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Isolation and Characterization of a Novel Bacterial Strain Rhodococcus sp. WUST-py for Pyridine Biodegradation with Special Application to Relatively Strong Alkaline Conditions

Jianzhong Liu1*; Yun Zhai1; Peng Tang1; Honglei Yi1; Xiaorong Qin1; Hao Huang1; Wu Xu2; Andrei Chistoserdov3; Rakesh K. Bajpai4; Ramalingam Subramaniam4*
1 School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan 430081, China
2 Department of Chemistry, University of Louisiana at Lafayette 70504, USA
3 Department of Biology, University of Louisiana at Lafayette 70504, USA
4 Department of Chemical Engineering, University of Louisiana at Lafayette 70504, USA

This paper reports successful isolation and characterization of a novel pyridine-degrading bacterial strain WUST-py from activated sludge out of an aeration tank of a coke plant in Wuhan, China. The experiments demonstrated that WUST-py is an aerobic, acidogenic, Gram-positive, sporulating, and typically rod-shaped bacterium. Partial 16S rDNA sequence alignment indicated that WUST-py belongs to the genus of Rhodococcus, with 99.0 % identity to the 16S rRNA gene sequence of a bacterial strain Rhodococcus sp. D-50. It mineralized up to 1800 mg L-1 pyridine within 120 h when the initial pH of the mineral salt medium (MSM) was 10.0, and 1600 mg L-1 pyridine within 108 h when the initial pH of MSM was 11.0, implying that this bacterial strain is ideally suited for pyridine biodegradation under strongly alkaline conditions. WUST-py can also consume phenol, alcohols, ether, and formic acid as primary carbon and energy sources. Preliminary experiments revealed that WUST-py could degrade 1000 mg L-1 phenol to undetectable levels at initial pH values between 8.0 and 11.0 within 44 h, implying the potential of co-biodegradation of pyridine and phenol. Antibiotic resistance results indicated that the strain WUST-py is highly sensitive to streptomycin, tetramycin, kanamycin, ampicillin, and chloromycetin.

Key words: WUST-py; Pyridine; Phenol; Biodegradation; Rhodococcus sp.; Bioremediation.

*Corresponding authors:

Jianzhong Liu, School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan 430081, China. Email: [email protected]

Ramalingam Subramaniam, Department of Chemical Engineering, University of Louisiana at Lafayette 70504, USA. Email: [email protected];[email protected]

