anaerobic conditions, NB and p-CNB could be biodegraded completely. However, the total amount of aniline from NB and p-CNB mixture
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Y. Ren et al. / Chemosphere 141 (2015) 13–18
0.9
0.9
(a) Anaerobic: NB
0.7 0.6 0.5
NB in influent NB in effluent AN in influent AN in effluent
0.4 0.3
(d) Aerobic: NB
0.8 0.7
Concentration (mM)
Concentration (mM)
0.8
0.2
0.6 0.5 NB in influent NB in effluent
0.4 0.3 0.2 0.1
0.1 0.0
0
14
28
42
56
70
84
0.0
98
0
14
28
0.9
0.9
(b) Anaerobic: NB+glucose
0.8 0.7 0.6 0.5
NB in influent NB in effluent AN in influent AN in effluent
0.4 0.3 0.2 0.1
70
84
98
(e) Aerobic: NB+glucose
0.7 0.6 0.5 NB in influent NB in effluent
0.4 0.3 0.2 0.1
0.0 0
14
28
42
56
70
84
0.0
98
0
14
28
56
70
84
98
0.9
0.9
(c) Anaerobic: NB+p-CNB+glucose
0.8
0.7
0.7
Concentration (mM)
0.8
0.6 0.5 0.4
AN in effluent p -CNB in influent p -CNB in effluent
NB in influent NB in effluent AN in influent
0.3 0.2 0.1 0.0 0
42
Time (d)
Time (d)
Concentration (mM)
56
0.8
Concentration (mM)
Concentration (mM)
42
Time (d)
Time (d)
(f) Aerobic: NB+p -CNB+glucose
0.6 NB in influent NB in effluent p-CNB in influent p-CNB in effluent
0.5 0.4 0.3 0.2 0.1
14
28
42
56
70
84
98
Time (h)
0.0 0
14
28
42
56
70
84
98
Time (h)
Fig. 1. Biodegradation of substrates (different carbon sources) in each SBR during 90 days of operation.
was approximately 20% less than that from NB only. It can be concluded that the degradation of NB was inhibited by p-CNB although aniline is also one of the anaerobic metabolic intermediates of p-CNB (Yang et al., 2013). In the aerobic system (Fig. 1(f)), the degradation of NB in the first 21 days after the addition of p-CNB was 80%. As the operation continued, the degradation of NB gradually decreased, until at 91 days the degradation of NB and p-CNB reached only approximately 20%. This finding indicates that p-CNB has a negative effect on the performance of the system under aerobic conditions. 3.4. The fate of NBA-1 in the mixed sludge system The fate of a pure culture strain exposed to wastewater treatment sludge is the main factor that determines the utility of the microorganism in the treatment process. Therefore, we sampled the sludge to evaluate the survival of strain NBA-1 and the variation of the microbial community under the different treatment conditions. Sludge samples after 0, 25, 49 and 89 days of operation were extracted for PCR, and the DNA segments obtained (220 bp) can be seen in Supplementary Material 4. The amplified DNA
segments were analyzed on DGGE gels to compare the bands resulting from different treatments. In Fig. 2, the NBA-1 lane represents the pure strain and the Day 0 lane represents the mixture of NBA-1 and the activated sludge. A1 (Day 25, 49 and 89) represents the anaerobic system using nitrobenzene as the sole source of carbon, nitrogen, and energy. It can be observed that some new bands appeared and/or the brightness of existing bands was enhanced. The addition of glucose changed the distribution of the bands, especially at the bottom of the lanes. The pairwise similarity coefficients are shown in Supplementary Material 5. Compared with glucose amendment (Fig. 2 – Lane A2), some bands with p-CNB amendment (Lane A3) began to fade away, suggesting that certain types of microbes were not adapting well to the environment containing p-CNB. In Fig. 2, O1, O2 and O3 represent the DGGE separation patterns when the experiments were run under aerobic conditions. After 89 days of operation, in the sludge with nitrobenzene as the sole carbon source (see O1), there were common attenuated signals of many bands including the dominant strain NBA-1. Additionally, in the case of the sludge with extra glucose added, some bands were intensified whereas some other bands faded away, with the
Y. Ren et al. / Chemosphere 141 (2015) 13–18
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Fig. 2. DGGE patterns of different reactors with different treatments and periods.
