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Isolation and characterization of aniline degradation slightly halophilic bacterium, Erwinia sp. Strain HSA 6
ARTICLE IN PRESS Microbiological Research 165 (2010) 418—426
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Isolation and characterization of aniline degradation slightly halophilic bacterium, Erwinia sp. Strain HSA 6 Junmin Li, Zexin Jin, Binbin Yu Institute of Ecology, Taizhou University, Linhai 317000, China Received 4 June 2009; received in revised form 9 September 2009; accepted 12 September 2009
KEYWORDS Erwinia amylovora; Aniline degradation; Salinity tolerant; Characteristics; Plasmid
Summary The isolated strain HSA6 is classified as Erwinia amylovora based on 16S rDNA sequence and the morphological and physiological properties. Strain HSA6 is the first reported E. amylovora in pure culture growing with aniline as sole electron donor and carbon source. The suitable pH for strain HSA6 is wide (from 5 to 11). Strain HSA6 is slightly halophilic with growth occurring at 0–10% (v/v) NaCl, and the suitable NaCl concentration for strain HSA6 is from 0% to 6%. The number of bacteria appeared to decrease with an increase in aniline concentration. The number of bacteria appeared to be constant as the wastewater concentration increased from 0% to 20%. However, the number of cells decreased with an increase in wastewater concentration from 30% to 50% and grew very slowly at 50%. The degradation rate of aniline was 100% at 0.5% aniline concentration after 24 h culture. The degradation rate of aniline was found to descend as the concentration of aniline increased from 0.5% to 3% and rose as the culture time increased. Strain HSA6 contains a plasmid with molecular weight higher than 42 kDA. Plasmid curing test and quantitative degradation test showed that strain requires the plasmid for aniline degradation. The gene cluster degrading aniline was determined in the plasmid by PCR amplification. & 2009 Elsevier GmbH. All rights reserved.
1. Introduction Corresponding author. Tel.: +86 57685137013;
fax: +86 57685137067. E-mail address: [email protected] (J. Li)
Aniline has toxicity to life and plant and is considered as an important environmental hazard and is subject to legislative control by the European Economic Community (EEC) directive and in the
0944-5013/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2009.09.003
ARTICLE IN PRESS Isolation and characterization of Erwinia sp. Priority Pollutant List of US Environmental Protection Agency (Federal Register 1979). While not as easily biodegraded as phenol and benzoate (Alexander and Lustigman 1966), aniline is none the less biodegradable and its biodegradation has been reported by several authors (Anson and Mackinnon 1984; Aoki et al. 1990; Lyons et al. 1984). Numbers of reports concerning the bacterial degradation of aniline had already been published, such as Delfria sp. (Liu et al. 2002; Zhang et al. 2008; Liang et al. 2005a), Pseudomonas sp. (Meyers 1992; Fukumori and Saint 1997; Bathe 2004), Acinetobacter sp. (Takeo et al. 1998; Fujii et al. 1997). O’Neill et al. (2000) isolated a consortium of bacteria capable of degrading aniline found in wastewaters produced by oil fields, in marine mud and in acid peat bog water and soils. Li et al. (2007) isolated strain PN1001, which is a member of the Pseudomonas species and it was capable of degrading pentyl amine and aniline. The effluents in pharmaceutical industries in which aniline occurs are frequently of elevated salinity (O’Neill et al. 2000), which could strongly inhibit the growth of the microorganisms (Kubo et al. 2001). The high salt content in the wastewaters can pose certain problems in treatment systems particularly on biological units. Salt-tolerant microorganisms, such as Staphylococcus sp. and Bacillus sp. were isolated to treat a high-salt-content wastewater (Kubo et al. 2001). At the pharmaceutical industry of interest to this study, wastewater is produced containing high salinity, aniline, chloride and chemical oxygen demand (COD). With the aim of obtaining microorganisms able to purify the wastewater, we attempted to isolate salt-tolerant microorganisms, which could degrade aniline and erase COD under salinity and chloride conditions. In addition, the effects of various culture medium parameters on the growth of bacteria, the removal of aniline and COD and the genetic characteristics of bacteria strain were explored.
419 0.316, and NaCl 0.25, pH 7.0. After autoclaving MSM, high salinity aniline-containing wastewater was added to obtain the desired final concentration as the sole carbon source. Luria Bertani (LB) medium contained (g l1): tryptone 10.0, NaCl 10.0, and yeast extracts 5.0.
2.2. Sources of wastewater and activated sludge Hypersalinity aniline-containing wastewater was collected from a pharmaceutical chemicals and intermediates industry. pH, salinity and chloride of the wastewater were analyzed according to the standard methods for the analysis of wastewater (State Environmental Protection Administration 2002). COD analyzed in a high dose of HgSO4 (at the ratio of HgSO4:Cl=10:1) was added to the samples to eradicate the chloride interference. The aniline compounds existed in the wastewater was determined by N-(1-naphthyl) ethylene diamine dihydrochloride spectrophotometric method (GB/T 15502-1995, http://english.mep.gov.cn/standards _reports/standards/Air_Environment/air_method/ 200809/t20080923_129249.htm). The wastewater was found to have pH lower than 1, with COD 19 400 mg l1, chloride13.69 g l1, aniline 2480 mg l1 and salinity 39.900 g l1. Subsequently, wastewater neutralized with NaOH was used for the isolation of microorganisms. Activated sludge was used as a starting material for the isolation of pure cultures. The activated sludge was collected from effluents treatment plants treating wastewater generated by pharmaceutical formulation industry. A liter of sludge sample was collected from the aerobic biological reactor. The supernatant was discarded and the biomass was pooled, mixed thoroughly and transported to the laboratory immediately.
