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Community analysis and metabolic pathway of halophilic bacteria for phenol degradation in saline environment
International Biodeterioration & Biodegradation 94 (2014) 115e120
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Community analysis and metabolic pathway of halophilic bacteria for phenol degradation in saline environment Zhong-zi Huang a, b, Ping Wang c, Hui Li a, *, Kuang-fei Lin a, Zhi-yan Lu a, Xiao-jue Guo a, Yong-di Liu a, * a
State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, State Key Laboratory of Biological Reactor Engineering, School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, PR China School of Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, PR China c School of Bioengineering, East China University of Science and Technology, Shanghai 200237, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 April 2014 Received in revised form 9 July 2014 Accepted 9 July 2014 Available online
A moderately halophilic bacterial enrichment was able to degrade 120 mg/L of phenol in the presence of 1e2 M of NaCl within 3 d or 2.5e3 M of NaCl within 6 d. The optimal degradation was achieved at 1.5 M of NaCl and 350 mg/L of phenol. PCR-DGGE profile of the enrichment showed that the Acidobacterium sp. and Chloroflexus sp. dominated the community. The phenol-biodegradation pathways consisted of an initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase and catechol 2,3-dioxygenase. Nuclear magnetic resonance (NMR) spectroscopy profiles showed that ectoine and hydroxyectoine were the main compatible solutes to adjust the bacterial osmotic pressure. This study provides further information on the understanding of phenol-degradation over a wide range of salinity and remediation of phenol as a pollutant in the environment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Phenol Biodegradation Moderately halophilics Metabolic pathway Compatible solutes
1. Introduction Phenol and phenolic compounds are hazardous pollutants widespread in the environment through wastewater discharges from a variety of industries including phenol-formaldehyde resin, oil refinery, coal conversion, coking plant, leather, textiles, pharmaceutical, etc (Arutchelvan et al., 2006). Because of their toxicity and persistence, they may be bioaccumulated and biomagnified in the food chain, and have adverse effect on aquatic life, plants and humans by inducing carcinogenicity and causing reproductive and developmental toxicity, neurotoxicity and acute toxicity (Paisio et al., 2013). Hence, phenol is one of the most important pollutants in the environment and considered as priority pollutants for control by many countries. In order to removal phenol, many microorganisms have been found to degrade phenol as a carbon and energy source (van Schie and Young, 2000), such as Pseudomonas putida (Movahedyan et al., 2009), and Pseudomonas aeruginosa (Wang et al., 2011). Moreover, the bioremediation of phenol contaminated sites has been performed with different bacterial
* Corresponding authors. Tel.: þ86 21 64253389; fax: þ86 21 64253988. E-mail addresses: [email protected] (H. Li), [email protected] (Y.-d. Liu). http://dx.doi.org/10.1016/j.ibiod.2014.07.003 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
species, such as Acinetobacter sp. and Bacillus cereus (Banerjee and Ghoshal, 2010). However, when pollution occurred in the high salinity environment, the biodegradation of the phenol often faces greater difficulties due to the high salinity, which inhibits the growth of microorganisms. Halophilic bacteria were isolated for degradation of phenol in the presence of high concentration of salts to improve the microbial activity and degrading efficiency (Afzal et al., 2007). In previous study, some halophilic bacterial were enriched to utilize phenol at concentrations of 320 mg/L in medium containing 10% NaCl (Peyton et al., 2002). Recently, more halophilic bacteria have been isolated from different saline environments to degrade phenol in hypersaline media, such as Halomonas organivorans, Arhodomo et al., 2013). In nas aquaeolei and Modicisalibacter tunisiensis (Bonfa particular, moderate halophiles are considered the most versatile group with a great potential for biological decontamination over a very wide range of salinity (de Lourdes Moreno et al., 2011) by members in the genus Halomonas (García et al., 2004). Normally, majority of the moderate halophiles employ the compatible solute to maintain the osmotic balance inside the microbial cells in saline environments (Shivanand and Mugeraya, 2011). Compatible solutes consist of a series of organic compounds synthesized and accumulated by halophilic bacteria, including betaine and ectoines
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(ectoine and hydroxyectoine), sugars (trehalose and sucrose), amino acids (glycine, alanine, proline, etc), polyols (glycerol, mannitol, sorbitol) (Brill et al., 2011). They play an important role in the mechanism of salt tolerance and resistance in halophiles to higher concentrations of saline. Up to now, only a limited reports documented the degradation of phenol by moderately halophilic bacterial community over a wide range of salinity. This study enriched a moderately halophilic bacterial community in degrading high concentration of phenol over a wide range of salinity (1e3 M). In addition, the dominant species of the microbial community, the key functional genes in the phenol-degrading pathway and the molecular mechanism of salinity tolerance of the bacterial community were revealed. 