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Isolation and Characterization of Methanethiol-Producing Bacteria from Agricultural Soils
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Title: Isolation and characterization of methanethiol-producing bacteria from agricultural soils
Author: LIU Hui, SHI Cheng-Fei, WU Ting, JIA Qi-Na, ZHAO Juan, WANG Xin-Ming
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S1002-0160(17)60411-9 10.1016/S1002-0160(17)60411-9 NA
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Please cite this article as: LIU Hui, SHI Cheng-Fei, WU Ting, JIA Qi-Na, ZHAO Juan, WANG Xin-Ming, Isolation and characterization of methanethiol-producing bacteria from agricultural soils, Pedosphere (2017), 10.1016/S1002-0160(17)60411-9.
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ACCEPTED MANUSCRIPT PEDOSPHERE Pedosphere ISSN 1002-0160/CN 32-1315/P
doi:10.1016/S1002-0160(17)60411-9
Isolation and characterization of methanethiol-producing bacteria from agricultural soils LIU Hui1, SHI Cheng-Fei1, WU Ting1, 2, JIA Qi-Na1, ZHAO Juan1, WANG Xin-Ming2, 1
College of Environmental Science and Engineering, Anhui Normal University, Wuhu 241000, China State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China
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ABSTRACT Methanethiol-producing bacteria (MPB) were enriched and isolated from agricultural soils in a modified basal medium (MBM) containing methionine (Met) as the sole carbon sources. The isolates were identified as Bacillus sp. WH-R1, WH-R2 and WH-R3, Arthrobacter sp. SZLB-W3 and Delftia sp. CHZG-R4 based on cell morphology, physiological and biochemical characteristics and 16S rRNA sequence analysis. Delftia sp. CHZG-R4 was reported as a novel strain to produce methanethiol (MT) using Met as precursor, and had the most active MT-producing potential, with the production of 21.8 μg at 30 ˚C and pH 7.0. Its optimal MT production occurred at 35 ˚C and pH 6.0, with 51.3% of sulfur content in Met converting into MT. Under these conditions, its MT production was changed by the supply of both carbon and nitrogen sources. The addition of 2 g L-1 starch and 2 g L-1 sucrose, 2 g L-1 urea and 2 g L-1 potassium nitrate into the MBM promoted above 10% of the MT production, while the provision of 2 g L-1 ammonia sulfate and 2 g L-1 peptone decreased 16% and 87% of the MT production, respectively. This is the first study to report Delftia sp. CHZG-R4 capable of producing MT, which might provide useful information for the microbial mechanism of MT production from agricultural soils and also contribute to the limited knowledge about the function of Delftia sp. CHZG-R4. Key words: agricultural soils, Delftia sp., methanethiol (MT), Methanethiol-producing bacteria (MPB), methionine (Met) INTRODUCTION Volatile organic sulfur compounds (VOSCs) produced from terrestrial soils can have important effects on atmospheric chemistry and ecosystem-level processes. In the atmosphere, they play an important role in acid precipitation, cloud formation, and global warming and sulfur budget (Andreae and Crutzen, 1997; Charlson et al., 1987; Cox and Sheppard, 1980; Watts, 2000). Methanethiol (MT) is one important member of the VOSC group. As a very reactive gas in the atmosphere, it is readily oxidized to methanesulfonic acid and SO2, and subsequently gives a rise to acid rain (Hatakeyama and Aklmoto, 1983). As an important intermediate, it can be further transformed to dimethyl sulfide (DMS) and thus contribute to the formation of
Corresponding author. E-mail address: [email protected] (T. Wu); [email protected] (X. Wang)