2.3. Isolation of bacteria
2. Materials and methods 2.1. Chemicals and media Aniline (C6H7N, 93.13MW) of high purity more than 99.5% was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All of other chemicals used in this study were of the highest analytical grad available. The mineral salt medium (MSM) contained (g l1): NH4NO3 1.0, (NH4)2SO4 0.25, kH2PO4 0.25, MgSO4
Aerobic cultures (20 ml culture media) were grown in 50 ml conical flasks with cotton wool bungs. Enrichment cultures were set up with LB medium received a 10% v/v activated sludge. After 48 h culture with shaking (150 rpm) at 30 1C, acclimation were set up using hypersaline anilinecontaining wastewater as the sole carbon and nitrogen source in MSM medium at a concentration from 5% to 40% at 5%. After 14 d culture with shaking (150 rpm) at 30 1C, subsequent subcultures received 10% inoculation from the preceding cultures.
ARTICLE IN PRESS 420 To isolate pure cultures, 100 ml cultures were plated onto LB agar plates containing 5% (w/v) hypersaline wastewater for single-colony isolation. Subsequently, colonies were isolated at random and sub-cultured twice. The single pure colony was picked and inoculated into LB medium containing 5% (w/v) hypersaline wastewater and the glycerol stocks of the selected isolates were prepared and preserved at 70 1C.
2.4. Strain identification The strains were identified by morphological and biochemical tests and its 16S ribosomal DNA (16S rDNA) sequence. Total genomic DNA extraction was performed according to the cetyltrimethyl ammonium bromide (CTAB)-lysozyme-proteinase K-freezing thaw lysing method reported earlier (Li and Jin 2006a). After overnight culture with shaking (150 rpm) at 30 1C, the pellet was collected by centrifugation at 10000 rpm for 10 min. The pellet was resuspended in CTAB-lysozyme buffer [100 mmol l1 Tris–HCl (pH 8.0), 100 mmol l1 EDTA 2Na (pH 8.0), 100 mmol l1 phosphate sodium buffer (pH 8.0), 1.5 mmol l1 NaCl, 1% CTAB, 2% CaCl2, 1 mg ml1 bovine serum albumin (BSA), 1 mg ml1 lysozyme] and incubated at 37 1C for 1 h, and then incubated at 65 1C after adding 100 mg ml1 protease K. DNA was purified using a chloroform/ isoamyl alcohol (1/1) solution, and precipitated by the addition of 50% (v/v) 25% PEG 8000 (w/v) and stored at 4 1C overnight. The DNA was determined and was diluted into 20 ng ml1 and stored at 20 1C. A partial gene sequencing of 16S rDNA was performed to identify the strains. The 1466 bp 16S rDNA was amplified with conserved primer (forward prime 27f, 50 -AGAGTTTGATYMTGGCTC AG-30 reversed primer 1492r, 50 -TACGGHTACCTTACG ACT-30 ) as reported earlier (Selvakumaran et al. 2008). The reaction mixture was prepared to a total volume of 20 ml containing 1 PCR buffer, 2.0 mmol l1 MgCl2, 10 ng template DNA, 0.2 mmol l1 4 dNTP, 1 mmol l1 forward primer and reversed primer, 1 U Taq polymerase (Shanghai Sangon Co. Ltd.). The reaction mixture was incubated in a thermal cycler (PTC 220, Bio-Rad Inc., USA) at 94 1C for 5 min for initial denaturation, followed by 35 cycles of 1 min at 94 1C, 1 min at 55 1C and 1.5 min at 72 1C, and a final extension at 72 1C for 5 min. PCR products were purified using UNIQ-10 EZ Spin Column PCR Production Purification Kit (Shanghai Sangon Co. Ltd.) and were sent to Jinsite Biotechnology Co., Ltd. (Nanjing, China) for sequencing. The sequence of 1466 bp of 16S rDNA was submitted to the NCBI
J. Li et al. (National Center for Biotechnology Information) and GenBank for obtaining accession numbers (GQ222272). The sequence was compared with 16S rRNA gene sequences available in the GenBank database by BLASTn search (http://www. ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignments of partial 16S rRNA gene sequences were aligned using CLUSTAL X, version 1.8. Phylogenetic analysis was performed by using the software package MEGA version 3.1 (Kumar et al. 2004). Distances (corrected by Kimura’s two-parameter model; Kimura 1980) were calculated and clustering was performed with the neighbor-joining method (Saitou and Nei 1987). Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by means of 1000 resamplings (Felsenstein 2002).
2.5. Strain characteristics Strain characterization experiments performed using LB medium as the basal medium. The pH of the medium was adjusted with 5 M HCl or 10 M KOH to obtain a range between 3 and 11. Different amounts of NaCl were directly weighed in flasks prior to dispensing 20 ml medium to obtain the desired NaCl concentration between 0 and 300 g l1 (at 50 g l1). Different volumes of aniline was directly added into 20 ml medium to obtain the desired aniline concentration between 0 and 3 g l1 at 0.5 g l1. Different volumes of wastewater were directly added into 20 ml medium to obtain the desired wastewater concentration between 0% and 50% at 10%. Bacterial growth was measured as optical density (OD) at 540 nm using a spectrophotometer (UV-PC 2401). For all the degradation experiments, MSM medium was used as the basal medium. Different volumes of aniline was directly added into 20 ml medium to obtain the desired aniline concentration between 0.5 and 3.0 g l1 at 0.5 g l1. A sample from each aerobic test culture was removed and the bacteria were harvested by centrifugation at 10000 rpm for 10 min. The supernatant was transferred into a new tube and the degradation of aniline was determined as mentioned above.
2.6. Plasmid isolation and curing For the isolation of plasmid of bacteria strain, a modified alkaline lysis method was used (Thomas et al. 1988). Plasmid DNA was electrophoresised on an agarose gel (0.6%) and stained in ethidium bromide (EB) and photographed in Gel Doc XR imaging system (Bio-Rad Inc., USA). For curing the
ARTICLE IN PRESS Isolation and characterization of Erwinia sp. plasmid from bacteria strain, sodium benzoate (Xu et al. 1997; Li and Jin 2006b) was added at a final concentration of 130 g l1 to LB medium inoculated with 1% bacteria strain. After 24 h incubation at 30 1C, the culture was diluted and plated onto LB agar plates. The colonies appearing on the plates were replicated onto MSA plates with aniline to examine the aniline-degradation ability.