Pyridine and its derivatives (including alkylpyridine compounds, pyridine chlorides, methylpyridine chlorides, pyridine fluorides, pyridine bromides, aminopyridines, cyanopyridine, pyridine amides, pyridine carboxylates, etc.?are either widely used as raw materials or produced by a variety of modern industries (pharmaceuticals, herbicides, pesticides, germicides, antiseptics, plant growth regulators, feed additives, spices, oil refineries, coke plants, rubber manufacturing, surfactants, catalysts, film sensitizers, explosives, dyes, etc.) (Huang et al., 2017; Scriven and Murugan, 2005). Millions of tons of wastewaters containing pyridine and/or pyridine derivatives are generated every day from these sources and pose serious environmental hazard as well as risks to living organisms. The typical pyridine concentration in real wastewaters is normally from 20-300 mg L-1. However, this level can be as high as 600-1000 mg L-1 during emergency spills (Huang et al., 2017; Zalat and Elsayed, 2013). These compounds possess high solubility, stability, and mobility through water and soil. Prolonged exposure, ingestion, and inhalation of these chemicals cause serious human discomfort and diseases symptomized by depression, gastrointestinal upset, liver and kidney damage, headache, nervousness, dizziness, insomnia, nausea, anorexia, frequent urination, and dermatitis (Mathur et al., 2008; Zalat and Elsayed, 2013). Furthermore, they are potentially carcinogenic (Uma and Sandhya, 1997).
Physicochemical methods, such as adsorption (Dhawan et al., 2015; Li et al., 1988) and oxidation (Singh and Lo, 2017) are available for treatment of these hazardous chemicals in aqueous systems, but they often suffer from drawbacks such as high initial investment and operational costs, and formation of secondary toxic byproducts (Huang et al., 2017; Jiang et al., 2018; Mudliar et al., 2008; Padoley et al., 2006). On the other hand, biodegradation is more environmentally friendly, cost effective, and can overcome the various drawbacks of physicochemical methods (Jiang et al., 2018). Several microorganisms are capable of degrading and utilizing pyridine and / or its derivatives as sole carbon, nitrogen, and energy sources; these include genera Rhizobium (Shen et al., 2015), Shinella (Bai et al., 2009), Streptomyces (Li et al., 2009), Paracoccus (Bai et al., 2008), Rhodococcus (Sun et al., 2011), Pseudomonas (Padoley et al., 2006), Bacillus (Chandra et al., 2009; Mathur et al., 2008; Uma and Sandhya, 1997), Shewanella (Mathur et al., 2008), Gordonia (Stobdan et al., 2008), Alcaligenes (Chandra et al., 2009). Several bacterial strains can biodegrade up to 1000 mg L-1 pyridine in water 7, 13-15. Recently, multiple research efforts have been undertaken that specifically aim at isolation and introduction of potent and robust pyridine-degraders to biological treatment systems (Liang et al., 2018). Stobdan et al. (Stobdan et al., 2008) have reported a microbial consortium of Gordonia, that degrades 70 mM (5,537 mg L-1) pyridine within 192 h. This, to our knowledge, is the highest concentration of pyridine degraded by a microbial consortium in open literature. Other reports included a microbial isolate of Paracoccus 10 capable of degrading 2614 mg L-1 pyridine within 49.5 h from an initial pH of 8.0, and a bacterial strain of Rhizobium NJUST18 11 degrading 2600 mg L-1 pyridine within 240 h at the initial pH of 7.0. Further research (Liu et al., 2015) showed that aerobic granules of Rhizobium NJUST18, developed in a sequencing batch reactor, can biodegrade up to 4000 mg L-1 pyridine within 7.5 h.
