Isolation and Characterization of a Novel Phenol Degrading Bacterial

Article pubs.acs.org/IECR

Isolation and Characterization of a Novel Phenol Degrading Bacterial Strain WUST-C1 Jianzhong Liu,†,§ Qian Wang,† Jiabao Yan,† Xiaorong Qin,† Lingling Li,† Wu Xu,‡ Ramalingam Subramaniam,§ and Rakesh K. Bajpai*,§ †

Hubei Coal Conversion and New Carbon Materials Key Laboratory; College of Chemical Engineering and Technology, Wuhan University of Science and Technology, Wuhan 430081, Hubei, China ‡ Department of Chemistry and §Department of Chemical Engineering, University of Louisiana at Lafayette, Lafayette, Louisiana 70504, United States ABSTRACT: This paper reports the successful isolation and characterization of a novel phenol-degrading bacterial strain WUST-C1 from activated sludge. WUST-C1 is an aerobic, Gram-negative, nonsporulating, and short-rod or peanut-shaped bacterium, which occurs singly, in pairs, but mainly in clusters. 16S rDNA sequence alignment demonstrated this strain belonged to the genus of pseudomonas, with a 99.9% identity to 16S rDNA sequences of Pseudomonas aeruginosa EU170480. It could mineralize up to 1200 mg L−1 phenol within 36 h (99%). Besides phenol, WUST-C1 could grow aerobically on pyrocatechol, alpha-naphthol, hydroquinone, naphthalene, isoquinoline, and indole. WUST-C1 was resistant to ampicillin and chloromycetin. The growth kinetics of strain WUST-C1 on phenol as a sole carbon and energy source at 35 °C can be described using Haldane equation. It has a maximum specific growth rate (μmax) of 2.47 h−1, a half-saturation constant (Ks) of 48.7 mg L−1, and a substrate inhibition constant (Ki) of 100.6 mg L−1 with a R2-value of 0.988. Strain WUST-C1 is tolerant to phenol at concentrations up to 1600 mg L−1. Hence, it could potentially be an excellent bacterial candidate for the biotreatment of high-strength phenolcontaining industrial wastewaters and for the in situ bioremediation of phenol-contaminated sites.

1. INTRODUCTION Phenol and its derivatives (halogenated phenol, nitro phenol, alkyl phenol, etc.) are either raw materials or products in a variety of modern industries (oil refineries, coke plants, phenol manufacturing, pharmaceuticals, resin, paint, dyeing, textile, leather, petrochemical plants, pulp mills, etc.). Millions of tons of wastewaters containing phenol or/and phenolic compounds are generated by these industries every day, and these pose serious risks to the environment as well as to the organisms living in it. The presence of phenol in water adversely affects germination and formation of seeds in field crops and causes the death of fish in lakes.1−3 Phenol is toxic to humans and aqueous species either by ingestion, contact, or by inhalation even at very low concentrations. WHO recommends a limit of 1 μg L−1 for phenol in drinking water.4 Phenols can be removed from industrial effluents by physicochemical methods5−9 or by microbial procedures.1,10−23 Conventional physical and chemical methods often suffer from drawbacks such as high initial investment and operational costs, and the formation of secondary toxic byproducts.10 On the other hand, biodegradation is more environmentally friendly and thorough. As a result, it has turned out to be a favorable alternative for phenols degradation. Microorganisms capable of degrading phenol and its derivatives are ubiquitous in nature.1,10−23 Of these, Pseudomonads13−15 exhibit high rates of degradation of phenol. Although phenol-contaminated waste waters are commonly treated at source to reduce the residual phenol concentrations, identifying and introducing potent and robust phenol-degraders in the treatment systems is highly desirable. In this paper, we report the taxonomic identification of a novel phenol-degrading bacterial strain isolated from activated © 2012 American Chemical Society

sludge of a coke plant in Wuhan, China, based on the homology of its 16S rRNA to its relatives in GenBank. Phenol degradation rates, cell growth kinetics of the isolate, carbon sources on which the strain could be grown, and pH changes during phenol degradation by the strain were also characterized. The strain’s resistance to different antibiotics was also determined as yet another measure of its characteristics. Growth of the isolated strain was monitored with different initial phenol concentrations in batch reactors and kinetics modeled using Haldane’s equation.