2.7. Cloning of aniline-degradation genes Primer are designed from Pseudomonas putida UCC22 plasmid pTDN1 genes for conversion of aniline to catechol (Genbank accession number: D85415.2) (Fukumori and Saint 1997) to clone aniline-degradation genes. The tdnQ gene was similar to various glutamine synthetase and was amplified with primers tdnQ1F (50 -TCCCTGGCTGGAG CCCGAAAC-30 ) and tdnQ1R (50 -TCCCGCGCCCTGAGTGACTG-30 ) and the expected size was 384 bp (Boon et al. 2001). The tpnA gene might encode a tranposase, which is not necessary for aniline conversation, and was amplified with primers tpnAF (50 -GCACCAAGTCTGGGAATGAT-30 ) and tpnAR (50 -TGTCAGAAGATGCCAAATCG-30 ) and the expected size was 730 bp. The tdnT gene was similar to amidotransferases and was amplified with primers tdnTF (50 -CGATTATTCAGGGCATGTCA30 ) and tdnTR (50 -GGTAGGCCATGAACTTCTGC-30 ) and the expected size was 454 bp. The tdnA1 gene could encode large subunits of a terminal dioxygenase and was amplified with primers tdnA1F (50 -CGTACAAGGGGTTCGTGTTT-30 ) and tdnA1R (50 -TCCACTGCGCGTAGT AGTTG-30 ) and the expected size was 828 bp. The tdnA2 gene could encode the small subunits of a terminal dioxygenase and was amplified with primers tdnA2F (50 -GGTCGCATCGTTCTACGACT-30 ) and tdnA2R (50 -TACGAGAGGTCGGGAACTG-30 ) and the expected size was 342 bp. PCR was performed in a reaction mixture containing (as final concentration): 1 Taq polymerase buffer [10 mmol l1 Tris–HCl (pH 9.0), 50 mmol l1 KCl, 0.1% Triton X-100], 2.0 mmol l1 MgCl2, 1 U Taq DNA polymerase (Jinsite Inc., Nanjing, China), 20 ng template DNA, 20 pmol forward primer and reverse primer, 2 mg ml1 bovine serum albumin (BSA), and 0.25 mmol l1 each of dATP, dCTP, dGTP or dTTP in the total 20 ml reaction volume. Amplification reaction was performed in a PTC 220 Thermal Cycler (Bio-Rad, Inc.). The touchdown cycle program included an initial 5 min denaturation at 94 1C, followed by 35 cycles of 30 s at 94 1C, 30 s at suitable temperature (the annealing temperature for genes tdnQ, tpnA, tdnT, tdnA1, and tdnA2 was 62.7, 61.0, 51.3, 56.4 and
421 62.7, respectively) and 30 s at 72 1C, and 5 min final extension at 72 1C.
3. Results 3.1. Characterization and identification of strain HSA6 16S rDNA sequence (accession No. in NCBI gene bank was GQ222272) showed that strain HSA6 was similar to Serratia sp., Yersinia sp. and Erwinia sp. Phylogenetic tree indicated that strain had the closest relationship with Serratia proteamaculans (Accession No. AB334771) (Figure 1). But the morphological and physiological properties at last identified that strain HSA6 is a species of Erwinia amylovora. Cells of strain HSA6 are gram negative, catalase positive, urease positive, oxidase negative, DNase negative, phenylalanine ammonia lyase negative, H2S negative, gelatin positive, indole negative and growth on arabinose, motile and aerobic rods.
3.2. The growth curve of strain HSA6 The growth curve of strain HSA6 at pH 7.0 was shown in Figure 2. The lag phase of strain HSA6 was very short, indicating that bacteria could adapt themselves to growth conditions quickly. The exponential phase was also very short and strain HSA6 spent little time to increase the number of cells. The stationary phase was long compared to other phases. The death phase begins after 48 h culture.
3.3. The effect of environmental factors on the growth of strain HSA6 Growth of strain HSA6 at a range of pH (3–13) was investigated and the results are shown in Figure 3a. The suitable pH for strain HSA6 was wide (from 5 to 11) and the optimal pH was 6. However, the mean generation time decreased significantly at pH 11. The OD540 was up to 2.391 at pH 11 after 40 h culture. Growth of strain HSA6 at a range of NaCl concentration (0–10%) was investigated and the results are shown in Figure 3b. The yield appeared to be largest at 1% NaCl (Figure 3b), then decreased with the increase in NaCl concentration. The strain was slightly halophilic (Kushner 1993), with growth occurring at 0–10% (v/v) NaCl, with the suitable NaCl concentration for strain HSA6 was from 0% to 6%. The OD540 was up to 2.046 at 6% NaCl after 24 h
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Figure 1. Neighbor-joining tree showing the position of strain HSA6 to a selected number of members of bacteria. Bootstrap values are indicated. Bar, 0.5% estimated sequence divergence.
Figure 2. The growth curve of E. amylovora strain HSA6.
culture. However, when the salinity increased from 5–9%, the mean generation time decreased significantly. For example, OD540 was up to 2.005 at 8% NaCl after 60 h culture. Growth of strain HSA6 at a range of aniline concentration (0–3 g l1) was investigated and the results are shown in Figure 3c. The number of bacteria appeared to decrease with an increase in aniline concentration. When the aniline
concentration increased from 2–3 g l1, the mean generation time decreased significantly. For example, OD540 was up to 1.339 at 3 g l1 aniline after 48 h culture. Growth of strain HSA6 at a range of wastewater concentration (0–50%) was investigated and the results are shown in Figure 3d. The number of bacteria appeared to be constant as the wastewater concentration increased from 0% to 20%.
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Figure 3. Effect of environmental factors on the growth of E. amylovora strain HSA6. (a) pH, (b) NaCl concentration, (c) aniline concentration and (d) wastewater concentration.
However, the number of cells decreased with the increase in wastewater concentration from 30% to 50% and grew very slowly at 50%. When the wastewater concentration increased from 30–40%, the mean generation time decreased significantly. For example, OD540 was up to 2.576 at 40% aniline after 48 h culture.