Nevertheless, there are still several issues awaiting to be addressed, in addition to enhancing the mineralizing capacities of microbes. For example, Liang et al. (2018) reported that biodegradation was inhibited when the initial pyridine concentration was greater than 300 mg L-1, because of the activity loss at a high pyridine loading rate by employing the species, Paracoccus denitrificans W12. Moreover, optimum pH values of the reported strains are predominantly neutral or slightly basic (pH 7~8), and some bacterial strains show adverse properties such as strong self-flocculation / aggregation in the culture media. The pH of wastewaters varies from source to source and we found that the pH fluctuates from 5 to 10 depending on source. Hence, isolation of pyridine degraders capable of handling a wider pH range to deal with some suddenly occurring circumstances of extreme pH changes, higher degrading efficiency, and in situ bioremediation of the contaminated sites after proliferation is necessary.
In this paper, we report the successful enrichment, isolation, and taxonomic identification of a pyridine-degrading bacterial strain from activated sludge out of an aeration tank of a coke plant in Wuhan, China. The homology of its 16S rRNA gene to 16S rRNA genes of its closest relatives in GenBank, suggests that it may be applicable to pyridine biodegradation under relatively strong alkaline conditions (8.0 to 11.0). Pyridine degradation rates, different carbon sources on which the strain could grow, and pH changes during pyridine degradation by the strain were also investigated. The strain’s resistance to streptomycin, tetramycin, kanamycin, ampicillin, and chloromycetin was evaluated, and growth of the microbe was monitored at different initial pyridine concentrations and at different initial pH values in batch reactors.
2.1 Chemicals and reagents:
All the chemicals and reagents used in bacterial cultivation and assay in this study were of analytical grade and all were purchased from dealers in Wuhan, China.
2.2 Media:
Luria broth (LB), mineral salt medium (MSM), and agar plate / slant media were used in this study. The LB consisted of 10.0 g L-1 tryptone, 5.0 g L-1 yeast extract, and 10.0 g L-1 NaCl. The MSM contained 0.5 g L-1 K2HPO4, 0.5 g L-1 KH2PO4, 0.1 g L-1 NaCl, 0.2 g L-1 MgSO4.7H2O, 0.5 g L-1 NH4Cl, 0.02 g L-1 CaCl2, and 2.0 mL L-1 of trace elements solution. The trace elements solution contained 0.1 g L-1 MnSO4.4H2O, 0.07 g L-1 ZnCl2, 0.035 g L-1 NaMoO4.2H2O, 0.06 g L-1 H3BO3, 0.2 g L-1 CoCl.6H2O, 0.029 g L-1 CuSO4.5H2O, 0.025 g L-1 NiCl2.6H2O, and 0.9 mL L-1 of 37 % HCl. The agar plate / slant media were obtained by adding 15.0 g L-1 agar in LB or MSM. pH of the LB was adjusted to 7.0-7.2 with NaOH, and pH of the MSM was adjusted from 5.0 to 11.0 with HCl or NaOH depending on experimental requirements. Media were sterilized by autoclaving at 121°C and 14.7 psi for 15 min. Pyridine was prepared as a stock solution (50 g L-1) and filter sterilized. Its concentration in media varied over the course of experiments.
2.3 Bacterial Enrichment, Acclimatization, and Isolation:
The activated sludge was collected from an aeration tank of a coke plant in Wuhan, China. 5 mL of the homogenized sludge sample were added to 95 mL of sterile LB medium in a 250 mL at 35°C and 150 rpm for bacteria enrichment. When an obvious OD increase was observed (OD ? 1.0 at around 96 h), 2 mL of culture broth were transferred to a second shake flask containing 98 mL of MSM and enough pyridine so that a final pyridine concentration of 100 mg L-1 was established in the medium. Pyridine consumption in the broth was monitored during the incubation until pyridine concentration was reduced by 95 % or more. Similar procedure was followed with different pyridine concentrations of 300 mg L-1, 500 mg L-1, and 600 mg L-1. The final broth from the 600 mg L-1 flask was serially diluted with phosphate-buffer, spread onto MSM agar plate containing 500 mg L-1 pyridine, and incubated at 35°C. The phosphate-buffer (pH 7.2) was obtained by mixing 28 mL of 0.2 mol L-1 NaH2PO4 solution and 72 mL of 0.2 mol L-1 Na2HPO4 solution. The visually different colonies were streaked for purity, and isolates capable of utilizing pyridine as a sole carbon and energy source were preserved at 4 °C on agar slants and at -80 °C in the LB medium supplemented with 20 % sterilized glycerol.