2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. The chemicals and reagents used in bacterial cultivation and assay in the study were of analytical grades and were obtained from Sinopharm Chemical Reagent Co., Ltd. 2.2. Media. LB medium, mineral salt medium (MSM), and agar plate/slant medium were used in this study. The composition of LB medium was 10 g L−1 tryptone, 5 g L−1 yeast extract, and 10 g L−1 NaCl. The MSM contained (in 1 L) 0.4 g K2HPO4, 0.4 g KH2PO4, 0.1 g NaCl, 0.2 g MgSO4·H2O, 0.01 g MnSO 4 ·H 2 O, 0.01 g Fe 2 (SO 4 ) 3 ·H 2 O, 0.01 g Na2MoO4·2H2O, and 1.0 g (NH4)2SO4. Agar plate/slant media consisted of LB or MSM with 15 g L−1 agar. The pH Special Issue: L. T. Fan Festschrift Received: Revised: Accepted: Published: 258

May 16, 2012 August 27, 2012 September 6, 2012 September 6, 2012 dx.doi.org/10.1021/ie3012903 | Ind. Eng. Chem. Res. 2013, 52, 258−265

Industrial & Engineering Chemistry Research

Article

fulfilled by Shanghai Sangon Biological Engineering Technology and Services Co.Ltd. A blast search of 16S rRNA 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).26 The ClustalW algorithm was applied to carry out pairwise and multiple sequence alignments, and a phylogenic tree was constructed using the neighbor-joining method.27 2.5. Growth Substrates. The ability of WUST-C1 to grow on chemicals frequently found in industrial wastewaters was measured by cultivating the cells at 35 °C in orbital shaker at 150 rpm with each chemical as sole carbon source. These experiments were conducted in 100 mL of MSM in 250 mL conical flasks. The chemicals were the following: aniline, dimethylbenzene, p-nitrophenol, pyrocatechol, α-naphthol, 1naphthylamine, hydroquinone, diphenylamine, naphthalene, quinoline, isoquinoline, pyridine, and indole. The concentrations of these compounds were fixed at 200 mg L−1. Cells grown on phenol up to the late exponential phase in MSM were used as inocula. All the cultivations were performed in triplicate. A flask containing MSM with 200 mg L−1 phenol was also inoculated with the cells as control. An increase in OD600 of at least 1 order of magnitude after 48 h was considered as true positive growth. 2.6. Antibiotics Resistance. Resistance of WUST-C1 to antibiotics (streptomycin, tetramycin, kanamycin, ampicillin, and chloromycetin) was investigated by plating phenol-grown cells (late exponential phase in MSM) on MSM agar plates containing 600 mg L−1 phenol and 50 mg L−1 of each antibiotic. The plates were incubated for 3 days at 35 °C. All the cultivations were performed in triplicate. The cells spread on antibiotic-free MSM agar plates (containing 600 mg L−1 phenol) were used as controls. 2.7. Preparation of Inocula. Seed cultures of the selected bacterial strains were grown in 200 mL MSM (containing 1000 mg L−1 phenol), contained in 500 mL flasks and incubated at 35 °C and 150 rpm until late exponential phase. Cells were recovered by centrifugation (8000g, 10 min), washed, and resuspended in MSM. The optical density of the suspension at 600 nm (OD600) was adjusted to 1.0 with phenol free MSM. A 5 mL portion of the suspension was used as a single inoculum for degradation studies, sole carbon source studies, and for cell growth kinetics. A 200 μL portion of the suspension was used for antibiotic resistance experiments. 2.8. Cell Biomass Measurement and Phenol Assay. Cell growth was monitored spectrophotometrically by measuring absorbance at 600 nm (Unico UV-200 spectrophotometer). The OD value was then converted to dry cell mass using a dry weight calibration curve. Dry cell mass density (mg L−1) was found to follow the regression equation: X (mg L−1) = 3.149 × OD600. Phenol assay was conducted by an improved 4-aminoantipyrine spectrophotometric method (4-AAP)28,29 in broth supernatants obtained by centrifugation at 12 000 rpm at 4 °C for 10 min. All the measurements were conducted in triplicate and the results are reported as average (points). 2.9. Degradation Experiments. The biodegradation experiments were conducted in a series of 500 mL sterile Erlenmeyer flasks. Five mL of inoculum was transferred aseptically to each flask containing 95 mL MSM supplemented with varying amounts of phenol (50−1800 mg L−1). Phenol