3.4. The aniline-degradation rate of strain HSA6 Strain HSA6 showed the ability of degrading aniline using aniline as sole carbon and nitrogen source. The effect of aniline concentration on the degradation rate of aniline in MSM medium was shown in Figure 4. The degradation rate of aniline was 100% at 0.5 g l1 aniline concentration after 24 h culture. The degradation rate of aniline was found to descend as the concentration of aniline increased from 0.5 to 3 g l1 and rise as the culture time increased. The degradation rate of aniline at
3 g l1 aniline concentration after 24, 48 and 72 h culture was 20.59%, 30.01% and 41.67%, respectively (Figure 4).
3.5. The genetic characteristics of strain HSA6 Plasmid extracted from strain HSA6 was shown in Figure 5a and the molecular weight of plasmid matched by Quantity One software (Bio-Rad Inc. USA) was higher than 42 kb. Plasmid was cured using sodium benzoate, and two strains without plasmid were isolated to determine the aniline degrading rate. Although the plasmid was cured, the strain could grow on MSM medium with aniline as the sole carbon and nitrogen source. But the aniline-degrading rate decreased significantly after the plasmid was cured. The degradation rate of aniline of two strains without plasmid at 3 g l1 aniline concentration after 72 h culture was 2.60%and 0.82%, respectively.
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Figure 4. Effect of aniline concentration on the aniline degradation rate of E. amylovora strain HSA6.
and tdnA1 with expected size could be detected from plasmid DNA of strain HSA6 but the amplification of tdnA2 gene with molecular weight 680 bp or so.
4. Discussion
Figure 5. Plasmid extracted from strain HSA6 and the PCR amplification results of genes degrading aniline in plasmid DNA. (a) Plasmid; Lane 1, plasmid; Lane M, lDNA/EcoR I molecular weight marker. (b) PCR amplification results of genes tdnQ and tpnA. Lane 1, amplification result with primer for gene tpnA; Lane 2, amplification result with primer for gene tdnQ; M: PCR marker. (c) PCR amplification results of genes tdnT and tdnA1. Lane 1, amplification result with primer for gene tdnA1; Lane 2, amplification result with primer for gene tdnT; M: PCR marker. (d) PCR amplification results of gene tdnA2. Lane 1, amplification result with primer for gene tdnA2; M: PCR marker.
The PCR amplification results of genes degrading aniline in plasmid were shown in Figure 5b–d. Amplification signals of four genes tdnQ, tpnA, tdnT
Since aniline is known to be toxic and carcinogenic to living organisms (Lyons et al. 1984), highly aniline-tolerant bacterial strains were isolated from various environments (Meyers 1992; Loidl et al. 1990; Konopka et al. 1989; Konopka 1993; Boon et al. 2001; Liu et al. 2002). E. amylovora strain HSA6 is similar to S. proteamaculans and S. plymuthica based on 16S rDNA sequence, but the DNase test is negative. The morphological and physiological properties indicated that strain HSA6 might be closely related to E. amylovora. E. amylovora is known as an important bacterial plant pathogen, which is the causative agent of fire blight (Geider 2006). As a member of the Enterobacteriaceae, it is related to Escherichia, Salmonella and Yersinia (http://www.sanger. ac.uk/Projects/E_amylovora/). Strain HSA6 is the first reported E. amylovora in pure culture growing with aniline as sole electron donor and carbon source. However, the possible pathway for degradation of aniline needs to be explored further. The treatment of aniline-containing wastewater under conditions of low pH and elevated salinity, which would dispense with the need for pretreatment would have obvious benefits. O’Neill et al. (2000) studied the degradation of aniline by
ARTICLE IN PRESS Isolation and characterization of Erwinia sp. bacterial consortia under aerobic, fermentative, nitrate-reducing and sulphate-reducing conditions and found that the rate of bacterial growth decreased with increasing salinity and the suitable salinity was 0.2% NaCl. E. amylovora strain HSA6 reported in the paper is slightly halophilic with growth occurring at 0–10% (v/v) NaCl, and the suitable NaCl concentration for strain HSA6 was from 0% to 6%. The degradation rate of aniline was 100% at 0.5 g l1 aniline concentration after 24 h culture. The degradation rate of aniline at 3 g l1 aniline concentration after 72 h culture was 41.67%. The salt-tolerant and aniline-degradation properties of strain HSA6 are encouraging and should be applicable to industrial effluents containing salt bioremediation. Catabolic plasmids play a certain role in aromatic amine degradation. Plasmid pTDN1 was discovered in a derivative of P. putida mt-2 (ATCC 33015) after its adaptation to growth on aniline (McClure and Venables 1987). Strain HSA6-containing plasmid and the plasmid curing test indicated that the plasmid is necessary in the degrading of aniline. The plasmid curing could significantly decrease the degradation rate of strain. Genes tdnQ, tpnA, tdnT, tdnA1 and tdnA2 could encode protein and enzyme related to the degrading of aniline in plasmid pTDN1 in P. putida UCC22 (Fukumori and Saint 1997). Gene tdnQ was similar to glutamine synthetases. Genes tdnA1 and tdnA2 could encode the large and small subunits of a terminal dioxygenase. TdnT might further transfer the amino group to an unknown substance or release ammonia. Gene tpnA encode transpose. It was determined that the gene cluster degrading aniline was carried in transposable element (Liang et al. 2005b). In strain HAS6, genes tdnQ, tpnA, tdnT, tdnA1 and tdnA2 with expected size could be detected in plasmid DNA indicating that the aniline-degrading gene might locate on plasmid. But the amplification of tdnA2 with molecular weight 680 bp or so, which was two fold larger than tdnA2 gene in P. putida UCC22 plasmid pTDN1, indicating that there might be some difference between the two plasmids. Transpose gene tpnA was amplified using PCR and this indicated that the gene encoded for aniline degradation might be located on transposable element.