2.4 Identification of the Bacterial Strains:
Cell morphology was investigated with a light microscope (SA3000, Beijing, China) after Gram and spore staining, and with a scanning electron microscope (Nova 400 NanoSEM, FEI, America). Gram and spore staining were done using standard protocols (Shen et al., 1999).
For taxonomic identification, the bacterial strain (WUST-py) was provided to Wuhan Qing Branch Innovation Biotechnology Co. Ltd. for partial nucleotide sequences of its 16S rRNA gene. The universal primers used to amplify the sequences were as follows: 27F (5?-AGAGTTTGATCCTGGCTCAG-3?) and 1492R (5?-TACGGCTACCTTGTTACGACTT -3?). A blast search of 16S rRNA gene nucleotide sequences was performed against the National Center for Biotechnology Information (NCBI) sequence database. The sequences were analyzed using Molecular Evolutionary Genetic Analysis (MEGA v.5) (Tamura et al., 2011). The CLUSTALW algorithm was applied to carry out pairwise and multiple sequence alignments, and a phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei, 1987).
2.5 Preparation of Inocula:
Seed cultures of the selected bacterial strain (WUST-py) were grown in 200 mL of MSM containing 1000 mg L-1 pyridine in 500 mL conical flasks at 35 °C and 150 rpm until the late exponential phase. Cells were recovered by centrifugation at 8000 x g for 5 min, washed twice with pyridine-free MSM and re-suspended in the pyridine-free MSM to the optical density of 1.0 at 600 nm (OD600) for use as inocula for degradation and growth substrate studies, as well as for antibiotic resistance experiments.