more dominant strains disappearing after 25 days. When p-CNB was added, the NBA-1 band and some others began to disappear after only 49 days of treatment. These data show that the presence of p-CNB can support the growth of nitrobenzene-degrading microbes (but not NBA-1) in an aerobic system at the same time that it has a strong inhibitory effect on other microbes (such as NBA-1) in the system. Again, it can be observed that p-CNB plays an important role in determining the bacterial community structure in an aerobic sludge system. To investigate the changes of the patterns in a greater detail, 14 bands were cut from the gel (see Fig. 2) to purify, clone for sequencing and identify their species and their evolutionary relationships (Supplementary material 6). Table 2 shows the band changes when the various lanes are compared with lanes A0 and O0, i.e., the initial microbial communities under anaerobic or aerobic conditions, respectively. Based on the similarity of the numbers of bands in each lane (see Supplementary material 5) and Table 2, it can be observed that the diversity changed during the operation of the system and that more bands were enhanced under anaerobic conditions whereas more bands were weakened under aerobic conditions. In particular, Bands 1, 6, 8 and 10 weakened or disappeared in all three treatments under aerobic conditions even though the target strain (Band 1) was still present in the system. 4. Discussion Strains of K. oxytoca in the Enterobacteriaceae are known for their ability of pollutants-degradation under aerobic (Wang et al., 2012) and anaerobic conditions (Chen et al., 2009; Chamkha et al., 2011). Bai et al. (2012) also reported the metabolism of pollutants under both conditions. Based upon our biodegradation data, it is clear that strain NBA-1 is a facultative bacterium that Table 2 The enhanced and faded bands in different treatment conditions. Lane
Enhanced
Faded
A1 A2 A3 O1 O2 O3
8, 4, 9, 3, 2, –
– – – 1, 6, 8, 10, 13 1, 6, 8, 10 1, 3, 6, 8, 10
9 9, 12, 14 12, 14 7, 9 9, 11, 12
proliferates faster under aerobic conditions but degrades nitrobenzene faster under anaerobic conditions (see Supplementary material 3). Aniline is the intermediate of NB degradation under both anaerobic and aerobic conditions (Wang et al., 2011; Wu et al., 2012), while the subsequent degradation efficiency of aniline is much higher under aerobic than under anaerobic condition (Kahng et al., 2000). Because the degradation rate of aniline under anaerobic conditions is very slow (Kahng et al., 2000), we assumed that aniline would not be degraded under anaerobic conditions and that the accumulation of aniline reflects the degradation rate of NB. Although aniline is also the intermediate of p-CNB degradation (Yang et al., 2013), we observed the decrease in aniline produced (Fig. 1c), which implied that the inhibitory effect of p-CNB on microbial activity was due to the inhibition of the reduction of NB. The addition of glucose had no obvious effect on the biodegradation of NB and the production of aniline. From observation of the sludge bacterial community, the dominant strains retained their competitive advantage in the anaerobic system, whereas in the aerobic system their dominant position was taken by other microbes. Other researchers have reported similar results when examining the effect of other environmental factors. Lapara et al. (2001) studied the bacterial community of a pharmaceutical wastewater at different temperatures and found that aerobic biological reactors suffered more adverse effects at elevated temperatures than did anaerobic reactors. Wei and Finneran (2011) demonstrated that the microbial community diversity decreased when TBA (tert-butyl alcohol) was mineralized with fuel-contaminated aquifer materials. In this study, the community structure of the aerobic system was more affected by the presence of the toxic p-CNB than was the case under anaerobic conditions. Throughout the course of the anaerobic sludge incubation, the added strains preserved their dominant position, both in the presence and absence of glucose, and there was a tendency that nearly all of the DGGE bands intensified over time during reactor operation. When the system containing p-CNB was operated under aerobic conditions, the native microorganisms in the pharmaceutical waste water were more adaptable to the stress than strain NBA-1, which was gradually eliminated from the sludge. This indicated that NBA-1 has limited competition ability when it is placed into an open system, although it can proliferate and degrade nitrobenzene under aerobic conditions.