Acknowledgements This research was financially supported by the project of Zhejiang Province Science and Technology Bureau, No. 2007C23052.
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www.elsevier.de/micres
Isolation and characterization of aniline degradation slightly halophilic bacterium, Erwinia sp. Strain HSA 6 Junmin Li, Zexin Jin, Binbin Yu Institute of Ecology, Taizhou University, Linhai 317000, China Received 4 June 2009; received in revised form 9 September 2009; accepted 12 September 2009
KEYWORDS Erwinia amylovora; Aniline degradation; Salinity tolerant; Characteristics; Plasmid
Summary The isolated strain HSA6 is classified as Erwinia amylovora based on 16S rDNA sequence and the morphological and physiological properties. Strain HSA6 is the first reported E. amylovora in pure culture growing with aniline as sole electron donor and carbon source. The suitable pH for strain HSA6 is wide (from 5 to 11). Strain HSA6 is slightly halophilic with growth occurring at 0–10% (v/v) NaCl, and the suitable NaCl concentration for strain HSA6 is from 0% to 6%. The number of bacteria appeared to decrease with an increase in aniline concentration. The number of bacteria appeared to be constant as the wastewater concentration increased from 0% to 20%. However, the number of cells decreased with an increase in wastewater concentration from 30% to 50% and grew very slowly at 50%. The degradation rate of aniline was 100% at 0.5% aniline concentration after 24 h culture. The degradation rate of aniline was found to descend as the concentration of aniline increased from 0.5% to 3% and rose as the culture time increased. Strain HSA6 contains a plasmid with molecular weight higher than 42 kDA. Plasmid curing test and quantitative degradation test showed that strain requires the plasmid for aniline degradation. The gene cluster degrading aniline was determined in the plasmid by PCR amplification. & 2009 Elsevier GmbH. All rights reserved.
1. Introduction Corresponding author. Tel.: +86 57685137013;
fax: +86 57685137067. E-mail address: [email protected] (J. Li)
Aniline has toxicity to life and plant and is considered as an important environmental hazard and is subject to legislative control by the European Economic Community (EEC) directive and in the
0944-5013/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2009.09.003
ARTICLE IN PRESS Isolation and characterization of Erwinia sp. Priority Pollutant List of US Environmental Protection Agency (Federal Register 1979). While not as easily biodegraded as phenol and benzoate (Alexander and Lustigman 1966), aniline is none the less biodegradable and its biodegradation has been reported by several authors (Anson and Mackinnon 1984; Aoki et al. 1990; Lyons et al. 1984). Numbers of reports concerning the bacterial degradation of aniline had already been published, such as Delfria sp. (Liu et al. 2002; Zhang et al. 2008; Liang et al. 2005a), Pseudomonas sp. (Meyers 1992; Fukumori and Saint 1997; Bathe 2004), Acinetobacter sp. (Takeo et al. 1998; Fujii et al. 1997). O’Neill et al. (2000) isolated a consortium of bacteria capable of degrading aniline found in wastewaters produced by oil fields, in marine mud and in acid peat bog water and soils. Li et al. (2007) isolated strain PN1001, which is a member of the Pseudomonas species and it was capable of degrading pentyl amine and aniline. The effluents in pharmaceutical industries in which aniline occurs are frequently of elevated salinity (O’Neill et al. 2000), which could strongly inhibit the growth of the microorganisms (Kubo et al. 2001). The high salt content in the wastewaters can pose certain problems in treatment systems particularly on biological units. Salt-tolerant microorganisms, such as Staphylococcus sp. and Bacillus sp. were isolated to treat a high-salt-content wastewater (Kubo et al. 2001). At the pharmaceutical industry of interest to this study, wastewater is produced containing high salinity, aniline, chloride and chemical oxygen demand (COD). With the aim of obtaining microorganisms able to purify the wastewater, we attempted to isolate salt-tolerant microorganisms, which could degrade aniline and erase COD under salinity and chloride conditions. In addition, the effects of various culture medium parameters on the growth of bacteria, the removal of aniline and COD and the genetic characteristics of bacteria strain were explored.
419 0.316, and NaCl 0.25, pH 7.0. After autoclaving MSM, high salinity aniline-containing wastewater was added to obtain the desired final concentration as the sole carbon source. Luria Bertani (LB) medium contained (g l1): tryptone 10.0, NaCl 10.0, and yeast extracts 5.0.
2.2. Sources of wastewater and activated sludge Hypersalinity aniline-containing wastewater was collected from a pharmaceutical chemicals and intermediates industry. pH, salinity and chloride of the wastewater were analyzed according to the standard methods for the analysis of wastewater (State Environmental Protection Administration 2002). COD analyzed in a high dose of HgSO4 (at the ratio of HgSO4:Cl=10:1) was added to the samples to eradicate the chloride interference. The aniline compounds existed in the wastewater was determined by N-(1-naphthyl) ethylene diamine dihydrochloride spectrophotometric method (GB/T 15502-1995, http://english.mep.gov.cn/standards _reports/standards/Air_Environment/air_method/ 200809/t20080923_129249.htm). The wastewater was found to have pH lower than 1, with COD 19 400 mg l1, chloride13.69 g l1, aniline 2480 mg l1 and salinity 39.900 g l1. Subsequently, wastewater neutralized with NaOH was used for the isolation of microorganisms. Activated sludge was used as a starting material for the isolation of pure cultures. The activated sludge was collected from effluents treatment plants treating wastewater generated by pharmaceutical formulation industry. A liter of sludge sample was collected from the aerobic biological reactor. The supernatant was discarded and the biomass was pooled, mixed thoroughly and transported to the laboratory immediately.