2.6 Degradation Experiments at Different Initial Pyridine Concentrations and at Different Initial pH values:
The pyridine biodegradation experiments were conducted in 250 mL sterile Erlenmeyer flasks. 5 mL of inocula, prepared as described in section 2.5 above, were transferred aseptically to each flask containing 95 mL MSM supplemented with varying amounts of 50 g L-1 filter-sterilized pyridine stock solution (final pyridine concentrations 100-2,500 mg L-1) at different initial pH values (5.0-11.0). Specific pH values of the MSM media were obtained by adding with acid (1 M HCl) or alkali (4 M NaOH) solutions. Three mL samples of broth were collected every 12 h for measurements of pH, cell growth, and residual pyridine concentrations. The controls consisted of flasks containing the same initial pyridine concentration and pH, but without any inoculation.
2.7 Antibiotics Resistance:
Resistance of WUST-py to antibiotics (streptomycin, tetramycin, kanamycin, ampicillin, and chloromycetin) was determined by spreading 200 µL cell suspension onto MSM agar plates containing 500 mg L-1 pyridine and 50 mg L-1 of each antibiotic listed above. Triplicate plates were incubated at 35 °C for 3 days. The cells spread on antibiotic-free MSM agar plates containing 500 mg L-1 pyridine were used as controls.
2.8 Growth of WUST-py cells on Different Substrates:
Chemicals frequently found in industrial wastewaters were investigated for their potential to serve as sole carbon and energy source for WUST-py cells growth. The chemicals tested were: benzene, o-xylene, p-xylene, n-octane, dibutyl phthalate, polyethylene, 1,4-butyrolactone, di-n-butylamine, octanol, formic acid, trimethylamine, ethyl acetate, quinoline, phenol, N,N-dimethylaniline, naphthalene, methylbenzene, phenylamine, pyrrole, phenylcarbinol, ethylbenzene, ethanol, and ether using the same experimental protocol as in section 2.6. The concentration of each chemical studied was 200 mg L-1. An increase of at least 1 order of magnitude in OD600 after 72 h was considered as true positive growth.
2.9 Measurement of Broth Concentrations of Cell Biomass and Pyridine, pH, and phenol:
Cell growth was monitored as absorbance of the culture broth at 600 nm using UV-Vis spectrophotometer (INESA UV-752N, Shanghai, China). OD values were converted to dry cell mass using a pre-prepared calibration curve relating dry weight density to OD values (Dry cell mass density (in g L-1) = 0.629 x OD600 – 0.039 (R2= 0.986)) in cell suspensions.
For assay of pyridine in cell suspensions, 1 mL suspensions were centrifuged at 12,000 rpm at 4 °C for 10 min, and the supernatants were transferred to sterile test tubes. Pyridine in the supernatant was then extracted with equal volume of ethyl acetate, and absorbance in the extract was measured at 256 nm with ethyl acetate as control. The measured OD256 were then converted into pyridine concentration in the cell suspensions using a calibration curve. All the measurements were done in triplicates and the results are reported as averages. pH values of the broth were measured with a pH meter (Sartorius, PB-10, Germany). Phenol concentration was measured by using 4-aminoantipyrine spectrophotometric method (Fiamegos et al., 2002).
3.1 Enrichment, Isolation and Characterization of Pyridine Degrading Isolates:
After several weeks of enrichment and adaptation to increasingly higher concentrations of pyridine to 600 mg L-1, followed by a serial dilution and plating on MSM agar plates containing 500 mg L-1 pyridine, streaking of cells adapted to 600 mg L-1 pyridine resulted in 100 colonies of single cells. Whereas cells adapted to 500 mg L-1 pyridine failed to grow in presence of 1000 mg L-1 pyridine, 13 colonies isolated from culture adapted to 600 mg L-1 pyridine showed promise. Initial shake flask experiments with a strain designated as WUST-py exhibited an outstanding pyridine-degrading capacity, and thus was further studied. Colonies of the strain WUST-py were spherical with sizes ranging 1.0-2.0 mm in diameters after 3 days of incubation at 35 °C, smooth and tidy edges, light brown in color due to production of pigments, and not transparent. WUST-py cells were typically rod-shaped (Fig. 1a and b), around 5.0 µm in length and 0.8 µm in width. No visible flagella were observed by either light or electron microscopy in cultures grown in liquid media. Gram stain (Fig. 1c), spore stain (Fig. 1d), and microscopic examination showed that WUST-py was Gram-positive. The homogenous distribution of cells under SEM indicated that the bacterial strain WUST-py is not self-flocculating. This contrasts it with the Rhizobium sp. NJUST18 strain reported by Shen et al. (Shen et al., 2015) whose self-flocculation was so strong that it was necessary to deflocculate the growth media by vortexing for 10 min when measuring the biomass. This property of WUST-py is particularly beneficial for bioremediation of pyridine contaminated waters and lands. The partial 16S rRNA gene sequence (1,393 bp) of the strain WUST-py were determined and deposited in the GenBank with an accession number KY658456. Fig. 2 illustrates the phylogenetic relationship of the isolate in this study and some of its relatives in Bacteria Kingdom including Actinobacteria, Firmicutes, Proteobacteria, Bacteroidetes, Cyanobacteria and Thermaceae. The results showed that the strain WUST-py is close to Rhodococcus sp. D-50 and Rhodococcus sp. WB1, demonstrating that it is a member of the genus Rhodococcus.