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cloud-condensation nuclei (CCN) and the change of global climate (Drotar et al., 1987; Bentley and Chasteen, 2004). There is also evidence that MT together with other VOSCs can influence biogeochemical processes within soils, altering the rates of carbon cycling by serving as substrates for microbial metabolism, inhibiting microbial processes associated with the nitrogen cycle, and either stimulating or inhibiting the growth of specific microbial taxa, and thus influence the growth of plants (Bremner and Bundy, 1974; Insam and Seewald, 2010; Saari and Martikainen, 2003). As one major component of the VOSC mixtures evolved from soils, MT originates mainly from the metabolism of sulfur-containing amino acids such as cysteine and methionine (Met) by microorganisms (Higgins et al., 2006). Met can be firstly deaminated and then demethiolated to produce MT by methionine γ-lyase catalysis in microorganisms (Bonnarme et al., 2000; Kadota and Ishida, 1972; Lomans et al., 2002; Segal and Starkey, 1969; Tanaka et al., 1985). A great variety of microorganisms have been isolated and identified to involve in this reaction in food industry, biosolid degradation, and freshwater and saline water ecosystem, including Achrobacter sp., Aspergillus sp., Aspergillus oryzae, Fusarium culmorum, Streptomyces griseus, Clostridium sp., Escherichia coil, Pseudomonas sp., Pseudomonas putifaciens, Pseudomonas fluorescens, Pseudomonas perolens and so on (Bonnarme et al., 2000; Kadota and Ishida, 1972; Rappert and Müller, 2005; Segal and Starkey, 1969). The area of agricultural fields worldwide is about 4912 million hectares, accounting for about 37.4% of the global land area (FAO, 2013). However, only a few bacteria have been reported to produce MT in agricultural soils, which include Parasporobacterium paucivorans, Holophaga foetida, Sporobacterium olearium and Rhodobacter capsulatus (Rappert and Müller, 2005 and its referencein). It would be of great importance to find more MT-producing bacterial species to provide useful information for understanding the microbial mechanism of MT production from agricultural soils. The aim of this study was to isolate, identify and characterize bacteria that can use Met as the sole carbon sources to produce MT in agricultural soils. The influence of several factors including temperature, pH, and carbon and nitrogen sources on MT production were also investigated. MATERIALS AND METHODS Media Enrichment, isolation and cultivation were carried out with the following medium. The modified basal medium (MBM) was prepared according to the literature (de Souza and Yoch, 1995) with minor modifications: KH2PO4, 0.6 g L-1; K2HPO4, 0.9 g L-1; MgSO4·7H2O, 0.2 g L-1; CaCl2·2H2O, 0.075 g L-1; Fe-EDTA, 0.0295 g L-1; MnSO4, 0.1599 g L-1; H3BO4, 0.28 g L-1; CuSO4·5H2O, 0.0079 g L-1; ZnSO4·7H2O, 0.024 g L-1; NaMoO4·2H2O, 0.126 g L-1; (NH4)2SO4, 0.66 g L-1; NaCl, 1.17 g L-1; yeast extract, 0.05 g L-1; methionine (Met), 0.0597 g L-1 (0.4 mmol L-1); vitamin B12, 2 μg L-1; p-aminobenzoic acid, 10 μg L-1; riboflavin, 10 μg L-1; vitamin B6, 10 μg L-1; niacin, 10 μg L-1; vitamin C, 10 μg L-1; thiamine, 10 μg L-1; and D-(+)-biotin, 10 μg L-1. The final pH was adjusted to 7.0. Agar (20 g L-1) was added into the liquid media to prepare solid medium plates. Nutrient Broth (NB) medium contained beef extract (3.0 g L-1), NaCl (5.0 g L-1) and peptone (10.0 g L-1) with pH 7.2-7.4. Enrichment, isolation and screening of MPB Soil samples used for enrichment and isolation of MT-producing strains were collected from two typical paddy rice fields in Wuhu and Chaohu and one typical wheat field in Suzhou from Anhui province in China. The basic characteristics of the soil samples were provided in Table I. Two grams of the soil were added to 50 mL MBM. The enrichment culture was incubated in a rotary shaker at 200 r min-1 at 30˚C for about 4 days. After shaking, dilutions of the sequential enrichment were spread onto the solidified agar MBM plates, and incubated at 30˚C. Then, colonies with distinct morphologies were picked and purified by the streaking method. The purified isolates were used for screening MPB. 3