2.3. Isolation of bacteria
2. Materials and methods 2.1. Chemicals and media Aniline (C6H7N, 93.13MW) of high purity more than 99.5% was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All of other chemicals used in this study were of the highest analytical grad available. The mineral salt medium (MSM) contained (g l1): NH4NO3 1.0, (NH4)2SO4 0.25, kH2PO4 0.25, MgSO4
Aerobic cultures (20 ml culture media) were grown in 50 ml conical flasks with cotton wool bungs. Enrichment cultures were set up with LB medium received a 10% v/v activated sludge. After 48 h culture with shaking (150 rpm) at 30 1C, acclimation were set up using hypersaline anilinecontaining wastewater as the sole carbon and nitrogen source in MSM medium at a concentration from 5% to 40% at 5%. After 14 d culture with shaking (150 rpm) at 30 1C, subsequent subcultures received 10% inoculation from the preceding cultures.
ARTICLE IN PRESS 420 To isolate pure cultures, 100 ml cultures were plated onto LB agar plates containing 5% (w/v) hypersaline wastewater for single-colony isolation. Subsequently, colonies were isolated at random and sub-cultured twice. The single pure colony was picked and inoculated into LB medium containing 5% (w/v) hypersaline wastewater and the glycerol stocks of the selected isolates were prepared and preserved at 70 1C.
2.4. Strain identification The strains were identified by morphological and biochemical tests and its 16S ribosomal DNA (16S rDNA) sequence. Total genomic DNA extraction was performed according to the cetyltrimethyl ammonium bromide (CTAB)-lysozyme-proteinase K-freezing thaw lysing method reported earlier (Li and Jin 2006a). After overnight culture with shaking (150 rpm) at 30 1C, the pellet was collected by centrifugation at 10000 rpm for 10 min. The pellet was resuspended in CTAB-lysozyme buffer [100 mmol l1 Tris–HCl (pH 8.0), 100 mmol l1 EDTA 2Na (pH 8.0), 100 mmol l1 phosphate sodium buffer (pH 8.0), 1.5 mmol l1 NaCl, 1% CTAB, 2% CaCl2, 1 mg ml1 bovine serum albumin (BSA), 1 mg ml1 lysozyme] and incubated at 37 1C for 1 h, and then incubated at 65 1C after adding 100 mg ml1 protease K. DNA was purified using a chloroform/ isoamyl alcohol (1/1) solution, and precipitated by the addition of 50% (v/v) 25% PEG 8000 (w/v) and stored at 4 1C overnight. The DNA was determined and was diluted into 20 ng ml1 and stored at 20 1C. A partial gene sequencing of 16S rDNA was performed to identify the strains. The 1466 bp 16S rDNA was amplified with conserved primer (forward prime 27f, 50 -AGAGTTTGATYMTGGCTC AG-30 reversed primer 1492r, 50 -TACGGHTACCTTACG ACT-30 ) as reported earlier (Selvakumaran et al. 2008). The reaction mixture was prepared to a total volume of 20 ml containing 1 PCR buffer, 2.0 mmol l1 MgCl2, 10 ng template DNA, 0.2 mmol l1 4 dNTP, 1 mmol l1 forward primer and reversed primer, 1 U Taq polymerase (Shanghai Sangon Co. Ltd.). The reaction mixture was incubated in a thermal cycler (PTC 220, Bio-Rad Inc., USA) at 94 1C for 5 min for initial denaturation, followed by 35 cycles of 1 min at 94 1C, 1 min at 55 1C and 1.5 min at 72 1C, and a final extension at 72 1C for 5 min. PCR products were purified using UNIQ-10 EZ Spin Column PCR Production Purification Kit (Shanghai Sangon Co. Ltd.) and were sent to Jinsite Biotechnology Co., Ltd. (Nanjing, China) for sequencing. The sequence of 1466 bp of 16S rDNA was submitted to the NCBI
J. Li et al. (National Center for Biotechnology Information) and GenBank for obtaining accession numbers (GQ222272). The sequence was compared with 16S rRNA gene sequences available in the GenBank database by BLASTn search (http://www. ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignments of partial 16S rRNA gene sequences were aligned using CLUSTAL X, version 1.8. Phylogenetic analysis was performed by using the software package MEGA version 3.1 (Kumar et al. 2004). Distances (corrected by Kimura’s two-parameter model; Kimura 1980) were calculated and clustering was performed with the neighbor-joining method (Saitou and Nei 1987). Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by means of 1000 resamplings (Felsenstein 2002).
2.5. Strain characteristics Strain characterization experiments performed using LB medium as the basal medium. The pH of the medium was adjusted with 5 M HCl or 10 M KOH to obtain a range between 3 and 11. Different amounts of NaCl were directly weighed in flasks prior to dispensing 20 ml medium to obtain the desired NaCl concentration between 0 and 300 g l1 (at 50 g l1). Different volumes of aniline was directly added into 20 ml medium to obtain the desired aniline concentration between 0 and 3 g l1 at 0.5 g l1. Different volumes of wastewater were directly added into 20 ml medium to obtain the desired wastewater concentration between 0% and 50% at 10%. Bacterial growth was measured as optical density (OD) at 540 nm using a spectrophotometer (UV-PC 2401). For all the degradation experiments, MSM medium was used as the basal medium. Different volumes of aniline was directly added into 20 ml medium to obtain the desired aniline concentration between 0.5 and 3.0 g l1 at 0.5 g l1. A sample from each aerobic test culture was removed and the bacteria were harvested by centrifugation at 10000 rpm for 10 min. The supernatant was transferred into a new tube and the degradation of aniline was determined as mentioned above.
2.6. Plasmid isolation and curing For the isolation of plasmid of bacteria strain, a modified alkaline lysis method was used (Thomas et al. 1988). Plasmid DNA was electrophoresised on an agarose gel (0.6%) and stained in ethidium bromide (EB) and photographed in Gel Doc XR imaging system (Bio-Rad Inc., USA). For curing the
ARTICLE IN PRESS Isolation and characterization of Erwinia sp. plasmid from bacteria strain, sodium benzoate (Xu et al. 1997; Li and Jin 2006b) was added at a final concentration of 130 g l1 to LB medium inoculated with 1% bacteria strain. After 24 h incubation at 30 1C, the culture was diluted and plated onto LB agar plates. The colonies appearing on the plates were replicated onto MSA plates with aniline to examine the aniline-degradation ability.