2. Materials and methods 2.1. Acclimating moderately halophilic bacterial community The upper sediment layers were collected from Qarhan Salt Lake using sterilized spatulas for the aerobic study. The samples were placed in sterile mason jars with ample headspace, and delivered to laboratory at 4 C. Microcosms were then prepared by using 10 g of sediment sample and 40 mL of autoclaved mineral salts medium (MSM) with pH 6.9 in serum bottles (125 mL capacity), amended with phenol as the sole carbon source in a range of salinity. Bottles were closed with butyl rubber stopper and shaken at 120 rpm in environmental chamber kept dark at 30 C. The enrichment cultures were propogated after showing significant degradation of the added phenol. The culture mediums were repeatedly amended with phenol 5 times to acclimatize the phenol-degrading bacteria, and the enrichments were transferred 4 times to obtain the sediment-free enrichment cultures. Then 30% of the slurry was aseptically transferred to freshly made and sterile MSM containing 1, 1.5, 2, 2.5 and 3 M of NaCl, respectively. 2.2. Phenol degradation assay The ability of the consortium to utilize phenol as the sole carbon source was determined by inoculating it into the mineral medium containing different concentrations of phenol from 50 to 350 mg/L. The cell suspensions were separated by centrifugation at 10,000 g for 15 min. The culture supernatant was filtered through a 0.45-mmpore-size filter for subsequent phenol quantification. The concentration of phenol was quantified by high-performance liquid chromatography (HPLC SHIMADZU) using a C18 column (4.6 250 mm). The mobile phase was composed of methanol and water (50:50, v/v) and the flow rate was 0.8 mL/min. Detection was made at 270 nm with a variable-wavelength UV detector, and quantification was made by peak integration using external standards. 2.3. Total DNA extraction, DGGE analysis and sequencing The total DNA from cells was extracted by a Fast DNA spin kit (ABigen, Beijing, China). The partial 16S rRNA genes were amplified by PCR using the universal primers 968F-GC and 1401R. PCR amplifications were performed in a 50 mL of reaction volume that contained 1 mL of template, 2 mL of each primer, 25 mL of PCR Taqmix, 20 mL of ddH2O. The PCR condition included: an initial denaturation at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C for 1 min, annealing at 55 C for 45 s, and extension at 72 C for 1 min, and a final extension at 72 C for 10 min. The PCR reactions were performed with a Mastercyle gradient (Eppendorf, US). The PCR products were run on a 1.0% agarose gel. The digital
images were obtained with a G:BOX system (Syngene, Cambridge, UK). DGGE analysis was performed on a DCode System (Bio-Rad, CA, US). PCR amplicons were loaded onto a 6% polyacrylamide gel with a denaturing acrylamide gradient ranging from 30% to 50%. A 100% denaturant solution was defined as 42 g of urea, 2 mL of 50 TAE buffer and 15 mL of 40% (v/v) acrylamide/bis-acrylamide (37.5:1), in 100 mL of DDW. Zero % denaturant solution was defined as 2 mL of 50 TAE buffer, 15 mL of 40% (v/v) acrylamide/bis-acrylamide, and 83 mL of DDW. The samples were electrophoresed at 200 V and 60 C for 6 h. The dominant bands were excised with a sterile knife blade and soaked in 40 mL of TE buffer overnight, and then 1 mL solution was used as the template for PCR amplification. The amplicons were purified by the DNA purification kit (V-gene, Shanghai, China) and then ligated to pMD 19-T vector according to the manufacturer's instructions (TaKaRa, Dalian, China). The combined plasmids were transformed into Escherichia coli DH5a. The insertion of 16S rRNA gene was retrieved by PCR amplification with the primer set of M1347 (50 -CGC CAG GGT TTT CCC AGT CAC GAC-30 ) and RV-M (50 -GAG CGG ATA ACA ATT TCA CAC AGG-30 ). The PCR products were sequenced (Sangon, Shanghai, China). After editing and checking sequences manually, the typical ones were identified the closest relatives by the BLAST software in GenBank database of NCBI (http:// www.ncbi.nlm.nih.gov). Phylogenetic trees were constructed by using the programs Clustal X (1.8) and Molecular Evolutionary Genetics Analysis (MEGA, version 5.05). Robustness for individual branches was estimated by bootstrapping based on 1000 replications. 2.4. Detection of functional genes involved in phenol biodegradation To detect the presence of three catabolic genes encoding key enzymes of the phenol metabolic pathways, PCR amplification from total DNA was performed using primers for phenol hydroxylase gene (Lph), catechol 1,2-dioxygenase (1,2-CTD) and catechol 2,3-dioxygenase (2,3-CTD). The genes encoding these enzymes were amplified by using the primers sets: Lphf (50 -CGCCAGAACCATTTATCGATC-30 ), Lphr (50 -AGGCATCAAGATCACCGACTG-30 ) (Xu et al., 2001); 1,2-CTDf (50 ACCATCGARGGYCCSCTSTAY-30 ), 1,2-CTDr (50 -GTTRATCTGGGTGGTSAG-30 ); and 2,3-CTDf (50 -GARCTSTAYGCSGAYAAGGAR-30 ), 2,3-CTDr (50 -RCCGCTSGGRTCGAAGAARTA-30 ) (García et al., 2006). 2.5. Compatible solutes determination with NMR method The enrichment culture was maintained at 30 C on MSM containing 0.2% yeast extract, 0.4% glucose and 15% NaCl. Cell growth was monitored by measuring turbidity at 600 nm spectrophotometrically. The cells were harvested by centrifugation (4000 g, 15 min) at late exponential phase and washed with isoosmotic solutions. The wet cell pellets were extracted the intracellular solutes, and the extraction was repeated twice then the solvent was removed by rotary evaporation. The residue was dissolved in CHCl3:CH3OH:H2O (5:10:4, v/v) until lipid components were eliminated (Li et al., 2012). The products were purified by a freeze dryer, and then suspended in D2O for detection of 1H NMR at 298 K on a Bruker Ultrashield 400 spectrometer operating at 400 MHz, using an inverse multinuclear probehead fitted with gradient along the z axis (Motta et al., 2004). 2.6. Nucleotide sequence accession numbers The GenBank accession numbers for bacterial 16S rRNA gene sequences are KC899112eKC899114, and for functional gene are KC899111, KF017271eKF017272.