of the media was adjusted from 7.0 to 7.2 with NaOH. Media were sterilized by autoclaving at 121 °C for 15 min. Phenol was prepared as a stock solution (50 000 mg L−1) and was filter sterilized. Its concentration in media was varied as required. 2.3. Bacterial Enrichment, Acclimatization, and Isolation. Activated sludge was sampled from the aeration tank of a coke plant in Wuhan, China. One milliliter of the homogenized sludge sample was added to 99 mL of sterile MSM supplemented with 300 mg L−1 phenol in a 500 mL Erlenmeyer flask and incubated at 35 °C in an orbital shaker operating at 150 rpm. After an obvious OD increase was observed (2−3 d), 1 mL of culture broth was transferred to the second shake flask containing 99 mL of MSM with 600 mg L−1 phenol. Subsequently, 1 mL culture broth from the second flask was used to inoculate the third containing 1000 mg L−1 phenol, and again from the third flask to a fourth containing 1500 mg L−1 phenol. The final broth from the fourth flask was serially diluted with 1× phosphate-buffered saline, spread onto MSM agar plates containing 1000 mg L−1 phenol, and incubated at 35 °C. All the resulting colonies were streaked for purity. Isolates were preserved at 4 °C on LB agar slants and at −80 °C in LB broth supplemented with 20% sterilized glycerol. More than 50 colonies were obtained. However, initial shake flask experiments showed that only one of the strains, WUST-C1, exhibited potent phenol degrading ability, and thus was investigated further. 2.4. Identification of the Bacterial Strain. Cell morphology was examined with a light microscope (SA3000, Beijing, China) after Gram and spore staining and with a scanning electron microscope (Nova 400 NanoSEM, FEI, Hong Kong) after preparation of samples as follows. Free cells were fixed on a glass slide with 2.5% glutaraldehyde (in phosphate buffer, pH 7.2) for 24 h at 4 °C, followed by three washings with phosphate buffer (for 5 min each time) and dehydration with increasing concentrations (50, 70, 85, 95, 100% vol/vol; 2 min at each concentration) of t-butyl alcohol; the final dehydration process was repeated twice. The dehydrated cells adhering to the glass slide were dried in a freeze-dryer (DZF-6020) and coated with gold before being viewed at 15.0 kV. Gram and spore staining was done using standard protocols.24 For taxonomic identification, total genomic DNA was extracted from cells using a standard protocol.25 16S rRNA gene was amplified from the extracted DNA using two universal primers, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1522R (5′-AAGGAGGTGATCCAGCC-3′). The target DNA was amplified in an automated thermal cycler (Mastercycler gradient, Eppendorf) using a 25 μL polymerase chain reaction (PCR) mixture containing 0.1 μg of template DNA, 2.5 μL of 10 × PCR buffer (containing Mg2+), 0.25 mM of dNTP, 0.1 μM of each primer, and 1U of taq DNA polymerase. PCR procedures consisted of denaturation at 95 °C for 5 min, 30 cycles of exposure for 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C, and a final extension step at 72 °C for 10 min. PCR products were separated by electrophoresis, and the band at approximately 1500 bp in the gel (1%) was excised and purified by a gel extraction kit (Omega Bio-Tek Co.) in accordance with the manufacturer’s instructions. The purified PCR products were ligated with pMD18-T vector (TaKaRa Co.) and used to transform competent cells (E. coli, DH5αTiangen Co.). The manipulation of transformation of plasmid and screening of recombinant plasmid followed the standard protocol,25 sequencing of the gene fragment was 259

dx.doi.org/10.1021/ie3012903 | Ind. Eng. Chem. Res. 2013, 52, 258−265


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Figure 1. Scanning electron microscope and light microscope pictures of strain WUST-C1: (a) SEM image of WUST-C1 at 10 000×, (b) SEM image at 30 000×, (c) Gram stain at 100×, and (d) spore stain at 100×.

was added as required from the stock solution (50 000 mg L−1, filter sterilized beforehand). A 3 mL portion of the culture was removed at periodic intervals from the flasks into a 10-mL graduated test tube for measurements of pH, cell biomass, and phenol concentration. pH of the broth was measured with a pH meter (Delta 320). Control experiments were conducted in the same manner as described above but without bacteria. All experiments were carried out at initial pH of 7.0 to 7.2, and flasks were incubated in a rotary shaker at 150 rpm and 35 °C.