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MPB were screened similarly to the method developed by previous studies (Drotar et al., 1987; Yoshimura et al., 2000; Tang and You, 2012). Briefly, the isolates were inoculated on NB medium and cultured on a rotary shaker at 200 r min-1 at 30˚C for about 20 h when they get to the late log phase with the optical density value at 600 nm (OD600 nm) of about 1.0 (approximately 108 CFU mL-1). After incubation, 5mL of cultures were harvested by centrifugation (TDZ5-WS centrifuge, Changsha centrifuge Co. Ltd., China) at 5000 r min-1 for 20 min, washed for triplicate and re-suspended with 5mL MBM not containing Met. Then, the suspension was transferred into a 22 mL headspace vial (PerkinElmer, USA), and 20 μL of 0.1 mol L-1 Met was added. The final pH was 7.0. The vials were sealed with a silicone/polytetrafluoroethylene (PTFE) septum and incubated at 30˚C for 4 h, and then the headspace gases were used to measure MT. Uninoculated solution of MBM containing Met was served as the negative control. The isolates producing MT were considered as potential MPB strains. The screened strains were preserved in 25% glycerol at -70˚C. Identification of MT-producing bacteria All the potential MPB strains were identified by morphology and physio-biochemistry, and 16S rRNA gene analyses. The cell morphology was observed under a microscopy (Olympus BX51, Japan). The physiological and biochemical characteristics were determined according to Bergey’s Manual of Determinative Bacteriology (8th Ed). The 16S rRNA gene was amplified by polymerase chain reaction (PCR) according to the method described by Zhao et al. (2012). Briefly, genomic DNA was extracted from a pure colony using Genomic DNA Extraction Kit for Bacteria (Sangon Biotech, Shanghai, China) as described in the manufacturer’s instructions. Then, the bacterial 16SrRNA was amplified by PCR with primers 5’-AGAGTTTGATCMTGGCTCAG-3’ (E.coli bases 8-27) and 5’-ACGGTTACCTTGTTACGACTT-3’ (E.coli bases 1,487-1,507). A 30 μL PCR mixture contained 2μL of dNTP at 2.5 μM, 5 μL of 10 × Taq buffer, 1 μL of 2.5 U Taq DNA polymerase, 1μL of each primer, 19 μL of sterile water and 1μL of template DNA. The PCR was performed in a PTC-200 Thermocycler (MJ Research Inc, Watertown, MA, USA). The PCR was programed as follows: an initial denaturation step at 94˚C for 5 min, and 30 cycles of denaturation at 94˚C for 45 s, primer annealing at 55˚C for 45 s and elongation at 72˚C for 1.5 min were performed, which was followed by a final elongation step at 72˚C for 10 min. The PCR product was subjected to electrophoresis on 1.5% (w/v) agarose gel with ethidium bromide under U.V. light, and then were excised and purified by AxyPrepTM PCR Cleanup Kit. (AXYGEN Biotech, Hangzhou, China) as recommended by the manufacturer. The PCR product was commercially sequenced by Sangon Biotech (Shanghai, China) Co., Ltd.. The sequences were obtained from the GenBank database on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) and EzBioCloud (http://www.ezbiocloud.net/) using the BLAST search program. Alignment of 16S rRNA gene sequences from GenBank database was performed using ClustalX 1.8.3 with default settings (Thompson et al., 1997). Phylogenesis was analyzed by MEGA (version 5.05). Distances were calculated using the Kimura two-parameter distance model. An unrooted tree was built by the neighbor-joining method. The dataset was boot-strapped 1000 times (Tamura et al., 2011). Determination of MT-producing ability and impact factors The five fastest-growing MPB strains were selected to determine the MT-producing capability. Strains were cultured in NB media to late log phase with OD600 nm of about 1.0 and cell concentration of approximately 108 CFU mL-1. Cells were harvested, washed, re-suspended and transferred into a 22 mL headspace vial containing Met, and then incubated and measured MT production in the same way as aforementioned. One MPB strain, named CHZG-R4, was used to study the influence of incubation conditions on MT 4