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3. Results and discussion 3.1. Enrichment of moderately halophilic bacterial community to degrade phenol After acclimated for 50 d, the enrichment was able to degraded phenol as the sole carbon source in MSM at a wide range of salt concentrations from 1 to 3 M (Fig. 1). The enrichment almost completely degraded 120 mg/L of phenol in less than 2 d in the presence of 1 or 1.5 M of NaCl, while roughly 3 d with 2 M of NaCl. It took 5 d to degrade 100 mg/L of phenol at higher salinity from 2.5 to 3 M of NaCl. Then the enrichment was acclimated for degradation of phenol from 50 to 350 mg/L of phenol at 1.5 M NaCl (Fig. 2) in 2 months. At the beginning, 50 mg/L of phenol were almost completely removed in 7 d. Through acclimation, the enrichment was able to completely degrade 150 mg/L of phenol in 3 d. Finally, the optimum degradation was achieved at 1.5 M of NaCl and 350 mg/L of phenol. The ability of the enrichment to degrade phenol under saline condition has potentially significant application, because phenol contamination can be found in saline-alkali soils or marine or brackish waters at oil exploration and industrial effluent discharging sites. This study showed that the ability of moderately halophilic microbial community to metabolize high concentration of phenol as the sole carbon source at a wide range of salinity from 1 to 3 M of NaCl. Though a few reports have documented the ability of halophiles or halotolerants in degrading hydrocarbons, including phenolic compounds (Abdelkafi et al., 2005; Afzal et al., 2007), the biodegradation of phenol by microbial communities over such a wide range of salinities has not been reported before. Gayathri and Vasudevan (2010) constructed a moderately halophilic bacterial consortium to degrade 50 mg/L of phenol at a range of salt concentrations from 10 to 150 g/L of NaCl (Gayathri and Vasudevan, 2010). Our study enriched a bacterial consortium to degrade higher concentration of phenol (350 mg/L) than previous study in a wider salinity (1e3 M of NaCl). In addition, the degradation rate
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and the tolerance of phenol by the enrichments were improved by continuous acclimation, and the degradation process was notably shortened. This study illustrated that the enrichments had advantage on not only tolerance of salinity and phenol concentration, but also high degradation rate, which may contribute to bio-remediate phenol as pollutant in the natural saline environments. 3.2. Moderately halophilic bacterial community structure The community structure of the enrichment growing on phenol at various salt concentration levels (1e3 M of NaCl) was investigated by PCR-DGGE (Fig. 3). BLAST analysis showed that the sequence of band 1, 2 and 3 had a high similarity of 98% to Acidobacterium sp. sw-xj59 (GQ302569), 96% to Chloroflexus sp. YNPSBC-BP4-B21 (HM448255.1), and 96% to Chloroflexi strain S26-95 (EU287395.1), respectively. Phylogenetic tree showed the dominant species in the enrichments were distributed in two phyla: Acidobacteria and Chloroflexi (Fig. 4), which might be the major functional populations in the bacterial communities. Acidobacterium sp. existed in 1, 1.5, 2 and 3 M of NaCl enrichments, indicating that this strain was dominant in the phenol-degrading enrichments. Previously, some members of Acidobacteria genus also were found as the most abundant groups in the wastewater treatment plants (Ahn et al., 2008), and in the BTEX contaminated groundwater (Lee et al., 2010). Chloroflexus sp. existed in all of the NaCl concentrations, which was the powerful PCB-degrading bacteria in contaminated marine sediments (Zanaroli et al., 2012) and bron et al., 2009). the major PAH degrader in contaminated soil (Ce The dominant species distribution indicated that the concentration of NaCl had no significant effects on bacterial community succession, the two species existed almost invariably at all the salinity conditions. Similarly, the species of Acidobacteria and Chloroflexi were also found in many geographically saline locations, such as the deep-sea sediments in Nankai Trough and the intertidal sediments in Yellow Sea (Shuang et al., 2006; Nunoura et al., 2012). Based on the adaptation by these strains to such a broad range of salinities, it indicated that they were most probably the moderately halophilic bacteria and with the ability to degrade the organic pollutants in the natural saline environments. 3.3. PCR detection of key functional genes in phenol-degrading pathway
Fig. 1. Biodegradation of phenol by the enrichment at wide salt concentrations including, -: 1 M NaCl, C: 1.5 M NaCl, :: 2 M NaCl, +: 2.5 M NaCl and A: 3 M NaCl. Microcosms containing 45 mL of mineral salts medium and different concentrations of NaCl were inoculated with 5 mL enrichment. No degradation occurred in autoclaved control bottles (;). Data were the mean of triplicate bottles, and bars indicate ± standard deviation.