3.2 Growth of WUST-py on pyridine.
The cell growth and pyridine consumption profiles and pH variations in a flask containing 1000 mg L-1 pyridine and pH of 10.0 initially are shown in Figure 3 (a) and (b). The profiles in a corresponding control flask are also presented here. Pyridine concentration in the experimental flask reduced to undetectable levels within 84 hours (Kaur and Pal, 2013). Pyridine consumption was associated with simultaneous reduction in pH of culture media.
During the course of biodegradation, some loss of pyridine through volatilization was observed. According to our observation, (as seen in the control flasks, Figure 3a, and we except the same happening in culture flasks as well) during the five days of incubation in flasks, the total loss of pyridine through volatilization was about 5 %. The pH of the medium dropped gradually from 10.0 to about 6.5 within 60 h and then slight fluctuation was observed. The pH reduction could be the result of formation of carbonic acid and other acidic intermediate metabolites, such as formic, acetic, succinic and glutaric acids (Li et al., 2009; Watson and Cain, 1975). The pH changes in this study are consistent with those in the Bai et al. (Bai et al., 2008) report, but quite different from those reported by Shen et al. (Shen et al., 2015) where pH increased during the whole cultivation process, and the extent of increment increased with the increasing initial pyridine concentrations.
3.3 Effects of initial Concentrations of Pyridine and initial pH values of Media:
Both initial pyridine concentrations and initial pH play crucial roles in the biodegradation process. The pyridine degradation and cell growth at several initial pyridine concentrations ranging from 100 to 2500 mg L-1 at different initial pH values from 5.0 to 11.0 are shown in Figure 4 (a-n).
As seen from Fig. 4 (a) and (b) for initial pH of 5.0, WUST-py could grow on pyridine concentrations up to 700 mg L-1 without showing any effect of pyridine concentration on cell growth rate. Initial pyridine concentrations of 100, 300, 500, and 700 mg L-1 were also reduced to undetectable levels within 36, 48, 60, and 84 h, respectively. A lag phase of 24 h was observed for all concentration of pyridine studied, reflecting time required for induction of the enzymes involved in pyridine degradation pathway. The biomass reached the peak in all the cases when almost all the pyridine was utilized by bacteria. However, no growth was observed when the cells were exposed to 1000 mg L-1 initial pyridine levels. From the results, it is evident that this bacterial strain has a potential to be used for the bioremediation of pyridine contaminated areas under acidic conditions. Attempts to study the potential of this strain for pyridine containing streams having highly acidic condition, e.g. pH 3.0, were not successful since the absorbance of pyridine at 256 nm in the MSM disappeared when pH of the medium was adjusted to 3.0 with HCl.
Data in Fig. 4 (c) and (d) suggest that the behavior of WUST-py cells at initial pH of 6.0 was almost identical to that at initial pH of 5.0 except when the initial pyridine concentration was 1000 mg L-1. With the initial pH of 6.0, the cells could reduce 1000 mg L-1 initial pyridine concentration to almost zero in 96 h. At low concentrations, the lag phase was the usual 24 h, but it extended to almost 48 h at 1000 mg L-1 pyridine level. After the lag phase, the biomass grew quickly and the pyridine was degraded progressively until it was completely consumed.
The neutral initial pH (7.0) of MSM was even more optimal for the bacterial strain to utilize both low as well as high concentrations of pyridine (Fig. 4 (e) and (f)). 1000 mg L-1 pyridine was reduced to undetectable level within 72 h at the neutral initial pH, whereas it took 96 h when the initial pH of MSM was 6.0. Furthermore, the cells could reduce 1400 and 1600 mg L-1 pyridine concentrations to non-detect levels within 96 and 120 h, respectively. In this case, a clear adverse effect of pyridine concentration on cell growth rate was also observed. During the experimental period of 120 h, the cells grew on 1800 mg L-1 pyridine, but did not reduce the concentration to zero. 2500 mg L-1 pyridine resulted in complete suppression of cell growth.
Increasing initial pH of the medium to 8.0 and above (up to 11.0, see Fig 4 g-l) resulted in slightly higher cell growth and faster rates of degradation of pyridine, but no further improvement in the threshold of pyridine degradation. At these initial pH values, the cells could handle pyridine concentrations up to 1800 mg L-1, but not the 2500 mg L-1. Initial pH 8.0 was found to be optimal, although further increase in initial pH resulted only in slightly increased lag periods.