3. RESULTS AND DISCUSSION 3.1. Enrichment, Isolation, and Characterization of Phenol Degrading Isolates. After several weeks of enrichment followed by isolation, more than 50 bacterial strains capable of growing on phenol as sole carbon and energy source were isolated from activated sludge. Two potent strains (named as WUST-C1 and WUST-L1, respectively) were eventually selected for further studies. These strains were in fact isolated from activated sludge from two separate coke plants. The findings of these studies indicated that their phenol degrading properties were quite similar and both belonged to Pseudomonas based on 16S rDNA sequences alignment. WUST-C1 was isolated before WUST-L1. Hence, only the strain WUST-C1 is described here in detail. Colonies of strain WUST-C1 were surface smooth with tidy edge, whitish, with sizes ranging 0.5−3 mm in diameters after 3 days of incubation

Figure 2. Electrophoresis results of genomic DNA from WUST-C1, PCR products, and recombinant plasmid: (lane M) DNA ladder marker; (lane 1) bacterial genomic DNA; (lane 2) PCR products; (lane 3) purified PCR products from gel; (lane 4) control plasmid (2692 bp); (lane 5) recombinant plasmid (about 4200 bp); (lane 6) control plasmid after digestion with endonuclease Eco RI; (lane 7) recombinant plasmid after digestion with endonuclease Eco RI.

at 35 °C. WUST-C1 cells were short-rod or peanut-shaped (Figure 1a and b). Cells were around 1 μL in length, and 0.5 μL 260



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Figure 3. Phylogenic analysis of 16S rRNA by ClustalW and Neighbor-Joining algorithms of MEGA 5. Genetic distance was calculated using Maximum Composite likelihood, a number of bootstrap replication (500) was selected, and complete deletion was chosen for gap/missing data treatment.

Table 1. Growth of WUST-C1 on Different Carbon Sources

a

substrate

growth

aniline dimethylbenzene p-nitrophenol pyrocatechol α-naphtol 1-Naphthylamine hydroquinone diphenylamine naphthalene quinoline isoquinoline pyridine indole

a b b a a b a b a b a b a

Growth. bNo growth.

Table 2. Resistance of WUST-C1 to Antibiotics

a

antibiotics

resistance

streptomycin tetramycin kanamycin ampicillin chloromycetin

B B B a a

Resistant. BNot resistant. Figure 4. Biodegradation of 1000 mg L−1 phenol in MSM by WUSTC1. (a) Cell growth and phenol biodegradation of WUST-C1 in MSM containing phenol (1000 mg L−1) in shake flask. (b) pH of broth during phenol degradation. Control experiments were conducted in the same manner but without bacteria.

in width, occurred mainly in clusters, but also as single cells and in pairs. No visible flagella were observed. Gram stain (Figure 1c), spore stain (Figure 1d), and microscopic examination showed that WUST-C1 was nonsporulating and Gramnegative. 261