In order to investigate the phenol-degrading pathway by the enrichment cultures, PCR detection of key functional genes in the biochemical pathway was performed by using three primer sets for targeting nucleotide sequences encoding for phenol hydroxylase involved in the initial oxidation of phenol, and catechol 1,2dioxygenase and catechol 2,3-dioxygenase involved in the aromatic ring meta-cleavage (García et al., 2006; Tuan et al., 2011). The PCR amplification of the three expected gene sequence fragments indicated the bacterial consortium possessed all three functional genes belonging to the catabolic pathway for phenol degradation. The BLAST analysis revealed that the sequences had 77%, 81%, 77% similarity to Lph, 1,2-CTD and 2,3-CTD genes referred to phenol degradation pathway, respectively (Table 1). The catechol 1,2dioxygenase and catechol 2,3-dioxygenase are the two major enzymes in the ring-cleavage step via ortho-pathway and metapathway (Tuan et al., 2011). Many strains degrade phenol by these two pathways, such as P. putida via catechol 2,3-dioxygenase, and Acinetobacter calcoaceticus ADP1 via catechol 1,2-dioxygenase (García et al., 2006). The initial oxidative step of phenol is the key metabolic reaction and the phenol hydroxylase plays an important role in the hydroxylation of aromatic ring during phenol degradation. Identification of these PCR products by cloning and
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Fig. 2. The enrichment was continuously acclimated the ability of biodegrading phenol (:) at 1.5 M NaCl in 2 months, incubated at 30 C. No degradation of phenol occurred in autoclaved control bottles (C). Data were the mean of three active bottles and two control bottles.
sequencing revealed that the enrichment in this study contained all the known pathways for phenol degradation, including phenol hydroxylase gene, catechol 1,2-dioxygenase gene and catechol 2,3dioxygenase gene, indicating the high efficiency and diversity of the phenol biodegradation by this enrichment culture.
3.4. Osmoprotection mechanism of the enriched bacterial community Halophilic bacteria usually synthesize or accumulate compatible solutes to maintain the osmotic equilibrium in response to the external high-salt condition. Compatible solutes are a series of low molecular organic solutes without a defined chemical class, being sugars, primary and secondary amino acids, polyols, organic sulfur compounds and phosphates. To identify the mechanism of salinity tolerance of the enriched bacterial community, the observed chemical shifts in NMR spectrogram peaks were compared with reference data (Motta et al., 2004). The NMR spectrogram showed that the compatible solutes were mainly ectoine and hydroxyectoine (Fig. 5). In previous studies, both ectoine and hydroxyectoine were reported as the main compatible solutes in the halophilic bacteria (Shivanand and Mugeraya, 2011). It was first discovered in Ectothiorhodospira halochloris, later a great variety of halophilic and halotolerant bacteria were found to produce this compound, often together with its 5-hydroxy derivative (Rothschild and Mancinelli, 2002). Up to now, a numbers of ectoines and its derivant have been detected in moderately halophilic bacteria such as Methylarcula marina (Roberts, 2005) and Halobacillus dabanensis (Zhao et al., 2006). This study showed that the phenol-degrading bacterial consortium adjusts the osmotic pressure by compatible solutes mainly ectoine and hydroxyectoine to allow them to grow in high salinity so to adapt the consortium to a wide range of salt concentrations (1e3 M of NaCl). 4. Conclusion
Fig. 3. DGGE gel image of amplicons of V6 partial sequence of bacterial 16S rDNA from the microcosms amended with 1e3 M NaCl. Each microcosm was filled with 50 mL of MSM amended phenol.
This study firstly enriched a bacterial consortium to degrade high concentration of phenol over a range of salinity (1e3 M of NaCl). The dominant bacteria included Acidobacterium sp.,
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Fig. 4. Phylogenetic tree of the V6 partial sequence of bacterial 16S rDNA from the microcosms amended with 1e3 M NaCl shown in boldface and the referred sequences in the EMBL database with the putative divisions listed to the right. The topology shown was calculated with the Neighbor-joining method. Bootstrap values n ¼ 1000 replicates of 50% were reported near the corresponding nodes. The scale bar represented 0.20 nuclear acid substitutions per nucleotide position.
Table 1 The BLAST results of phenol degradation gene sequences. Primer
Amplicon size (bp)
The most relatives sequences
Similarity (%)
Lph
575
77
1,2-CTD
388
2,3-CTD
417
Alcaligenes faecalis phenol-degradation regulator protein, phenol hydroxylase subunit (EF540866.1) Halomonas organivorans partial catA gene for catechol 1,2-dioxygenase (FN997643.1) Arhodomonas sp. Seminole catechol 2,3-dioxygenase gene, complete cds (JX311713.1)
81
77
Chloroflexus sp. and Chloroflexus sp. three dominant members by 16S rRNA gene sequence analysis. The moderately halophilic bacterial consortium was able to rapidly degrade 100e120 mg/L of phenol at different salt concentrations. The optimum degradation was achieved at 1.5 M of NaCl and 350 mg/L phenol via the catalysis steps of phenol hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase. The salt-tolerant mechanism of the bacterial consortium mainly depended on ectoine and hydroxyectoine. These observations contribute to information for bioremediation of phenol contamination in various saline environments. Acknowledgments Supported jointly by National Natural Science Foundation of China (51378208, 41273109, 41003031), Specialized Research Fund for the Doctoral Program of Higher Education (20110074130002), Program for New Century Excellent Talents in University (NCET-130797), Fok Ying Tung Education Foundation (141077), Shanghai Rising-Star Program (12QA1400800), Innovation Program of Shanghai Municipal Education Commission (14ZZ059), Fundamental Research Funds for the Central Universities (222201313008). We also would like to thank the anonymous referees for their helpful comments on this paper. References
Fig. 5. 1H spectrum of extraction of the enrichment grown in MSM with 0.4% glucose and 0.2% yeast extract with 15% NaCl.