The results presented here suggest that the strain WUST-py is a considerably superior strain compared to those reported in Literature when it comes to capacity to degrade pyridine in alkaline streams. The optimal pH for most of the pyridine degrading strains published in the literature was neutral or slightly basic (pH 7.0-8.0), (Bai et al., 2009; Chandra et al., 2009; Chandra et al., 2010; Mathur et al., 2008; Shen et al., 2015) and relatively strong alkaline conditions severely inhibited their growth and degrading capabilities. Li et al. (Li et al., 2009) reported that the bacterial strain Streptomyces sp. HJ02 degraded 250 mg L-1 pyridine within 7 d at an initial pH of 10.0. Mathur et al. (Mathur et al., 2008) tested the degrading ability of bacterial strains S. putrefaciens and B. sphaericus at initial pH of 10.0, and the results revealed that the two bacterial strains could remove only about 40 % (the former) and 25 % (the latter) of 100 mg L-1 pyridine within 150 h, respectively. Thus the potential of WUST-py strain to degrade pyridine at the initial pH 10.0 is tens or even hundreds times greater than those reported by Li et al., (Li et al., 2009) and Mathur et al (Mathur et al., 2008). Similarly, Mathur et al. (Mathur et al., 2008) reported the degrading activities of strains S. putrefaciens and B. sphaericus at an initial pyridine concentration of 100 mg L-1 and at the initial pH of 11.0, and demonstrated that the two bacterial strains degraded less than 20 % of the pyridine within 150 h. On the other hand, WUST-py strain robustly degraded pyridine at pH values up to 11.0. Therefore, the bacterial strain described in this study has a huge potential that can be applied to pyridine contaminated waters and lands for in situ bioremediation under relatively strong alkaline conditions. A possible mechanism which could explain the WUST-py ability to grow at high pHs is that it yields relatively large amount of acids during the biodegradation, which neutralizes the basic conditions of media and provides a more suitable environment for cells to grow and utilize pyridine.
Further investigation demonstrated that bacterial strain WUST-py could even degrade 1000 mg L-1 pyridine to an undetectable level within 192 h starting at the initial pH of 11.5. The biodegradation of pyridine and biomass growth of bacterial strain WUST-py at the initial pH of 11.5 and with different initial concentrations are shown in Fig. 4 (m) and (n), respectively. However, its lag phase was significantly extended at this high pH. At the initial pH of 12.0 WUST-py failed to grow in MSM for all the concentrations tested.
3.4 Growth on different Carbon Sources:
The growth of WUST-py on different carbon sources given in section 2.8 was studied. An increase of at least 1 order of magnitude in OD600 after 72 h incubation was considered as true positive growth of cells. The bacterial strain WUST-py can grow on phenol, formic acid, ethanol, and diethylether, at the individual concentration of 200 mg L-1 but it failed to grow on the rest of the carbon sources studied.
To prove the potential of WUST-py to degrade phenol, 5 mL portion of inocula prepared beforehand using phenol as substrate was inoculated into 95 mL MSM containing a final phenol concentration of 1000 mg L-1 at the initial pH values of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively. One thousand mg L-1 of phenol in MSM could be completely mineralized by WUST-py within 44 h, at the initial pH values of between 8.0 and 11.0, but it failed to degrade phenol at the initial pH values of 5.0, 6.0, and 12.0 (Fig. 5). At the initial pH of 7.0, WUST-py cells began to degrade phenol effectively within 32 h following the inoculation. By the 52th h of incubation, about 75 % of phenol was utilized by the strain at the exponential rate. From 52 to 60 h, the degrading rates slowed down progressively but almost 80 % of phenol was utilized by 60 h. Then the consumption of phenol stopped. It was postulated that the slowing of the cell growth was caused by the accumulated acids. pH changes during the phenol degradation process starting with the initial pH of 7.0 was monitored and shown in Fig. 6. When some NaOH solution was supplemented to the broth aseptically, the biodegradation restored quickly and the residual phenol was completely consumed within the following 8 h (Fig. 7). Separate experiments conducted at pH 5 and 6 showed that WUST-py could degrade low concentration of phenol (300 mg L-1) to undetectable levels at the initial pH of 6.0, but it failed to degrade it at the initial pH of 5.0. Nevertheless, it could degrade 700 mg L-1 pyridine within 84 h at the initial pH of 6.0. This difference can possibly be explained by production of different metabolites from phenol and pyridine by WUST-py. Several researchers (Bai et al., 2008; Padoley et al., 2006; Shen et al., 2015) dealing with the metabolic mechanisms of pyridine degradation have suggested that the nitrogen atom in the pyridine ring is ultimately released as ammonia that in turn could partially neutralize acids produced and thus contribute to the growth of cells at low pH values.