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(JN797509 for strain WUST-L1). Figure 3 illustrates the phylogenic relationships of our isolates with some of the phenol-degrading strains described in literature; these demonstrate that strains WUST-C1 and WUST-L1 are typical members of the genus Pseudomonas. The strain WUST-C1 was closely related to Pseudomonas aeruginosa EU170480 with 99.9% sequence identity and strain WUST-L1 was 99.50% identical to Pseudomonas aeruginosa GQ926936. WUST-C1 and WUST-L1 are slightly different from Pseudomonas putida and pseudomonas fluorescence. In accordance with these data, the two strains are both referred as Pseudomonas aeruginosa. As previously described, WUST-C1 and WUST-L1 were isolated from sludge collected from two separate coke plants during two different seasons (WUST-C1 in Fall and WUST-L1 in the following Spring). This implied that the microorganisms of Pseudomonas play the main roles of phenol degradation in this region. 3.2. Growth on Carbon Sources. Table 1 shows the growth of strain WUST-C1 on 13 carbon sources. The results demonstrated that WUST-C1 could be grown on pyrocatechol, alpha-naphtol, hydroquinone, naphthalene, isoquinoline, and indole. It is interesting that isoquinoline was its favorite substrate, but quinoline was not. The biomass density of the cells grown on isoquinoline was as high as density of cells grown on phenol. 3.3. Antibiotic Resistance. Pseudomonas aeruginosa is increasingly recognized as an emerging opportunistic pathogen of clinical relevance. Several different epidemiological studies indicate Pseudomonas aeruginosa is a notoriously difficult organism to control with antibiotics or disinfectants.30 Since phylogenetic study (Figure 3) indicated that WUST-C1 is a species of Pseudomonas aeruginosa, antibiotic resistance of WUST-C1 was investigated. Another reason for testing the antibiotic resistance/sensitivity of WUST-C1 is the prevalence of antibiotics in sewage systems.31,32 Antibiotics are widely used in human and animal disease control and treatment, as well as in animal and poultry feed as additives. Most of them are eliminated by specific tissues and organs (e.g., liver, kidney, and spleen), but they are usually not degraded completely inside human and animal bodies. As the result, parts of them are inevitably discharged into soil or water via urine or feces.31 These residues are also regarded as pollutants.32 Screening of bacterial strains with phenol-degrading capability and resistance to antibiotics will undoubtedly be useful for bioremediation of environments. Table 2 shows the resistance of WUST-C1 to five antibiotics. The results showed that the strain is resistant to ampicillin and chloromycetin, but not to streptomycin, tetramycin, and

Figure 5. Cell growth (a) and phenol degradation (b) profiles at different initial phenol concentrations: (⧫) 200, (■) 600, (▲) 1000, (×) 1400, (●) 1800 mg L−1.

Figure 6. Cell-specific growth rate and fitting of the Haldane equation.

The genomic DNA isolated from the bacterial strain WUSTC1, amplification products with the universal oligonucleotide primers, and the products of endonuclease digestion of the recombinant plasmid with Eco RI were shown in Figure 2. The partial sequences of the 16S rDNA of strain WUST-C1 (1,529 bases) (and those of the WUST-L1) were determined and deposited in the GenBank under number JN180124

Table 3. Haldane Parameters for Different Bacteria Grown on Phenol Haldane’s model bacterial strain Pseudomonas WUST-C1 Pseudomonas putida Pseudomonas putida MTCC 1194 Acinetobacter Candida albicans Alcaligenes faecalis mixed culture

−1

conc range (mg L )

−1

μmax (h )

Ks (mg L−1)

Ki (mg L−1)

ref

0−1600

2.5

48.7

100.6

This work

25−800 0−1000

0.9 0.305

6.93 36.3

284.3 129.8

35 36

60−350 0−1800 10−1400 23.5−659

0.8−0.85 0.315 0.15 0.309

1.2−1.5 19.5 2.22 74.6

188−315 208.6 245.4 648.1

37 20 38 34

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mineralize 1200 mg L−1 phenol within 36 h and 200 mg L−1 phenol within 4 h. These results suggested that WUST-C1 is more tolerant to phenol, and its phenol degrading efficiency is much higher than that of strains in published reports.21,33 The phenol concentration in the effluent of the coke plant, from where activated sludge for this experiment was collected, was around 300 mg L−1, and this level of phenol could be eliminated by WUST-C1 in less than 4 h (results are not shown). 3.6. Kinetics of Growth of WUST-C1. The knowledge of growth kinetics is essential for understanding the capacities of the microorganisms to degrade phenol as well as the operation of the treatment units. Almost total inhibition of cell growth is evident in Figure 5 at initial phenol concentration of 1800 mg L−1. Haldane kinetic equation (eq 1) is frequently used to describe cell growth rates on inhibitory substrates such as phenol in pure or mixed cultures, and it was used to describe the growth behavior of WUST-C1 on phenol.