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Community analysis and metabolic pathway of halophilic bacteria for phenol degradation in saline environment Zhong-zi Huang a, b, Ping Wang c, Hui Li a, *, Kuang-fei Lin a, Zhi-yan Lu a, Xiao-jue Guo a, Yong-di Liu a, * a
State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, State Key Laboratory of Biological Reactor Engineering, School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, PR China School of Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, PR China c School of Bioengineering, East China University of Science and Technology, Shanghai 200237, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 April 2014 Received in revised form 9 July 2014 Accepted 9 July 2014 Available online
A moderately halophilic bacterial enrichment was able to degrade 120 mg/L of phenol in the presence of 1e2 M of NaCl within 3 d or 2.5e3 M of NaCl within 6 d. The optimal degradation was achieved at 1.5 M of NaCl and 350 mg/L of phenol. PCR-DGGE profile of the enrichment showed that the Acidobacterium sp. and Chloroflexus sp. dominated the community. The phenol-biodegradation pathways consisted of an initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase and catechol 2,3-dioxygenase. Nuclear magnetic resonance (NMR) spectroscopy profiles showed that ectoine and hydroxyectoine were the main compatible solutes to adjust the bacterial osmotic pressure. This study provides further information on the understanding of phenol-degradation over a wide range of salinity and remediation of phenol as a pollutant in the environment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Phenol Biodegradation Moderately halophilics Metabolic pathway Compatible solutes
1. Introduction Phenol and phenolic compounds are hazardous pollutants widespread in the environment through wastewater discharges from a variety of industries including phenol-formaldehyde resin, oil refinery, coal conversion, coking plant, leather, textiles, pharmaceutical, etc (Arutchelvan et al., 2006). Because of their toxicity and persistence, they may be bioaccumulated and biomagnified in the food chain, and have adverse effect on aquatic life, plants and humans by inducing carcinogenicity and causing reproductive and developmental toxicity, neurotoxicity and acute toxicity (Paisio et al., 2013). Hence, phenol is one of the most important pollutants in the environment and considered as priority pollutants for control by many countries. In order to removal phenol, many microorganisms have been found to degrade phenol as a carbon and energy source (van Schie and Young, 2000), such as Pseudomonas putida (Movahedyan et al., 2009), and Pseudomonas aeruginosa (Wang et al., 2011). Moreover, the bioremediation of phenol contaminated sites has been performed with different bacterial
* Corresponding authors. Tel.: þ86 21 64253389; fax: þ86 21 64253988. E-mail addresses: [email protected] (H. Li), [email protected] (Y.-d. Liu). http://dx.doi.org/10.1016/j.ibiod.2014.07.003 0964-8305/© 2014 Elsevier Ltd. All rights reserved.
species, such as Acinetobacter sp. and Bacillus cereus (Banerjee and Ghoshal, 2010). However, when pollution occurred in the high salinity environment, the biodegradation of the phenol often faces greater difficulties due to the high salinity, which inhibits the growth of microorganisms. Halophilic bacteria were isolated for degradation of phenol in the presence of high concentration of salts to improve the microbial activity and degrading efficiency (Afzal et al., 2007). In previous study, some halophilic bacterial were enriched to utilize phenol at concentrations of 320 mg/L in medium containing 10% NaCl (Peyton et al., 2002). Recently, more halophilic bacteria have been isolated from different saline environments to degrade phenol in hypersaline media, such as Halomonas organivorans, Arhodomo et al., 2013). In nas aquaeolei and Modicisalibacter tunisiensis (Bonfa particular, moderate halophiles are considered the most versatile group with a great potential for biological decontamination over a very wide range of salinity (de Lourdes Moreno et al., 2011) by members in the genus Halomonas (García et al., 2004). Normally, majority of the moderate halophiles employ the compatible solute to maintain the osmotic balance inside the microbial cells in saline environments (Shivanand and Mugeraya, 2011). Compatible solutes consist of a series of organic compounds synthesized and accumulated by halophilic bacteria, including betaine and ectoines
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(ectoine and hydroxyectoine), sugars (trehalose and sucrose), amino acids (glycine, alanine, proline, etc), polyols (glycerol, mannitol, sorbitol) (Brill et al., 2011). They play an important role in the mechanism of salt tolerance and resistance in halophiles to higher concentrations of saline. Up to now, only a limited reports documented the degradation of phenol by moderately halophilic bacterial community over a wide range of salinity. This study enriched a moderately halophilic bacterial community in degrading high concentration of phenol over a wide range of salinity (1e3 M). In addition, the dominant species of the microbial community, the key functional genes in the phenol-degrading pathway and the molecular mechanism of salinity tolerance of the bacterial community were revealed. 