2.7. Cloning of aniline-degradation genes Primer are designed from Pseudomonas putida UCC22 plasmid pTDN1 genes for conversion of aniline to catechol (Genbank accession number: D85415.2) (Fukumori and Saint 1997) to clone aniline-degradation genes. The tdnQ gene was similar to various glutamine synthetase and was amplified with primers tdnQ1F (50 -TCCCTGGCTGGAG CCCGAAAC-30 ) and tdnQ1R (50 -TCCCGCGCCCTGAGTGACTG-30 ) and the expected size was 384 bp (Boon et al. 2001). The tpnA gene might encode a tranposase, which is not necessary for aniline conversation, and was amplified with primers tpnAF (50 -GCACCAAGTCTGGGAATGAT-30 ) and tpnAR (50 -TGTCAGAAGATGCCAAATCG-30 ) and the expected size was 730 bp. The tdnT gene was similar to amidotransferases and was amplified with primers tdnTF (50 -CGATTATTCAGGGCATGTCA30 ) and tdnTR (50 -GGTAGGCCATGAACTTCTGC-30 ) and the expected size was 454 bp. The tdnA1 gene could encode large subunits of a terminal dioxygenase and was amplified with primers tdnA1F (50 -CGTACAAGGGGTTCGTGTTT-30 ) and tdnA1R (50 -TCCACTGCGCGTAGT AGTTG-30 ) and the expected size was 828 bp. The tdnA2 gene could encode the small subunits of a terminal dioxygenase and was amplified with primers tdnA2F (50 -GGTCGCATCGTTCTACGACT-30 ) and tdnA2R (50 -TACGAGAGGTCGGGAACTG-30 ) and the expected size was 342 bp. PCR was performed in a reaction mixture containing (as final concentration): 1 Taq polymerase buffer [10 mmol l1 Tris–HCl (pH 9.0), 50 mmol l1 KCl, 0.1% Triton X-100], 2.0 mmol l1 MgCl2, 1 U Taq DNA polymerase (Jinsite Inc., Nanjing, China), 20 ng template DNA, 20 pmol forward primer and reverse primer, 2 mg ml1 bovine serum albumin (BSA), and 0.25 mmol l1 each of dATP, dCTP, dGTP or dTTP in the total 20 ml reaction volume. Amplification reaction was performed in a PTC 220 Thermal Cycler (Bio-Rad, Inc.). The touchdown cycle program included an initial 5 min denaturation at 94 1C, followed by 35 cycles of 30 s at 94 1C, 30 s at suitable temperature (the annealing temperature for genes tdnQ, tpnA, tdnT, tdnA1, and tdnA2 was 62.7, 61.0, 51.3, 56.4 and
421 62.7, respectively) and 30 s at 72 1C, and 5 min final extension at 72 1C.
3. Results 3.1. Characterization and identification of strain HSA6 16S rDNA sequence (accession No. in NCBI gene bank was GQ222272) showed that strain HSA6 was similar to Serratia sp., Yersinia sp. and Erwinia sp. Phylogenetic tree indicated that strain had the closest relationship with Serratia proteamaculans (Accession No. AB334771) (Figure 1). But the morphological and physiological properties at last identified that strain HSA6 is a species of Erwinia amylovora. Cells of strain HSA6 are gram negative, catalase positive, urease positive, oxidase negative, DNase negative, phenylalanine ammonia lyase negative, H2S negative, gelatin positive, indole negative and growth on arabinose, motile and aerobic rods.
3.2. The growth curve of strain HSA6 The growth curve of strain HSA6 at pH 7.0 was shown in Figure 2. The lag phase of strain HSA6 was very short, indicating that bacteria could adapt themselves to growth conditions quickly. The exponential phase was also very short and strain HSA6 spent little time to increase the number of cells. The stationary phase was long compared to other phases. The death phase begins after 48 h culture.
3.3. The effect of environmental factors on the growth of strain HSA6 Growth of strain HSA6 at a range of pH (3–13) was investigated and the results are shown in Figure 3a. The suitable pH for strain HSA6 was wide (from 5 to 11) and the optimal pH was 6. However, the mean generation time decreased significantly at pH 11. The OD540 was up to 2.391 at pH 11 after 40 h culture. Growth of strain HSA6 at a range of NaCl concentration (0–10%) was investigated and the results are shown in Figure 3b. The yield appeared to be largest at 1% NaCl (Figure 3b), then decreased with the increase in NaCl concentration. The strain was slightly halophilic (Kushner 1993), with growth occurring at 0–10% (v/v) NaCl, with the suitable NaCl concentration for strain HSA6 was from 0% to 6%. The OD540 was up to 2.046 at 6% NaCl after 24 h
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Figure 1. Neighbor-joining tree showing the position of strain HSA6 to a selected number of members of bacteria. Bootstrap values are indicated. Bar, 0.5% estimated sequence divergence.
Figure 2. The growth curve of E. amylovora strain HSA6.
culture. However, when the salinity increased from 5–9%, the mean generation time decreased significantly. For example, OD540 was up to 2.005 at 8% NaCl after 60 h culture. Growth of strain HSA6 at a range of aniline concentration (0–3 g l1) was investigated and the results are shown in Figure 3c. The number of bacteria appeared to decrease with an increase in aniline concentration. When the aniline
concentration increased from 2–3 g l1, the mean generation time decreased significantly. For example, OD540 was up to 1.339 at 3 g l1 aniline after 48 h culture. Growth of strain HSA6 at a range of wastewater concentration (0–50%) was investigated and the results are shown in Figure 3d. The number of bacteria appeared to be constant as the wastewater concentration increased from 0% to 20%.
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Figure 3. Effect of environmental factors on the growth of E. amylovora strain HSA6. (a) pH, (b) NaCl concentration, (c) aniline concentration and (d) wastewater concentration.
However, the number of cells decreased with the increase in wastewater concentration from 30% to 50% and grew very slowly at 50%. When the wastewater concentration increased from 30–40%, the mean generation time decreased significantly. For example, OD540 was up to 2.576 at 40% aniline after 48 h culture.