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production. The effects of different incubation temperatures (20˚C, 25˚C, 30˚C, 35˚C, 40˚C, 45˚C and 50˚C, at pH 6.0), pH values (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, at 35˚C) on the MT production were studied. All these experiments were performed in triplicate. Since some bacteria required other organic compounds to support their growth and/or provide energy when they decomposed Met (Segal and Starkey, 1969; Sreekumar et al., 2009), the effects of different carbon and nitrogen sources on MT production were investigated under optimal condition (35˚C, pH 6.0). The effects of carbon and nitrogen sources, respectively, were studied by adding different carbon sources (control without addition, 2 g L-1 glucose, 2 g L-1 starch, 2 g L-1 sucrose and 2 g L-1 carboxymethylcellulose sodium (CMC)) and nitrogen-containing materials (control without addition, 2 g L-1 ammonia sulfate, 2 g L-1 urea, 2 g L-1 potassium nitrate and 2 g L-1 peptone) into cell suspension in headspace vial, and then incubated and measured MT production as described above. All these experiments were performed in triplicate. MT Analysis MT was analyzed by a TurboMatrix 16 headspace (PerkinElmer, USA) coupled with a Shimazu QP2010 gas chromatography-mass selective detector (GC-MSD, Shimazu, Japan). Details about sample analysis, standard preparation and calibration were similar to those presented previously (Lei and Boatright, 2001; Zhang et al., 2013). Briefly, MT in the headspace gas was injected using a TurboMatrix headspace. 5 μL of ethanethiol (100 μg mL-1) was added to each vial as internal standard before analysis. The headspace injector conditions were vial pressure of 19 psi, oven temperature of 80˚C, loop temperature of 90˚C, transfer line temperature of 100˚C, vial equilibration for 15 min, vial pressurization for 1 min, loop fill for 0.15 min, loop equilibration for 0.5 min, sample injection for 0.04 min, and GC cycle for 30 min. After headspace, MT was transferred into the GC-MS system for analysis. The mixture was firstly separated by a HP-1 capillary column (30 m × 0.25 mm i.d. × 0.25 μm film, Agilent Technologies, USA) with helium as carrier gas. The GC oven temperature was programmed to be initially at 30˚C, holding for 3 min, increasing to 250˚C at 20˚C min-1 and holding for 10 min. The MSD was used in selected ion monitoring (SIM) mode with the target ion of m/z 47 and 48 for MT and m/z 62 and 47 for ethanethiol, and the ionization method was electron impacting (EI). MT was identified based on its retention time, and quantified by multi-point internal calibration method. To prepare calibration curves, standard MT liquid in propanediol (10% w/w, Shanghai Xiangjie Perfumery Plant, China) was diluted with MBM not containing Met to 0 (only MBM), 10, 100, 500, 1000, 2000 and 5000 ng mL-1. The diluted MT standards were analyzed in the same way as the MPB samples. Good dose-response correlation (R > 0.99) was found in the range 0-5000 ng mL-1. The analytical system was challenged daily with a one-point (typically 500 ng mL-1) calibration before running MPB samples. If the response was beyond ± 10% of the initial calibration curve, recalibration was performed. The method detection limits (MDLs) for MT was 2 ng mL-1 with a sample volume of 5 mL. The relative standard deviations (RSDs) were less than 6% after 10 replicate analysis of a standard (500 ng mL-1) in 10 consecutive days. Data analysis All date reported were means of at least three replicates and were analyzed by one-way analysis of variance (ANOVA) with a LSD test. All statistical significance was accepted at p < 0.05. RESULTS AND DISCUSSION Isolation and identification of MPB A number of colonies with MT-producing potential were successfully enriched and isolated from agricultural soils in the MBM with Met as sole carbon source. Among them, five fastest-growing strains were selected for further study, and they were designated WH-R1, WH-R2, WH-R3, SZLB-W3 and 5