2. Materials and methods 2.1. Acclimating moderately halophilic bacterial community The upper sediment layers were collected from Qarhan Salt Lake using sterilized spatulas for the aerobic study. The samples were placed in sterile mason jars with ample headspace, and delivered to laboratory at 4 C. Microcosms were then prepared by using 10 g of sediment sample and 40 mL of autoclaved mineral salts medium (MSM) with pH 6.9 in serum bottles (125 mL capacity), amended with phenol as the sole carbon source in a range of salinity. Bottles were closed with butyl rubber stopper and shaken at 120 rpm in environmental chamber kept dark at 30 C. The enrichment cultures were propogated after showing significant degradation of the added phenol. The culture mediums were repeatedly amended with phenol 5 times to acclimatize the phenol-degrading bacteria, and the enrichments were transferred 4 times to obtain the sediment-free enrichment cultures. Then 30% of the slurry was aseptically transferred to freshly made and sterile MSM containing 1, 1.5, 2, 2.5 and 3 M of NaCl, respectively. 2.2. Phenol degradation assay The ability of the consortium to utilize phenol as the sole carbon source was determined by inoculating it into the mineral medium containing different concentrations of phenol from 50 to 350 mg/L. The cell suspensions were separated by centrifugation at 10,000 g for 15 min. The culture supernatant was filtered through a 0.45-mmpore-size filter for subsequent phenol quantification. The concentration of phenol was quantified by high-performance liquid chromatography (HPLC SHIMADZU) using a C18 column (4.6 250 mm). The mobile phase was composed of methanol and water (50:50, v/v) and the flow rate was 0.8 mL/min. Detection was made at 270 nm with a variable-wavelength UV detector, and quantification was made by peak integration using external standards. 2.3. Total DNA extraction, DGGE analysis and sequencing The total DNA from cells was extracted by a Fast DNA spin kit (ABigen, Beijing, China). The partial 16S rRNA genes were amplified by PCR using the universal primers 968F-GC and 1401R. PCR amplifications were performed in a 50 mL of reaction volume that contained 1 mL of template, 2 mL of each primer, 25 mL of PCR Taqmix, 20 mL of ddH2O. The PCR condition included: an initial denaturation at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C for 1 min, annealing at 55 C for 45 s, and extension at 72 C for 1 min, and a final extension at 72 C for 10 min. The PCR reactions were performed with a Mastercyle gradient (Eppendorf, US). The PCR products were run on a 1.0% agarose gel. The digital
images were obtained with a G:BOX system (Syngene, Cambridge, UK). DGGE analysis was performed on a DCode System (Bio-Rad, CA, US). PCR amplicons were loaded onto a 6% polyacrylamide gel with a denaturing acrylamide gradient ranging from 30% to 50%. A 100% denaturant solution was defined as 42 g of urea, 2 mL of 50 TAE buffer and 15 mL of 40% (v/v) acrylamide/bis-acrylamide (37.5:1), in 100 mL of DDW. Zero % denaturant solution was defined as 2 mL of 50 TAE buffer, 15 mL of 40% (v/v) acrylamide/bis-acrylamide, and 83 mL of DDW. The samples were electrophoresed at 200 V and 60 C for 6 h. The dominant bands were excised with a sterile knife blade and soaked in 40 mL of TE buffer overnight, and then 1 mL solution was used as the template for PCR amplification. The amplicons were purified by the DNA purification kit (V-gene, Shanghai, China) and then ligated to pMD 19-T vector according to the manufacturer's instructions (TaKaRa, Dalian, China). The combined plasmids were transformed into Escherichia coli DH5a. The insertion of 16S rRNA gene was retrieved by PCR amplification with the primer set of M1347 (50 -CGC CAG GGT TTT CCC AGT CAC GAC-30 ) and RV-M (50 -GAG CGG ATA ACA ATT TCA CAC AGG-30 ). The PCR products were sequenced (Sangon, Shanghai, China). After editing and checking sequences manually, the typical ones were identified the closest relatives by the BLAST software in GenBank database of NCBI (http:// www.ncbi.nlm.nih.gov). Phylogenetic trees were constructed by using the programs Clustal X (1.8) and Molecular Evolutionary Genetics Analysis (MEGA, version 5.05). Robustness for individual branches was estimated by bootstrapping based on 1000 replications. 2.4. Detection of functional genes involved in phenol biodegradation To detect the presence of three catabolic genes encoding key enzymes of the phenol metabolic pathways, PCR amplification from total DNA was performed using primers for phenol hydroxylase gene (Lph), catechol 1,2-dioxygenase (1,2-CTD) and catechol 2,3-dioxygenase (2,3-CTD). The genes encoding these enzymes were amplified by using the primers sets: Lphf (50 -CGCCAGAACCATTTATCGATC-30 ), Lphr (50 -AGGCATCAAGATCACCGACTG-30 ) (Xu et al., 2001); 1,2-CTDf (50 ACCATCGARGGYCCSCTSTAY-30 ), 1,2-CTDr (50 -GTTRATCTGGGTGGTSAG-30 ); and 2,3-CTDf (50 -GARCTSTAYGCSGAYAAGGAR-30 ), 2,3-CTDr (50 -RCCGCTSGGRTCGAAGAARTA-30 ) (García et al., 2006). 2.5. Compatible solutes determination with NMR method The enrichment culture was maintained at 30 C on MSM containing 0.2% yeast extract, 0.4% glucose and 15% NaCl. Cell growth was monitored by measuring turbidity at 600 nm spectrophotometrically. The cells were harvested by centrifugation (4000 g, 15 min) at late exponential phase and washed with isoosmotic solutions. The wet cell pellets were extracted the intracellular solutes, and the extraction was repeated twice then the solvent was removed by rotary evaporation. The residue was dissolved in CHCl3:CH3OH:H2O (5:10:4, v/v) until lipid components were eliminated (Li et al., 2012). The products were purified by a freeze dryer, and then suspended in D2O for detection of 1H NMR at 298 K on a Bruker Ultrashield 400 spectrometer operating at 400 MHz, using an inverse multinuclear probehead fitted with gradient along the z axis (Motta et al., 2004). 2.6. Nucleotide sequence accession numbers The GenBank accession numbers for bacterial 16S rRNA gene sequences are KC899112eKC899114, and for functional gene are KC899111, KF017271eKF017272.