3.5 Antibiotic Resistance:
The main reason to investigate the antibiotic resistance/sensitivity of WUST-py in this study is the prevalence of antibiotics in modern sewage systems (Hirsch et al., 1998; Martinez, 2009). It’s well known that antibiotics are widely applied to the control and treatment of diseases in humans and domestic animals. They are also widely used as additives in animal and poultry feed industries. Although most of them are metabolized by specific tissues and organs, such as liver, kidney, and spleen, etc., usually they are not metabolized completely. As the results, some quantities of antibiotics are inevitably discharged into soil and water via urine and feces (Hirsch et al., 1998). These residues are also regarded as pollutants (Martinez, 2009). Theoretically, the resistance of the strain WUST-py to antibiotics would be beneficial for its bioremediation purposes, but the results demonstrated that the strain is highly sensitive to Streptomycin, Tetramycin, Kanamycin, Ampecillin and Chloromycetin. No single colony was found on agar plates with any of the antibiotics applied at 50 mg L-1 after three days of cultivation at 35 °C. This antibiotic sensitivity to some extent would lower the proliferation of the strain in the antibiotics contaminated areas, but it also would reduce the risk of spreading antibiotic resistance to opportunistic pathogens during in situ bioremediation of soil and water contaminated by pyridine such as Pseudomonas aeruginosa (Hancock, 1998; Martinez, 2009).
WUST-py strain degrades pyridine under relatively strong alkaline conditions. It could mineralize pyridine efficiently under a wide pH range (5.0-11.0). It mineralized up to 700 mg L-1 pyridine to undetectable levels within 84 h at the initial pH of 5.0; up to 1000 mg L-1 pyridine within 96 h at the initial pH of 6.0; up to 1600 mg L-1 pyridine within 120 h at the initial pH of 7.0; up to 1800 mg L-1 pyridine within 108 h at the initial pH of 8.0; up to 1800 mg L-1 pyridine at the initial pH of 9.0 and 10.0 within 120 h; up to 1600 mg L-1 pyridine within 108 h at the initial pH of 11.0. pH 8.0 was found to be the optimum pH for to mineralization of pyridine at higher concentration. An increase in alkalinity (pH 11.0) did not inhibit strain degrading activities significantly, implying that this strain is also applicable to the in situ bioremediation of pyridine contaminated waters and lands under relatively strong alkaline conditions. Experiments with different substrates revealed that the strain WUST-py can grow on phenol, alcohol, diethyl ether, and formic acid. Preliminary phenol biodegradation experiments showed that WUST-py could mineralize 1000 mg L-1 phenol to an undetectable levels within 40 h at the initial pH values of 9.0 and 10.0, and within 44 h, at the initial pH values of 8.0 and 11.0, implying a substantial potential of co-biodegradation of pyridine and phenol. The strain WUST-py is highly sensitive to five tested antibiotics, including streptomycin, tetramycin, kanamycin, ampicillin, and chloromycetin.


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Figure 1. Scanning electron microscope and light microscope images of strain WUST-py: (a) SEM image of WUST-py at 10,000×, (b) SEM image of WUST-py at 5000×, (c) Gram stain at 100×, and (d) spore stain at 100×.

Figure 2. A phylogenetic study of WUST-py strain using partial nucleotide sequences of its 16S rDNA gene by CLUSTALW and Neighbor-Joining algorithms of MEGA 5.

Figure 3. Biodegradation of 1000 mg L-1 pyridine by WUST-py at initial pH of 10.0. (a) Cell growth and pyridine biodegradation at 35°C and 150 rpm (b) pH changes during pyridine degradation and in the control flasks.









Figure 5 Biodegradation of phenol at initial concentration of 1000 mg L-1 by WUST-py at different initial pH values

Figure 6. Biodegradation of phenol in MSM at initial concentration of 1000 mg L-1 and at initial pH of 7.0

Figure 7. The effects of pH adjustment of the broth at 60 h upon inoculation on the biodegradation of the remnants of phenol

Abstract Graphics: Isolation and Characterization of a Novel Bacterial Strain Rhodococcus sp. WUST-py for Pyridine Biodegradation with special application for Relatively Strong Alkaline Conditions.

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