3.4. The aniline-degradation rate of strain HSA6 Strain HSA6 showed the ability of degrading aniline using aniline as sole carbon and nitrogen source. The effect of aniline concentration on the degradation rate of aniline in MSM medium was shown in Figure 4. The degradation rate of aniline was 100% at 0.5 g l1 aniline concentration after 24 h culture. The degradation rate of aniline was found to descend as the concentration of aniline increased from 0.5 to 3 g l1 and rise as the culture time increased. The degradation rate of aniline at
3 g l1 aniline concentration after 24, 48 and 72 h culture was 20.59%, 30.01% and 41.67%, respectively (Figure 4).
3.5. The genetic characteristics of strain HSA6 Plasmid extracted from strain HSA6 was shown in Figure 5a and the molecular weight of plasmid matched by Quantity One software (Bio-Rad Inc. USA) was higher than 42 kb. Plasmid was cured using sodium benzoate, and two strains without plasmid were isolated to determine the aniline degrading rate. Although the plasmid was cured, the strain could grow on MSM medium with aniline as the sole carbon and nitrogen source. But the aniline-degrading rate decreased significantly after the plasmid was cured. The degradation rate of aniline of two strains without plasmid at 3 g l1 aniline concentration after 72 h culture was 2.60%and 0.82%, respectively.
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Figure 4. Effect of aniline concentration on the aniline degradation rate of E. amylovora strain HSA6.
and tdnA1 with expected size could be detected from plasmid DNA of strain HSA6 but the amplification of tdnA2 gene with molecular weight 680 bp or so.
4. Discussion
Figure 5. Plasmid extracted from strain HSA6 and the PCR amplification results of genes degrading aniline in plasmid DNA. (a) Plasmid; Lane 1, plasmid; Lane M, lDNA/EcoR I molecular weight marker. (b) PCR amplification results of genes tdnQ and tpnA. Lane 1, amplification result with primer for gene tpnA; Lane 2, amplification result with primer for gene tdnQ; M: PCR marker. (c) PCR amplification results of genes tdnT and tdnA1. Lane 1, amplification result with primer for gene tdnA1; Lane 2, amplification result with primer for gene tdnT; M: PCR marker. (d) PCR amplification results of gene tdnA2. Lane 1, amplification result with primer for gene tdnA2; M: PCR marker.
The PCR amplification results of genes degrading aniline in plasmid were shown in Figure 5b–d. Amplification signals of four genes tdnQ, tpnA, tdnT
Since aniline is known to be toxic and carcinogenic to living organisms (Lyons et al. 1984), highly aniline-tolerant bacterial strains were isolated from various environments (Meyers 1992; Loidl et al. 1990; Konopka et al. 1989; Konopka 1993; Boon et al. 2001; Liu et al. 2002). E. amylovora strain HSA6 is similar to S. proteamaculans and S. plymuthica based on 16S rDNA sequence, but the DNase test is negative. The morphological and physiological properties indicated that strain HSA6 might be closely related to E. amylovora. E. amylovora is known as an important bacterial plant pathogen, which is the causative agent of fire blight (Geider 2006). As a member of the Enterobacteriaceae, it is related to Escherichia, Salmonella and Yersinia (http://www.sanger. ac.uk/Projects/E_amylovora/). Strain HSA6 is the first reported E. amylovora in pure culture growing with aniline as sole electron donor and carbon source. However, the possible pathway for degradation of aniline needs to be explored further. The treatment of aniline-containing wastewater under conditions of low pH and elevated salinity, which would dispense with the need for pretreatment would have obvious benefits. O’Neill et al. (2000) studied the degradation of aniline by
ARTICLE IN PRESS Isolation and characterization of Erwinia sp. bacterial consortia under aerobic, fermentative, nitrate-reducing and sulphate-reducing conditions and found that the rate of bacterial growth decreased with increasing salinity and the suitable salinity was 0.2% NaCl. E. amylovora strain HSA6 reported in the paper is slightly halophilic with growth occurring at 0–10% (v/v) NaCl, and the suitable NaCl concentration for strain HSA6 was from 0% to 6%. The degradation rate of aniline was 100% at 0.5 g l1 aniline concentration after 24 h culture. The degradation rate of aniline at 3 g l1 aniline concentration after 72 h culture was 41.67%. The salt-tolerant and aniline-degradation properties of strain HSA6 are encouraging and should be applicable to industrial effluents containing salt bioremediation. Catabolic plasmids play a certain role in aromatic amine degradation. Plasmid pTDN1 was discovered in a derivative of P. putida mt-2 (ATCC 33015) after its adaptation to growth on aniline (McClure and Venables 1987). Strain HSA6-containing plasmid and the plasmid curing test indicated that the plasmid is necessary in the degrading of aniline. The plasmid curing could significantly decrease the degradation rate of strain. Genes tdnQ, tpnA, tdnT, tdnA1 and tdnA2 could encode protein and enzyme related to the degrading of aniline in plasmid pTDN1 in P. putida UCC22 (Fukumori and Saint 1997). Gene tdnQ was similar to glutamine synthetases. Genes tdnA1 and tdnA2 could encode the large and small subunits of a terminal dioxygenase. TdnT might further transfer the amino group to an unknown substance or release ammonia. Gene tpnA encode transpose. It was determined that the gene cluster degrading aniline was carried in transposable element (Liang et al. 2005b). In strain HAS6, genes tdnQ, tpnA, tdnT, tdnA1 and tdnA2 with expected size could be detected in plasmid DNA indicating that the aniline-degrading gene might locate on plasmid. But the amplification of tdnA2 with molecular weight 680 bp or so, which was two fold larger than tdnA2 gene in P. putida UCC22 plasmid pTDN1, indicating that there might be some difference between the two plasmids. Transpose gene tpnA was amplified using PCR and this indicated that the gene encoded for aniline degradation might be located on transposable element.
Acknowledgements This research was financially supported by the project of Zhejiang Province Science and Technology Bureau, No. 2007C23052.
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