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3. Results and discussion 3.1. Enrichment of moderately halophilic bacterial community to degrade phenol After acclimated for 50 d, the enrichment was able to degraded phenol as the sole carbon source in MSM at a wide range of salt concentrations from 1 to 3 M (Fig. 1). The enrichment almost completely degraded 120 mg/L of phenol in less than 2 d in the presence of 1 or 1.5 M of NaCl, while roughly 3 d with 2 M of NaCl. It took 5 d to degrade 100 mg/L of phenol at higher salinity from 2.5 to 3 M of NaCl. Then the enrichment was acclimated for degradation of phenol from 50 to 350 mg/L of phenol at 1.5 M NaCl (Fig. 2) in 2 months. At the beginning, 50 mg/L of phenol were almost completely removed in 7 d. Through acclimation, the enrichment was able to completely degrade 150 mg/L of phenol in 3 d. Finally, the optimum degradation was achieved at 1.5 M of NaCl and 350 mg/L of phenol. The ability of the enrichment to degrade phenol under saline condition has potentially significant application, because phenol contamination can be found in saline-alkali soils or marine or brackish waters at oil exploration and industrial effluent discharging sites. This study showed that the ability of moderately halophilic microbial community to metabolize high concentration of phenol as the sole carbon source at a wide range of salinity from 1 to 3 M of NaCl. Though a few reports have documented the ability of halophiles or halotolerants in degrading hydrocarbons, including phenolic compounds (Abdelkafi et al., 2005; Afzal et al., 2007), the biodegradation of phenol by microbial communities over such a wide range of salinities has not been reported before. Gayathri and Vasudevan (2010) constructed a moderately halophilic bacterial consortium to degrade 50 mg/L of phenol at a range of salt concentrations from 10 to 150 g/L of NaCl (Gayathri and Vasudevan, 2010). Our study enriched a bacterial consortium to degrade higher concentration of phenol (350 mg/L) than previous study in a wider salinity (1e3 M of NaCl). In addition, the degradation rate
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and the tolerance of phenol by the enrichments were improved by continuous acclimation, and the degradation process was notably shortened. This study illustrated that the enrichments had advantage on not only tolerance of salinity and phenol concentration, but also high degradation rate, which may contribute to bio-remediate phenol as pollutant in the natural saline environments. 3.2. Moderately halophilic bacterial community structure The community structure of the enrichment growing on phenol at various salt concentration levels (1e3 M of NaCl) was investigated by PCR-DGGE (Fig. 3). BLAST analysis showed that the sequence of band 1, 2 and 3 had a high similarity of 98% to Acidobacterium sp. sw-xj59 (GQ302569), 96% to Chloroflexus sp. YNPSBC-BP4-B21 (HM448255.1), and 96% to Chloroflexi strain S26-95 (EU287395.1), respectively. Phylogenetic tree showed the dominant species in the enrichments were distributed in two phyla: Acidobacteria and Chloroflexi (Fig. 4), which might be the major functional populations in the bacterial communities. Acidobacterium sp. existed in 1, 1.5, 2 and 3 M of NaCl enrichments, indicating that this strain was dominant in the phenol-degrading enrichments. Previously, some members of Acidobacteria genus also were found as the most abundant groups in the wastewater treatment plants (Ahn et al., 2008), and in the BTEX contaminated groundwater (Lee et al., 2010). Chloroflexus sp. existed in all of the NaCl concentrations, which was the powerful PCB-degrading bacteria in contaminated marine sediments (Zanaroli et al., 2012) and bron et al., 2009). the major PAH degrader in contaminated soil (Ce The dominant species distribution indicated that the concentration of NaCl had no significant effects on bacterial community succession, the two species existed almost invariably at all the salinity conditions. Similarly, the species of Acidobacteria and Chloroflexi were also found in many geographically saline locations, such as the deep-sea sediments in Nankai Trough and the intertidal sediments in Yellow Sea (Shuang et al., 2006; Nunoura et al., 2012). Based on the adaptation by these strains to such a broad range of salinities, it indicated that they were most probably the moderately halophilic bacteria and with the ability to degrade the organic pollutants in the natural saline environments. 3.3. PCR detection of key functional genes in phenol-degrading pathway
Fig. 1. Biodegradation of phenol by the enrichment at wide salt concentrations including, -: 1 M NaCl, C: 1.5 M NaCl, :: 2 M NaCl, +: 2.5 M NaCl and A: 3 M NaCl. Microcosms containing 45 mL of mineral salts medium and different concentrations of NaCl were inoculated with 5 mL enrichment. No degradation occurred in autoclaved control bottles (;). Data were the mean of triplicate bottles, and bars indicate ± standard deviation.