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CHZG-R4 (Table II), respectively. The strains of WH-R1, WH-R2 and WH-R3 showed pale yellow, opaque colonies, whereas CHZG-R4 and SZLB-W3 showed white, translucent and yellow, translucent colony, respectively. All the colonies were smooth and moist. SZLB-W5 was coccus-shaped, while the other four strains were rod-shaped. Both CHZG-R4 and SZLB-W5 were gram-negative bacteria, while WH-R1, WH-R2 and WH-R3 were gram-positive. Detail morphological and physio-biochemical characteristics were listed in Table II. The 16S rRNA gene sequences of WH-R1, WH-R2 and WH-R3 exhibited 99% similarity to Bacillus sp., while SZLB-W3 and CHZG-R4 had 100% and 99% similarity to Arthrobacter sp. and Delftia sp., respectively. The results were consistent with the physiologic and biochemical characteristics of these strains (Table II). Therefore, these strains of WH-R1, WH-R2, WH-R3, SZLB-W3 and CHZG-R4 were named Bacillus sp. WH-R1, WH-R2 and WH-R3, Arthrobacter sp. SZLB-W3 and Delftia sp. CHZG-R4, respectively. The sequences of five representative strains were deposited in the GenBank nucleotide sequence date library under the following accession numbers: KJ492943 (WH-R3, Bacillus sp.), KJ492944 (WH-R1, Bacillus sp.), KJ492945 (WH-R2, Bacillus sp.), KJ492946 (CHZG-R4, Delftia sp.), KJ492947 (SZLB-W3, Arthrobacter sp.). The identification of the five selected strains based on 16S rRNA sequence and their phylogenetic tree were presented in Fig. 1. Pure cultures of various bacteria, actinomycetes and fungi can produce MT ( Buzzini et al., 2002; Kadota and Ishida, 1972; Segal and Starkey, 1969; Sreekumar et al., 2009). The genera Bacillus and Arthrobacter have been reported to convert Met into MT in several previous studies (Bonnarme et al., 2000; Burra et al., 2010; Rappert and Müller, 2005; Ting et al., 2010). However, to the best of our knowledge, the genus Delftia was found to produce MT from Met for the first time, although it was isolated from various environment (Wen et al., 1999) and had a capability to oxidize thiosulfate to sulfate (Graff and Stubner, 2003), degrade aniline into TCA-cycle intermediates (Liu et al., 2002) and decompose di-n-butylphthalate into intermediates of monobutylphthalate, phthalate and protocatechuate (Patil et al., 2006). Ability to produce MT of different strains As shown in Table III, all the five strains had the high MT-producing ability, with the production ranging from 0.07 to 21.0 μg. Substantial differences in MT-producing ability existed between Delftia sp. CHZG-R4 and other four strains (p < 0.05). Delftia sp. CHZG-R4 strain had the highest MT-producing capability at a rate of 21.0 μg for 4 h incubation at 30˚C and pH 7.0, which was 2-3 orders of magnitude greater than those of other four strains. There was no significant difference among Bacillus sp. WH-R1, WH-R2, WH-R3 and Arthrobacter sp. SZLB-W3, although the MT-forming rate of Arthrobacter sp. SZLB-W3 strain was one order of magnitude lower than those of three Bacillus sp. SZLB-W3 strains. The results were consistent with the fact that 21.8% of sulfur content added as Met was converted into MT by Arthrobacter sp. SZLB-W3, while less than 1% of sulfur was transferred into MT by other four strains. Differences in MT production levels of five strains are likely attributed to differences in the presence or activity of amino acid transporters and/or catabolic enzymes (Sreekumar et al., 2009). Many literatures have already studied the MT-producing ability of various microorganisms. For example, Drotar et al. (1987) reported that 40 strains of heterotrophic bacteria utilized ammonium sulfide as Met-breakdown inducer to formed MT at a rate ranging from 0-300 pmol min-1 per mL of headspace per 1010 cells. Buzzini et al. (2005) investigated the VOSC-producing capability of 37 basidiomycetous yeasts grown on media containing L-cysteine or L-Met as sole nitrogen sources, and found that only 10 strains were able to grow on media containing L-Met and produce MT at a rate of 0.01-0.12 mg L-1 culture. However, the MT-producing capabilities of microbes in these studies cannot be compared with those in our study because of the difference in the culture conditions of strains and the analysis methods of MT. It is further noted that the MT-producing ability of MPB strains may be not the same at different growth phase 6