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The addition of p-CNB to the reactor system did not have a significant effect on the composition of the bacterial community under anaerobic conditions. One of the possible reasons for the adaptability of strain NBA-1 is that it was isolated from a pharmaceutical sludge that contains many toxic compounds and this environment selected for its competitive ability to survive. Moreover, advanced studies of the enzyme system changes and the degradation mechanisms should be further developed. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant Nos. 20977035, 51178190) and the Research Fund of SIT of Guangzhou (Grant No. 2013J4100107). We would like to thank Dr. Max M. Häggblom, Dr. Weilin Huang, and Dr. Donald G. Barnes for linguistic revision and useful suggestions. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2015.05.098. References Arora, P.K., Sasikala, C., Ramana, C.V., 2012. Degradation of chlorinated nitroaromatic compounds. Appl. Microbiol. Biotechnol. 93, 2265–2277. Bai, L.P., Wu, X.B., Jiang, L.J., Liu, J., Long, M.N., 2012. Hydrogen production by overexpression of hydrogenase subunit in oxygen-tolerant Klebsiella oxytoca HP1. Int. J. Hydrogen Energy 37, 13227–13233. Basheer, F., Isa, M.H., Farooqi, I.H., 2012. Biodegradation of o-nitrophenol by aerobic granules with glucose as co-substrate. Water Sci. Technol. 65, 2132–2139. Batista, S.B., Mounteer, A.H., Amorim, F.R., Totola, M.R., 2006. Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites. Bioresour. Technol. 97, 868–875. Chamkha, M., Trabelsi, Y., Mnif, S., Sayadi, S., 2011. Isolation and characterization of Klebsiella oxytoca strain degrading crude oil from a Tunisian off-shore oil field. J. Basic Microbiol. 51, 580–589. Chao, W., Yi, L., Zhigang, L., Peifang, W., 2009. Bioremediation of nitrobenzenepolluted sediments by Pseudomonas putida. Bull. Environ. Contam. Toxicol. 83, 865–868. Chen, C.Y., Kao, C.M., Chen, S.C., Chen, T.Y., 2009. Biodegradation of tetracyanonickelate by Klebsiella oxytoca under anaerobic conditions. Desalination 249, 1212–1216. Fang, H.H.P., Liu, H., Zhang, T., 2002. Characterization of a hydrogen-producing granular sludge. Biotechnol. Bioeng. 78, 44–52. Gilbride, K.A., Frigon, D., Cesnik, A., Gawat, J., Fulthorpe, R.R., 2006. Effect of chemical and physical parameters on a pulp mill biotreatment bacterial community. Water Res. 40, 775–787.
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
The fate of a nitrobenzene-degrading bacterium in pharmaceutical wastewater treatment sludge Yuan Ren a,b,c,⇑, Juan Yang a, Shaoyi Chen a a
School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Panyu District, Guangzhou 510006, PR China The Key Laboratory of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, PR China c The Key Laboratory of Environmental Protection and Eco-Remediation of Guangdong Regular Higher Education Institutions, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
NBA-1 can grow and biodegrade
nitrobenzene under anaerobic and aerobic conditions. There was no obvious difference of NB degradation when glucose was added. NB biodegradation rate decreased when was added under aerobic condition. NBA-1 and some microbes are weakened when p-CNB was added under aerobic condition. The bacterial community was stable when p-CNB was added under anaerobic condition.
a r t i c l e
i n f o
Article history: Received 29 December 2014 Received in revised form 9 May 2015 Accepted 29 May 2015
Keywords: Nitrobenzene-degrading bacterium Fate Pharmaceutical sludge PCR-DGGE Bacterial community
a b s t r a c t This paper describes the fate of a nitrobenzene-degrading bacterium, Klebsiella oxytoca NBA-1, which was isolated from a pharmaceutical wastewater treatment facility. The 90-day survivability of strain NBA-1 after exposure to sludge under anaerobic and aerobic conditions was investigated. The bacterium was inoculated into sludge amended with glucose and p-chloronitrobenzene (p-CNB) to compare the bacterial community variations between the modified sludge and nitrobenzene amendment. The results showed that glucose had no obvious effect on nitrobenzene biodegradation in the co-metabolism process, regardless of the presence/absence of oxygen. When p-CNB was added under anaerobic conditions, the biodegradation rate of nitrobenzene remained unchanged although p-CNB inhibited the production of aniline. The diversity of the microbial community increased and NBA-1 continued to be one of the dominant strains. Under aerobic conditions, the degradation rate of both nitrobenzene and p-CNB was only 20% of that under anaerobic conditions. p-CNB had a toxic effect on the microorganisms in the sludge so that most of the DGGE (denaturing gradient gel electrophoresis) bands, including that of NBA-1, began to disappear under aerobic conditions after 90 days of exposure. These data show that the bacterial community was stable under anaerobic conditions and the microorganisms, including NBA-1, were more resistant to the adverse environment. Ó 2015 Elsevier Ltd. All rights reserved.