kanamycin. These antibiotic resistance traits of WUST-C1 may be genetically encoded by specific plasmids. Efforts to isolate the plasmids are undergoing currently. 3.4. Growth of WUST-C1 on Phenol. The cell growth and phenol consumption profiles are presented in Figure 4. Since pH plays a critical role in determining the rate as well as the extent of biodegradation, pH changes were also monitored and these too are shown in the figure. The initial concentration of phenol was 1000 mg L−1. Phenol concentration in the broth was reduced to nondetectable levels within 36 h of inoculation. No lag phase was observed, and rapid growth of cells started immediately upon inoculation. The rapid growth phase lasted 12 h during which phenol concentration also decreased by about 50%. However, cell growth reduced considerably after the first 12 h. This is suspected to be a result of reduced pH of the broth (Figure 4b). Reductions in pH of cultural media were noticed in all the experiments except control and, in most occasions, pH was then stabilized above 4. This pH reduction could be a result of formation of carbonic acid that could also cause growth inhibition. Besides, it has been observed that biomass concentrations increased at the end of batch cultures even after phenol was completely metabolized (not shown). As a result, it can be postulated that phenol was first biotransformed into metabolites (not measured in this experiment), and the metabolites served as substrates until these are consumed by the bacteria. 3.5. Effect of initial Concentration of Phenol. Initial phenol concentration plays an important role in the biodegradation process. The phenol degradation and cell growth profiles at several initial phenol concentrations ranging from 200 to 1800 mg L−1 are presented in Figure 5a and b. The results showed that the higher the initial concentration of phenol the longer time it takes for it to be degraded completely. At phenol concentrations below 800 mg L−1, no or very short lag phase was observed for cell growth and phenol consumption. Beyond this concentration, the lag period increased with increasing phenol concentration. WUST-C1 had a lag phase of 12 h at 1600 mg L−1 phenol, and only 62% phenol removal was achieved after testing for 36 h. Bacterial growth was completely inhibited, and no biodegradation was observed for initial phenol concentration of 1800 mg L−1. Once the lag phase was over, exponential growth occurred during which significant increases in cell biomass occurred, and phenol was consumed at high rates at each of the initial phenol concentration. Once past the exponential growth, the phenol degradation rate decreased too. In most cases (initial phenol concentrations 400 mg L−1 or more), cell growth stopped at around 300 mg dry cells L−1 which may be indicative of limitation of growth by a nutrient different than carbon source (phenol), following the depletion of which the cells still metabolized phenol but with altered metabolism. In such a situation, the initial phenol concentration may be considered to be responsible for the measured specific growth rate of cells. Geng et al.33 isolated a bacterial strain EDP3 from an industrial activated sludge. This strain could grow on phenol up to a concentration of 1000 mg L−1, and no growth was observed at an initial concentration of 1500 mg L−1. It took nearly 60 h for the bacterial strain to degrade 500 mg L−1 phenol to nondetectable levels and over 220 h for 1000 mg L−1 initial phenol concentration. It took 30 h for the bacteria strain AS1, isolated by El-sayed et al.,21 to degrade 100 mg L−1 phenol to nondetect level. In contrast, WUST-C1 could grow on phenol at concentrations as high as 1600 mg L−1; It could

μx =

μmax Csi K s + Csi +

Csi 2 Ki

(1)