In order to investigate the phenol-degrading pathway by the enrichment cultures, PCR detection of key functional genes in the biochemical pathway was performed by using three primer sets for targeting nucleotide sequences encoding for phenol hydroxylase involved in the initial oxidation of phenol, and catechol 1,2dioxygenase and catechol 2,3-dioxygenase involved in the aromatic ring meta-cleavage (García et al., 2006; Tuan et al., 2011). The PCR amplification of the three expected gene sequence fragments indicated the bacterial consortium possessed all three functional genes belonging to the catabolic pathway for phenol degradation. The BLAST analysis revealed that the sequences had 77%, 81%, 77% similarity to Lph, 1,2-CTD and 2,3-CTD genes referred to phenol degradation pathway, respectively (Table 1). The catechol 1,2dioxygenase and catechol 2,3-dioxygenase are the two major enzymes in the ring-cleavage step via ortho-pathway and metapathway (Tuan et al., 2011). Many strains degrade phenol by these two pathways, such as P. putida via catechol 2,3-dioxygenase, and Acinetobacter calcoaceticus ADP1 via catechol 1,2-dioxygenase (García et al., 2006). The initial oxidative step of phenol is the key metabolic reaction and the phenol hydroxylase plays an important role in the hydroxylation of aromatic ring during phenol degradation. Identification of these PCR products by cloning and
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Fig. 2. The enrichment was continuously acclimated the ability of biodegrading phenol (:) at 1.5 M NaCl in 2 months, incubated at 30 C. No degradation of phenol occurred in autoclaved control bottles (C). Data were the mean of three active bottles and two control bottles.
sequencing revealed that the enrichment in this study contained all the known pathways for phenol degradation, including phenol hydroxylase gene, catechol 1,2-dioxygenase gene and catechol 2,3dioxygenase gene, indicating the high efficiency and diversity of the phenol biodegradation by this enrichment culture.
3.4. Osmoprotection mechanism of the enriched bacterial community Halophilic bacteria usually synthesize or accumulate compatible solutes to maintain the osmotic equilibrium in response to the external high-salt condition. Compatible solutes are a series of low molecular organic solutes without a defined chemical class, being sugars, primary and secondary amino acids, polyols, organic sulfur compounds and phosphates. To identify the mechanism of salinity tolerance of the enriched bacterial community, the observed chemical shifts in NMR spectrogram peaks were compared with reference data (Motta et al., 2004). The NMR spectrogram showed that the compatible solutes were mainly ectoine and hydroxyectoine (Fig. 5). In previous studies, both ectoine and hydroxyectoine were reported as the main compatible solutes in the halophilic bacteria (Shivanand and Mugeraya, 2011). It was first discovered in Ectothiorhodospira halochloris, later a great variety of halophilic and halotolerant bacteria were found to produce this compound, often together with its 5-hydroxy derivative (Rothschild and Mancinelli, 2002). Up to now, a numbers of ectoines and its derivant have been detected in moderately halophilic bacteria such as Methylarcula marina (Roberts, 2005) and Halobacillus dabanensis (Zhao et al., 2006). This study showed that the phenol-degrading bacterial consortium adjusts the osmotic pressure by compatible solutes mainly ectoine and hydroxyectoine to allow them to grow in high salinity so to adapt the consortium to a wide range of salt concentrations (1e3 M of NaCl). 4. Conclusion
Fig. 3. DGGE gel image of amplicons of V6 partial sequence of bacterial 16S rDNA from the microcosms amended with 1e3 M NaCl. Each microcosm was filled with 50 mL of MSM amended phenol.
This study firstly enriched a bacterial consortium to degrade high concentration of phenol over a range of salinity (1e3 M of NaCl). The dominant bacteria included Acidobacterium sp.,
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Fig. 4. Phylogenetic tree of the V6 partial sequence of bacterial 16S rDNA from the microcosms amended with 1e3 M NaCl shown in boldface and the referred sequences in the EMBL database with the putative divisions listed to the right. The topology shown was calculated with the Neighbor-joining method. Bootstrap values n ¼ 1000 replicates of 50% were reported near the corresponding nodes. The scale bar represented 0.20 nuclear acid substitutions per nucleotide position.
Table 1 The BLAST results of phenol degradation gene sequences. Primer
Amplicon size (bp)
The most relatives sequences
Similarity (%)
Lph
575
77
1,2-CTD
388
2,3-CTD
417
Alcaligenes faecalis phenol-degradation regulator protein, phenol hydroxylase subunit (EF540866.1) Halomonas organivorans partial catA gene for catechol 1,2-dioxygenase (FN997643.1) Arhodomonas sp. Seminole catechol 2,3-dioxygenase gene, complete cds (JX311713.1)
81
77
Chloroflexus sp. and Chloroflexus sp. three dominant members by 16S rRNA gene sequence analysis. The moderately halophilic bacterial consortium was able to rapidly degrade 100e120 mg/L of phenol at different salt concentrations. The optimum degradation was achieved at 1.5 M of NaCl and 350 mg/L phenol via the catalysis steps of phenol hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase. The salt-tolerant mechanism of the bacterial consortium mainly depended on ectoine and hydroxyectoine. These observations contribute to information for bioremediation of phenol contamination in various saline environments. Acknowledgments Supported jointly by National Natural Science Foundation of China (51378208, 41273109, 41003031), Specialized Research Fund for the Doctoral Program of Higher Education (20110074130002), Program for New Century Excellent Talents in University (NCET-130797), Fok Ying Tung Education Foundation (141077), Shanghai Rising-Star Program (12QA1400800), Innovation Program of Shanghai Municipal Education Commission (14ZZ059), Fundamental Research Funds for the Central Universities (222201313008). We also would like to thank the anonymous referees for their helpful comments on this paper. References
Fig. 5. 1H spectrum of extraction of the enrichment grown in MSM with 0.4% glucose and 0.2% yeast extract with 15% NaCl.
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