⇑ Corresponding author at: School of Environment and Energy, South China University of Technology, Higher Education Mega Center, Panyu District, Guangzhou 510006, PR China. E-mail address: [email protected] (Y. Ren). http://dx.doi.org/10.1016/j.chemosphere.2015.05.098 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
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1. Introduction Nitrobenzene and its chlorinated compounds are important organic intermediates for the production of organic pigments, azo dyes, pesticides, and medicines. Wastewater from aniline-producing factories, waste gas from chemical factories, and transport accidents are the main sources of nitrobenzene pollution. For treatment of moderate and high concentrations of nitrobenzene-containing wastewater, extraction, absorption, advanced oxidation, and airlift processes can be used to separate water and the pollutant with high efficiency. Biological treatment for nitrobenzene-containing wastewater is practical for lower nitrobenzene concentrations or for effluent from physical and chemical pretreatment. Many studies have screened and isolated nitrobenzene-degrading microorganisms from different samples, while the adaptability of bacterial pure culture to salinity, co-substrates and heavy metals still needs more consideration (Chao et al., 2009; Zheng et al., 2009; Lin et al., 2011; Li et al., 2012; Wang et al., 2012). The fate of a given bacterial species is a crucial aspect for its application in wastewater treatment because competition among microorganisms is very common in the environment. Generally, a single organic compound is often used as the sole carbon source and energy source to screen for the most effective microorganisms to use in the biodegradation of waste (Arora et al., 2012). When two or more species of bacteria exist in the same system, the competition for carbon sources, oxygen, temperature and other nutritional factors can affect the survival of the dominant strain (Polymenakou and Stephanou, 2005; Giorni et al., 2009; Chamkha et al., 2011). Some studies have been conducted to examine the variations of some specific bacterial communities with different types of molecular biological technologies that do not rely on the traditional culturing methods (Fang et al., 2002; Gilbride et al., 2006; O’Reilly et al., 2009; Meynet et al., 2012). Compared with the papers published in the field of effective microorganism isolation, identification and determination of other characteristics (Song et al., 2005; Batista et al., 2006; Hassanshahian et al., 2012; Li et al., 2012; Wang et al., 2012), less work has been conducted to investigate the fate of the microbes in full or laboratory scale wastewater treatment facilities (Sahlstrom et al., 2008; Harrison, 2013). Although some composite microbial preparations, such as effective microorganisms and growth-promoting microorganisms, are commercialized in food additives and wastewater treatment (Glick et al., 1999), many of these new bacterial agents have to be added regularly during the process to compensate for loss or inactivation of the amended strains. The dominant strain or clones that are responsible for the degradation have different fates due to their responses to carbon sources, oxygen availability and many other environmental factors. Therefore, the results of adaptability studies using pure culture strains have important applications in both production and environmental situations. As a case in point, to understand the fate of a pure strain in pharmaceutical wastewater, a nitrobenzene-degrading stain was isolated and added to lab scale nitrobenzene-containing wastewater treatment sludge amended with nitrobenzene and/or p-CNB as carbon sources. The concentrations of nitrobenzene, p-chloronitrobenzene (p-CNB) and aniline (a prominent degradation product of nitrobenzene) were monitored under anaerobic or aerobic conditions while the bacterial community structure was studied using the PCR-DGGE/cloning method. The diversity changes associated with the different treatments were assessed to examine the survivability of the pure cultures and gather additional data that will be useful in judging the utility of the microorganism in the treatment of pharmaceutical wastewater. Moreover, we hope to obtain more