Here μx is the specific growth rate (h−1), μmax is the maximum specific growth rate (h−1), Csi is the initial substrate concentration (mg L−1), Ks is the half-saturation constant of growth kinetics (mg L−1), and Ki is the inhibition constant (mg L−1). In order to obtain the kinetic model parameters of cell growth, cell concentrations were measured over time at different initial phenol concentrations ranging from 50 to 1600 mg L−1 and the specific growth rates, μx, were estimated by performing a linear least-squares regression of the semilogarithmic plot of biomass concentration against cultivation time during the exponential growth phase. The specific growth data at different initial phenol concentrations are plotted in Figure 6. Nonlinear least-squares regression analysis was used to estimate the Haldane kinetic parameters for WUST-C1, and these were the following: μx = 2.47 h−1, Ks = 48.7 mg L−1, Ki = 100.6 mg L−1. These parameters resulted in a high correlation (R2 = 0.988) between the Haldane kinetics and experimental data. Predictions of specific growth rate using the Haldane equation are also shown in Figure 6 as a continuous curve. It should be noted that the maximum in specific growth rate is predicted at approximately 70 mg L−1 phenol, below which cell growth seemed to be less optimal due to substrate limitation, and above which cell growth was inhibited increasingly due to substrate inhibition. According to Bajaj et al.,34 high phenol concentrations result in damaged cells with decreased metabolic activity; death of the cells may also contribute to apparent inhibition of growth at high phenol concentrations. Therefore, the model should be modified to account for cell death at very high substrate concentrations. The values of Haldane parameters for phenol biodegradation by several bacterial strains reported in literature are listed in Table 3. The values of inhibition constant (Ki) and the saturation constant (Ks) for WUST-C1 strain are similar to that for several other strains, especially those obtained by Arinjay et al.35 However, its value for μmax was much higher than those for most strains previously reported,20,34−39 implying that the WUST-C1 could grow fast on high-strength phenol-containing industrial wastewaters and thus degrade phenol quickly. 263



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(6) Banat, F. A.; Al-Bashir, B.; Al-Asheh, S.; Hayajneh, O. Adsorption of Phenol by Bentonite. Environ. Pollut. 2000, 107, 391. (7) Comninellis, C.; Pulgarin, C. Anodic Oxidation of Phenol for Wastewater Treatment. J. Appl. Electrochem. 1991, 21, 703. (8) Comninellis, C.; Pulgarin, C. Electrochemical Oxidation of Phenol for Wastewater Treatment Using SnO2 Anodes. J. Appl. Electrochem. 1993, 23, 108. (9) Pardeshi, S. K.; Patil, A. B. A Simple Route for the Photocatalytic Degradation of Phenol in Aqueous Zinc Oxide Suspension Using Solar Energy. Solar Energy 2008, 82, 700. (10) Li, Y.; Li, J.; Wang, C.; Wang, P. F. Growth Kinetics and Phenol Biodegradation of Psychrotrophic Pseudononas putida LY1. Bioresour. Technol. 2010, 101, 6740. (11) Baek, S. H.; Yin, C. R.; Lee, S. T. Aerobic Nitrate Respiration by a newly Isolated Phenol-degrading Bacterium, Alcaligenes Strain P5. Biotechnol. Lett. 2001, 23, 627. (12) Essam, T.; Amin, M. A.; Tayeb, O. E.; Mattiasson, B.; Guieysse, B. Kinetics and Metabolic Versatility of highly Tolerant Phenol Degrading Strain TW1. J. Haz. Mat. 2010, 173, 783. (13) Wang, Y. J.; Song, J.; Zhao, W.; He, X. L.; Chen, J.; Xiao, M. In situ Degradation of Phenol and Promotion of Plant Growth in Contaminated Environment by a Single pseudomonas aeruginosa Strain. J. Haz. Mat. 2011, 192, 354. (14) Chung, T. P.; Tseng, H. Y.; Juang, R. S. Mass Transfer Effect and Intermediate Detection for Phenol Degradation in Immobilized pseudomonas putida Systems. J. Process Biochem. 2003, 38, 1497. (15) Afzal, M.; Iqbal, S.; Raul, S.; Khalid, Z. M. Characteristics of Phenol Biodegradation in Saline Solutions by Monocultures of pseudomonas aeruginosa and pseudomonas pseudomallei. J. Haz. Mat. 2007, 149, 60. (16) Bourque, D.; Bisaillon, J. G.; Beaudet, R.; Sylvestre, M.; Ishaque, M.; Morin, A. Microbiological Degradation of Malodorous Substances of Swine Waste under Aerobic Conditions. Appl. Environ. Microbiol. 1987, 53, 137. (17) Arutchelvan, V.; Kanakasabai, V.; Nagarajam, S.; Muralikrishnan, V. Isolation and Identification of Novel High Strength Phenol Degrading Bacterial Strains from Phenol-formaldehyde Resin Manufacturing Industrial Wastewater. J. Haz. Mat. 2005, 127, 238. (18) Prieto, M. B.; Hidalgo, A.; Serra, J. L.; Llama, M. J. Degradation of Phenol by Rhodococcus erythropolis UVP-1 Immobilized on Biolite in a Packed-bed Reactor. J. Biotechnol. 2002, 97, 1. (19) Paller, G.; Hommel, R. K.; Kleber, H.-P. Phenol Degradation by Acinetobacter Calcoaceticus NCIB 8250. J. Basic Microbiol. 1995, 35, 325. (20) Wei, G.; Yu, J; Zhu, Y.; Chen, W.; Wang, L. Characterization of Phenol Degradation by Rhizobium sp CCNWTB 701 Isolated from Astragalus chrysopteru in Mining Tailing Region. J. Haz. Mat. 2008, 151, 111. (21) El-sayed, W. S.; Ibrahim, M. K.; Abu-shady, M.; El-beih, F.; Ohmura, N.; Saiki, H.; Ando, A. Isolation and Identification of a Novel Strain of the Genus Ochrobactrum with Phenol-degrading Activity. J. Biosci. Bioeng. 2003, 96, 310. (22) Jiang, Y.; Wen, J.; Liu, H.; Yang, S.; Hu, Z. The Biodegradation of Phenol at High Initial Concentration by the Yeast Candida tropicalis. Biochem. Eng. J. 2005, 24, 243. (23) Adav, S. S.; Chen, M. Y.; Lee, D. J.; Ren, N. Q. Degradation of Phenol by Aerobic Granules and Isolated Yeast Candida tropicalis. Biotechnol. Bioeng. 2006, 96, 844. (24) Sheng, P.; Fan, X.; Li, G. Microbiological Experiments; Higher Education Press: Beijing, 1999. (25) Cao, Y. Practical Manipulation Guide for Molecular Biology; People’s Medical Publishing House: Beijing, 2003. (26) Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 2011, 28, 2731. (27) Saitou, N.; Nei, M. The Neighbor-joining Method: a New Method for Reconstructing Phylogenetic Trees. Mol. Biol. Evol. 1987, 4, 406.