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and that microorganism species generally have the maximum metabolic activity at late logarithmic phase. So we will determine the growth curve of each analyzed bacteria species as well as their MT-producing ability at different growth phase in the future work. Effect of temperature and pH on MT production As a novel and most active MT-producing strain, Delftia sp. CHZG-R4 may be important for sulfur cycle in agricultural soil, so it was selected for further study. The effects of temperature and pH on MT production were investigated with Met as sole carbon source. As shown in Fig. 2, suitable temperature and pH favored the MT production of Delftia sp. CHZG-R4. The highest MT production of Delftia sp. CHZG-R4 was measured at 35˚C, with a rate of 32.8 μg, followed by 40˚C (31.0 μg). The production of MT was still significantly increased from 25˚C to 30˚C, but was seriously inhibited at 20˚C and above 45˚C. Quite similar, Patil et al. (2006) also found Delftia sp. growth occurred on mineral salts medium supplemented with di-n-butylphthalate between 20˚C and 40˚C, with optimum growth at 34˚C. The optimal pH for MT production of Delftia sp. CHZG-R4 was 6.0 (32.8 μg), with a wide stable range from 5.0 to 10.0. When pH was 4.0 and 11.0, the production of MT decreased sharply to 0.07 and 0.03 μg, respectively. This result was inconsistent with previous studies, in which Delftia sp. growth occurred at a narrow range of 6.0-8.0, with optimum growth at pH 7.0 (Liu et al., 2002; Patil et al., 2006). This could be explained by the change of the suspension pH in the culture process in the vials. The initial pH values above 6.0 decreased at the end of culture, while those below 6.0 increased (Fig. 3). Particularly, the suspensions at initial pH 5.0-10.0 finally get to approximately 6.0-8.0 during the incubation. The Delftia sp. CHZG-R4 strain could simultaneously metabolize Met to α-ketobutyrate and ammonia (Lomans et al., 2002), which had a neutralization and made this strain thrive in such a wide pH range. It’s worth noting that MT production by Delftia sp. CHZG-R4 at its optimal temperature (35˚C) and pH (6.0) accounted for 51.3% of sulfur content added as Met, supporting that the genera Delftia bacteria had the high MT-producing capability. Effect of carbon and nitrogen source on MT production The effects of different carbon sources on the MT production of Delftia sp. CHZG-R4 strain were shown in Fig. 4A. Even though the strain CHZG-R4 grew well in MBM with Met as the sole carbon source, the addition of starch, sucrose and CMC into culture medium had a positive influence on MT production. Sucrose was the strongest in promoting the MT production, followed by starch and CMC. When sucrose, starch and CMC were added, the MT productions get to 36.1 , 35.0 and 32.8 μg MT after 4 h incubation at 35˚C and pH 6.0, respectively, 1.17 , 1.13 and 1.05 times those in the control treatment (31.0 μg). For glucose, its supply only slightly enhanced the MT production, with a value of 32.4 μg. The increasing of production might be attributed to the reason that the available organic carbon sources could promote the growth and/or activity of Delftia sp. CHZG-R4 strain and thus enhance its uptake of Met, so that more Met were converted into MT. Quite similar, Arfi et al. (2003) also found the addition of glycerol and glucose significantly increased the production of volatile sulfur compounds from L-Met by Geotrichum candidum, because the carbon sources had a dramatic effect on the activity of aminotransferase, a key enzyme in L-Met catabolism. Fig. 4B showed the significant differences of MT production between control treatment (without other nitrogen source addition) and each experiment treatment (with other nitrogen source addition). The addition of urea and potassium nitrate obviously promoted the MT production, while the addition of ammonia sulfate and peptone notably decreased the MT production (p < 0.05). The MT productions in the treatments with the addition of urea and potassium nitrate were 34.5 and 33.7 μg, respectively, after 4 h incubation at 35˚C and pH 6.0, which were 1.11 and 1.10 times those in the control treatment (31.0 μg). One explanation for the enhancement of production was that the soluble organic nitrogen sources could promote the growth and/or 7