scientific data regarding the characteristics and factors that influence the bioactivity of the pure cultures. 2. Materials and methods 2.1. Chemicals and cultures Nitrobenzene (>99.0%) and p-CNB (99.0%) were purchased from Fuchen Chemical Reagents Co. Ltd. (Tianjin, China). All other chemicals used in this study for culture medium were obtained from Kermel Chemical Reagents Co. Ltd. (Tianjin, China). The reagents for HPLC flow phase were all HPLC grade pure. The inorganic culture medium contained (w/v): 0.5% NaCl; 0.05% MgSO4; 0.5% K2HPO4: 0.3% KH2PO4; and a mixture of 1.0% trace elements which contained (g L1): 0.19 CoCl26H2O, 0.024 NiCl26H2O, 0.03 CuCl22H2O, 0.05 ZnCl2, 0.05 H3BO3, 0.5 MnCl24H2O and 0.09 (NH4)6Mo7O242H2O. The isolation medium contained (w/v): inorganic culture medium, 1.5% agar with nitrobenzene as the sole source of carbon, nitrogen and energy. Na2S9H2O 0.2 mL L1 (stock solution 500 g L1) was added as an oxygen scavenger when anaerobic medium was being prepared. Enrichment cultures were established using a medium containing (w/v) inorganic culture medium, 1.0% trace elements mix (see content above), 1.0% yeast extract and 1.0% peptone. Nitrobenzene was added after sterilization. 2.2. Molecular biological analysis of strains in pharmaceutical sludge The sludge sample was taken from a pharmaceutical wastewater treatment plant (Guangzhou, Guangdong, China) and screened for nitrobenzene-degrading microorganisms using a gradient flow under anaerobic/aerobic conditions. The detailed screening, isolation and molecular identification process can be found in Supplementary Material 1. After enrichment, the screened strains were added to a sequencing batch reactor (SBR) with nitrobenzene and other substrates to investigate the variation of microbial community and the competitive relationships between the screened strains and the indigenous flora. 2.3. The addition method of pure culture A 2.5 L sealable plastic bottle was used as an anaerobic batch reactor. After enrichment, the supernatant solution obtained by centrifugation was discarded. The screened microbes were washed three times with sterile water and then placed into 2 L of nitrobenzene-containing wastewater, which was then mixed with 500 mL anaerobic sludge obtained from a pharmaceutical wastewater treatment plant. Nitrogen gas was used for 20–30 min to purge the dissolved oxygen to achieve anaerobic conditions. After screwing the cap, the Anaerobic Sequencing Batch Reactor (ASBR) was fastened on a horizontal shaker at room temperature. The liquid was sampled every seven days to determine the degradation of
Table 1 Type of substrates and additional carbon sources in the systems. Label
Respiration types
Substrates
Co-substrates
A1 A2 A3
Anaerobic
Nitrobenzene
None Glucose Glucose and p-CNB
O1 O2 O3
Aerobic
Nitrobenzene
None Glucose Glucose and p-CNB
Y. Ren et al. / Chemosphere 141 (2015) 13–18
nitrobenzene and the slurries were taken at the same time for subsequent molecular analysis. The liquid supernatant was discarded after the settling and the fresh culture media was added to the ASBR bottle. For the aerobic process, the screened strains were put in a 2.5 L SBR with 300 mL aerobic sludge. The substrates in the anaerobic and aerobic reactors are shown in Table 1. 2.4. Analytical methods Nitrobenzene, p-CNB and aniline were analyzed using an HPLC system (Shimadzu LC20A; SPD-M20A UV–Vis Detector; SIL-20A Auto sampler; Agilent HC-C18 column, 4.6 mm 250 mm, 5 lm). A 1.5–2.0 mL sample of the cultures was collected for analysis. The liquid was centrifuged at 4 °C for 15 min at 13,000 rpm. The supernatant was collected and filtered through a 0.45 lm membrane filter before HPLC analysis. Methanol:water = 70:30 (v/v) was the mobile phase at a flow rate of 1.0 mL min1. The UV detector was set at 245 nm. The retention times for aniline, nitrobenzene and p-CNB were 3.92, 5.38 and 7.12 min, respectively. The recoveries of NB, 4-CNB, and aniline are 88–105%, 91–115%, and 91–106%, respectively. All experiments and measurements were performed in triplicate, and the arithmetic averages were taken for additional calculations and data analysis. 2.5. DNA extraction from sludge and subsequent PCR Total DNA of 1.5 mL sludge sample was extracted with OMEGA DNA kits, following the manufacturer’s instructions. The extracted and purified DNA was used as the template with F34l (50 -ACTCCT ACGGGAGGCAGCAG-30 ) (GC clamp: 50 -CGCCCGCCGCGCCCCGCGC CCGTCCCGCCGCCCCCGCCCG-30 ) as the forward primer sequence and R518 (50 -ATTACCGCGGCTGCTGGC-30 ) as the reverse primer. The PCR reaction system and procedures were the same as that described in Supplementary Material 2. 2.6. DGGE, gel segmentation, cloning and sequencing Samples of the PCR products (20 mL) were mixed with 2 DGGE dye and loaded onto 8% polyacrylamide gels in 1 Tris–acetate EDTA (TAE) buffer using a DCode universal mutation detection system (Bio-Rad, USA). The polyacrylamide gels were made with a linear denaturing gradient from 35% at the top of the gel to 70% denaturant at the bottom. The electrophoresis was run for 14 h at 60 °C and 60 V, after which the gels were stained with ethidium bromide and photographed on a UV transilluminator. DGGE bands of interest were cut and immersed overnight in 50 mL of MilliQÒH2O at 4 °C. After centrifuging the prepared bands, DNA in the supernatant was amplified using universal bacterial primers (F341 and R534). The PCR reaction system contained 5 lL 10 TE buffer, 4 lL dNTPs, 1 lL F341 and 1 lL R534 primers (10 lM), 3 lL MgCl2 (25 mM), 5 lL DNA template and 1.5 U Ex Taq (TaKaRa, Dalian, China). The mixture was then diluted to 50 lL with sterilized water. The PCR amplification was conducted using the following protocol: initial denaturation for 10 min at 95 °C and 40 cycles of denaturation for 1 min at 93 °C, 48 °C for 1 min, and 72 °C for 1 min 10 s, followed by a final extension for 3 min 50 s at 72 °C, after which the DNA was stored at 4 °C. A TaKaRa MiniBEST DNA Fragment Purification Kit was used to purify the PCR products. The DNA was purified following the instructions of the manufacturer. The PCR products were cloned into the pMD18-T vector using a pMD 18-T Simple Vector kit (TaKaRa Code: 103, TaKaRa, Dalian, China) according to the manufacturer’s instructions. The cloned PCR products were sequenced using a 16-capillary ABI PRISM 3100 Genetic Analyzer. Homology