4. CONCLUSIONS A bacterial strain capable of degrading phenol up to 1600 mg L−1 was isolated from activated sludge of a coke plant in Wuhan, China. The strain was nonsporulating and Gramnegative, and it was identified as Pseudomonas aeruginosa based on the 16S rDNA sequence alignment. The strain was named Pseudomonas aeruginosa WUST-C1. The strain could utilize pyrocatechol, alpha-naphtol, hydroquinone, naphthalene, isoquinoline, and indole also as sole carbon sources. WUST-C1 was resistant to ampicillin and chloromycetin, but not to streptomycin, kanamycin, and tetramycin. WUST-C1 was able to completely biodegrade up to 1200 mg L−1 phenol within 36 h. The cell growth kinetics of WUST-C1 with phenol as sole carbon and energy source was investigated at initial phenol concentrations ranging from 50 to 1600 mg L−1 at 35 °C. The inhibitory effects of phenol could be described by the Haldane equation and the Haldane parameters for WUST-C1 grown on phenol were μmax = 2.47 h−1, Ks = 48.7 mg L−1, and Ki = 100.6 mg L−1 (R2 = 0.988). The maximum in the specific rate of cell growth was observed as a phenol concentration of 70 mg L−1. All in all, WUST-C1 is possibly an excellent bacterial candidate for the biotreatment of high-strength phenol-containing industrial wastewaters and for the in situ bioremediation of phenol-contaminated sites.

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AUTHOR INFORMATION

Corresponding Author

*Corresponding author: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Cui Huiming, a personnel manager at Wuhan Coke Plant, for permitting collection of activated sludge samples and for his constant support for the project. We also want to express our sincere appreciation to Professor Wang Guanghui, Dean of the College of Chemical Engineering and Technology, Wuhan University of Science and Technology, for his constructive suggestions towards this research. We thank Dr. Andrei Chistoserdov of the Department of Biology, University of Louisiana at Lafayette, for his beneficial suggestions on the phylogenic tree and Mr. Sharif Rahman, of the Bioprocessing Research Laboratory, Department of Chemical Engineering, UL at Lafayette, for his help with the manuscript.

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Isolation and Characterization of a Novel Phenol Degrading Bacterial

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