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searches were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). MEGA 4 (Build# 4026) was used to align the sequences and compare their homologies to known bacterial 16S rRNA gene sequences. 2.7. Accession number of all the isolates After sequencing, all the sequences were submitted to Genbank with accession numbers of KC336083 and KF895813 for strains NBA-1 and NBO-1, respectively. The accession numbers of the sequences obtained from DGGE gel are KC315784–KC315797 for Band 1 to Band 14. 3. Results 3.1. Isolation and morphological identification After acclimation for several generations and repeated smear cultures, two bacterial strains were isolated based on their utilization of nitrobenzene as the sole carbon, nitrogen and energy source under either anaerobic or aerobic conditions. The strains were designated NBA-1 and NBO-1, respectively. The number of colonies that appeared under anaerobic conditions was more than that under aerobic conditions (see Supplementary Material 1), suggesting the preferential growth of nitrobenzene-degrading bacteria under anaerobic than that under aerobic conditions. 3.2. The molecular identification of pure strains NBA-1 and NBO-1 were purified by re-streaking single colonies three times (see Supplementary Material 2). Their 16S rRNA genes were amplified and sequenced and they were both identified as Klebsiella oxytoca strains by BLAST comparison, with >99% similarity to the type strain of the species. NBA-1 was used in the subsequent experiments because it was more efficient in degradation of nitrobenzene (see Supplementary Material 3). The sequence of NBA-1 was submitted to GenBank under accession number KC336083 and the strain is deposited in the Guangdong Culture Collection Centre of Microbiology (collection number: GIMCC1.724). 3.3. Reactor operation To investigate the fate of the screened strain, NBA-1 was introduced into nitrobenzene-containing wastewater as described above. Fig. 1 shows the concentrations of nitrobenzene, aniline, and p-CNB in the influent and effluent over different operating periods. As shown in Fig. 1(a) and (b), 0.7 mM NB could be degraded within 7 days by strain NBA-1 under anaerobic conditions. Similar results were obtained under aerobic conditions (Fig. 1(d) and (e)) which indicated that NBA-1 can degrade NB both with and without oxygen. So, we could detect the presence of aniline under anaerobic biodegradation conditions (in Fig. 1(a)–(c)). No aniline was detected under aerobic conditions (Fig. 1(d)–(f)) because it is readily degraded. The addition of glucose (Fig. 1(b)) had no impact on the performance of the system. The co-metabolic influence of glucose is prevalent in the degradation of PAHs and other aromatic compounds (Basheer et al., 2012; Li et al., 2012; Wang et al., 2012). Glucose provides energy and nutrients that are useful for the proliferation of microorganisms without selectivity, so there was no obvious variation of NB biodegradation when glucose was added to this system. When 20 mg L1 p-CNB was added (Fig. 1(c)) under anaerobic conditions, NB and p-CNB could be biodegraded completely. However, the total amount of aniline from NB and p-CNB mixture
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0.9
0.9
(a) Anaerobic: NB
0.7 0.6 0.5
NB in influent NB in effluent AN in influent AN in effluent
0.4 0.3
(d) Aerobic: NB
0.8 0.7
Concentration (mM)
Concentration (mM)
0.8
0.2
0.6 0.5 NB in influent NB in effluent
0.4 0.3 0.2 0.1
0.1 0.0
0
14
28
42
56
70
84
0.0
98
0
14
28
0.9
0.9
(b) Anaerobic: NB+glucose
0.8 0.7 0.6 0.5
NB in influent NB in effluent AN in influent AN in effluent
0.4 0.3 0.2 0.1
70
84
98
(e) Aerobic: NB+glucose
0.7 0.6 0.5 NB in influent NB in effluent
0.4 0.3 0.2 0.1
0.0 0
14
28
42
56
70
84
0.0
98
0
14
28
56
70
84
98
0.9
0.9
(c) Anaerobic: NB+p-CNB+glucose
0.8
0.7
0.7
Concentration (mM)
0.8
0.6 0.5 0.4
AN in effluent p -CNB in influent p -CNB in effluent
NB in influent NB in effluent AN in influent
0.3 0.2 0.1 0.0 0
42
Time (d)
Time (d)
Concentration (mM)
56
0.8
Concentration (mM)
Concentration (mM)
42
Time (d)
Time (d)
(f) Aerobic: NB+p -CNB+glucose
0.6 NB in influent NB in effluent p-CNB in influent p-CNB in effluent
0.5 0.4 0.3 0.2 0.1
14
28
42
56
70
84
98
Time (h)
0.0 0
14
28
42
56
70
84
98
Time (h)
Fig. 1. Biodegradation of substrates (different carbon sources) in each SBR during 90 days of operation.
was approximately 20% less than that from NB only. It can be concluded that the degradation of NB was inhibited by p-CNB although aniline is also one of the anaerobic metabolic intermediates of p-CNB (Yang et al., 2013). In the aerobic system (Fig. 1(f)), the degradation of NB in the first 21 days after the addition of p-CNB was 80%. As the operation continued, the degradation of NB gradually decreased, until at 91 days the degradation of NB and p-CNB reached only approximately 20%. This finding indicates that p-CNB has a negative effect on the performance of the system under aerobic conditions. 3.4. The fate of NBA-1 in the mixed sludge system The fate of a pure culture strain exposed to wastewater treatment sludge is the main factor that determines the utility of the microorganism in the treatment process. Therefore, we sampled the sludge to evaluate the survival of strain NBA-1 and the variation of the microbial community under the different treatment conditions. Sludge samples after 0, 25, 49 and 89 days of operation were extracted for PCR, and the DNA segments obtained (220 bp) can be seen in Supplementary Material 4. The amplified DNA
segments were analyzed on DGGE gels to compare the bands resulting from different treatments. In Fig. 2, the NBA-1 lane represents the pure strain and the Day 0 lane represents the mixture of NBA-1 and the activated sludge. A1 (Day 25, 49 and 89) represents the anaerobic system using nitrobenzene as the sole source of carbon, nitrogen, and energy. It can be observed that some new bands appeared and/or the brightness of existing bands was enhanced. The addition of glucose changed the distribution of the bands, especially at the bottom of the lanes. The pairwise similarity coefficients are shown in Supplementary Material 5. Compared with glucose amendment (Fig. 2 – Lane A2), some bands with p-CNB amendment (Lane A3) began to fade away, suggesting that certain types of microbes were not adapting well to the environment containing p-CNB. In Fig. 2, O1, O2 and O3 represent the DGGE separation patterns when the experiments were run under aerobic conditions. After 89 days of operation, in the sludge with nitrobenzene as the sole carbon source (see O1), there were common attenuated signals of many bands including the dominant strain NBA-1. Additionally, in the case of the sludge with extra glucose added, some bands were intensified whereas some other bands faded away, with the
Y. Ren et al. / Chemosphere 141 (2015) 13–18
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Fig. 2. DGGE patterns of different reactors with different treatments and periods.
more dominant strains disappearing after 25 days. When p-CNB was added, the NBA-1 band and some others began to disappear after only 49 days of treatment. These data show that the presence of p-CNB can support the growth of nitrobenzene-degrading microbes (but not NBA-1) in an aerobic system at the same time that it has a strong inhibitory effect on other microbes (such as NBA-1) in the system. Again, it can be observed that p-CNB plays an important role in determining the bacterial community structure in an aerobic sludge system. To investigate the changes of the patterns in a greater detail, 14 bands were cut from the gel (see Fig. 2) to purify, clone for sequencing and identify their species and their evolutionary relationships (Supplementary material 6). Table 2 shows the band changes when the various lanes are compared with lanes A0 and O0, i.e., the initial microbial communities under anaerobic or aerobic conditions, respectively. Based on the similarity of the numbers of bands in each lane (see Supplementary material 5) and Table 2, it can be observed that the diversity changed during the operation of the system and that more bands were enhanced under anaerobic conditions whereas more bands were weakened under aerobic conditions. In particular, Bands 1, 6, 8 and 10 weakened or disappeared in all three treatments under aerobic conditions even though the target strain (Band 1) was still present in the system. 4. Discussion Strains of K. oxytoca in the Enterobacteriaceae are known for their ability of pollutants-degradation under aerobic (Wang et al., 2012) and anaerobic conditions (Chen et al., 2009; Chamkha et al., 2011). Bai et al. (2012) also reported the metabolism of pollutants under both conditions. Based upon our biodegradation data, it is clear that strain NBA-1 is a facultative bacterium that Table 2 The enhanced and faded bands in different treatment conditions. Lane
Enhanced
Faded
A1 A2 A3 O1 O2 O3
8, 4, 9, 3, 2, –
– – – 1, 6, 8, 10, 13 1, 6, 8, 10 1, 3, 6, 8, 10
9 9, 12, 14 12, 14 7, 9 9, 11, 12
proliferates faster under aerobic conditions but degrades nitrobenzene faster under anaerobic conditions (see Supplementary material 3). Aniline is the intermediate of NB degradation under both anaerobic and aerobic conditions (Wang et al., 2011; Wu et al., 2012), while the subsequent degradation efficiency of aniline is much higher under aerobic than under anaerobic condition (Kahng et al., 2000). Because the degradation rate of aniline under anaerobic conditions is very slow (Kahng et al., 2000), we assumed that aniline would not be degraded under anaerobic conditions and that the accumulation of aniline reflects the degradation rate of NB. Although aniline is also the intermediate of p-CNB degradation (Yang et al., 2013), we observed the decrease in aniline produced (Fig. 1c), which implied that the inhibitory effect of p-CNB on microbial activity was due to the inhibition of the reduction of NB. The addition of glucose had no obvious effect on the biodegradation of NB and the production of aniline. From observation of the sludge bacterial community, the dominant strains retained their competitive advantage in the anaerobic system, whereas in the aerobic system their dominant position was taken by other microbes. Other researchers have reported similar results when examining the effect of other environmental factors. Lapara et al. (2001) studied the bacterial community of a pharmaceutical wastewater at different temperatures and found that aerobic biological reactors suffered more adverse effects at elevated temperatures than did anaerobic reactors. Wei and Finneran (2011) demonstrated that the microbial community diversity decreased when TBA (tert-butyl alcohol) was mineralized with fuel-contaminated aquifer materials. In this study, the community structure of the aerobic system was more affected by the presence of the toxic p-CNB than was the case under anaerobic conditions. Throughout the course of the anaerobic sludge incubation, the added strains preserved their dominant position, both in the presence and absence of glucose, and there was a tendency that nearly all of the DGGE bands intensified over time during reactor operation. When the system containing p-CNB was operated under aerobic conditions, the native microorganisms in the pharmaceutical waste water were more adaptable to the stress than strain NBA-1, which was gradually eliminated from the sludge. This indicated that NBA-1 has limited competition ability when it is placed into an open system, although it can proliferate and degrade nitrobenzene under aerobic conditions.
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The addition of p-CNB to the reactor system did not have a significant effect on the composition of the bacterial community under anaerobic conditions. One of the possible reasons for the adaptability of strain NBA-1 is that it was isolated from a pharmaceutical sludge that contains many toxic compounds and this environment selected for its competitive ability to survive. Moreover, advanced studies of the enzyme system changes and the degradation mechanisms should be further developed. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant Nos. 20977035, 51178190) and the Research Fund of SIT of Guangzhou (Grant No. 2013J4100107). We would like to thank Dr. Max M. Häggblom, Dr. Weilin Huang, and Dr. Donald G. Barnes for linguistic revision and useful suggestions. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2015.05.098. References Arora, P.K., Sasikala, C., Ramana, C.V., 2012. Degradation of chlorinated nitroaromatic compounds. Appl. Microbiol. Biotechnol. 93, 2265–2277. Bai, L.P., Wu, X.B., Jiang, L.J., Liu, J., Long, M.N., 2012. Hydrogen production by overexpression of hydrogenase subunit in oxygen-tolerant Klebsiella oxytoca HP1. Int. J. Hydrogen Energy 37, 13227–13233. Basheer, F., Isa, M.H., Farooqi, I.H., 2012. Biodegradation of o-nitrophenol by aerobic granules with glucose as co-substrate. Water Sci. Technol. 65, 2132–2139. Batista, S.B., Mounteer, A.H., Amorim, F.R., Totola, M.R., 2006. Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites. Bioresour. Technol. 97, 868–875. Chamkha, M., Trabelsi, Y., Mnif, S., Sayadi, S., 2011. Isolation and characterization of Klebsiella oxytoca strain degrading crude oil from a Tunisian off-shore oil field. J. Basic Microbiol. 51, 580–589. Chao, W., Yi, L., Zhigang, L., Peifang, W., 2009. Bioremediation of nitrobenzenepolluted sediments by Pseudomonas putida. Bull. Environ. Contam. Toxicol. 83, 865–868. Chen, C.Y., Kao, C.M., Chen, S.C., Chen, T.Y., 2009. Biodegradation of tetracyanonickelate by Klebsiella oxytoca under anaerobic conditions. Desalination 249, 1212–1216. Fang, H.H.P., Liu, H., Zhang, T., 2002. Characterization of a hydrogen-producing granular sludge. Biotechnol. Bioeng. 78, 44–52. Gilbride, K.A., Frigon, D., Cesnik, A., Gawat, J., Fulthorpe, R.R., 2006. Effect of chemical and physical parameters on a pulp mill biotreatment bacterial community. Water Res. 40, 775–787.
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