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activity of Delftia sp. CHZG-R4 strain and thus increase its consumption of Met, so that more MT were produced. However, when ammonia sulfate and peptone was added into the MBM, the MT productions were 26.0 and 4.01 μg, respectively, that is, 0.84 and 0.13 times those in the control treatment. This result could account for the reason that Delftia sp. CHZG-R4 strain prefer to ammonia sulfate and peptone as nitrogen sources or that the growth and/or activity of Delftia sp. CHZG-R4 strain was suppressed by ammonia sulfate and peptone, so that transformation of Met into MT by this strain was inhibited. Qiao et al. (2000) also found that the addition of ammonia nitrogen decreased the production of VOSCs from rice paddy soils. CONCLUSIONS Five MT-producing strains of WH-R1, WH-R2, WH-R3, SZLB-W3 and CHZG-R4 were isolated and characterized, utilizing Met as the sole carbon source for growth. WH-R1, WH-R2 and WH-R3 belong to the genus Bacillus, and SZLB-W3 and CHZG-R4 were identified as Arthrobacter sp. SZLB-W3 and Delftia sp. CHZG-R4, respectively. The strain of Delftia sp. CHZG-R4 was reported to produce MT for the first time, and had the highest capability in producing MT among five strains. Its optimal MT production appeared at 35 °C and pH 6.0. Under these conditions, Delftia sp. CHZG-R4 transferred 51.3% of sulfur content in Met into MT, and its MT production was increased above 10% by the addition of 2 g L-1 starch and 2 g L-1 sucrose, 2 g L-1 urea and 2 g L-1 potassium nitrate after 4 h incubation, but decreased 16% and 87% by the provision of 2 g L-1 ammonia sulfate and 2 g L-1 peptone, respectively. The results might provide useful information for the development of microbial resources and the microbial mechanism of MT production from agricultural soils. ACKNOWLEDGEMENTS REFERENCES Andreae, M. O. and Crutzen, P. J. 1997. Atmospheric aerosols: biogeochemical source and roles in atmospheric chemistry. Science. 276: 1052-1058. Arfi, K., Tâche, R., Spinnler, H. E. and Bonnarme, P. 2003. Dual influence of the carbon source and L-methionine on the synthesis of sulphur compounds in the cheese-ripening yeast Geotrichum candidum. Appl Microbiol Biotechnol. 61: 359-365. Bentley, R. and Chasteen, T. G. 2004. Environmental VOSCs-formation and degradation of dimethyl sulfide, methanethiol and related materials. Chemosphere. 55: 291-317. Bonnarme, P., Psoni, L. and Spinnler, H. E. 2000. Diversity of L-methionine catabolism pathways in cheese-ripening bacteria. Appl Environ Microbiol. 66: 5514-5517. Bremner, J. M. and Bundy, L. G. 1974. Inhibition of nitrification in soils by volatile sulfur compounds. Soil Biol Biochem. 6: 161-165. Burra, R., Pradenas, G. A., Montes, R. A., Vásquez, C. C. and Chasteen, T. G. 2010. Production of dimethyl triselenide and dimethyl diselenenyl sulfide in the headspace of metalloid-resistant Bacillus species grown in the presence of selenium oxyanions. Anal Biochem. 396: 217-222. Buzzini, P., Romano, S., Turchetti, B., Vaughan, A., Pagnoni, U. M. and Davoli, P. 2005. Production of volatile organic sulfur compounds VOSCs by basidiomycetous yeasts. FEMS Yeast Res. 5: 379-385. Charlson, R. J., Lovelock, J. E., Andreae, M.O. and Warren, S. G. 1987. Oceanic phytoplankton, atmospheric sulphur, cloud albedo and climate. Nature. 326: 655-661. Cox, R. A.and Sheppard, D. 1980. Reactions of OH radicals with gaseous sulphur compounds. Nature. 284: 330-331. de Souza, M. P. and Yoch, D. C. 1995. Purification and characterization of dimethylsulfoniumpropionate lyase from an Alcaligenes-like dimethyl sulfide-producing marine isolate. Appl Environ Microbiol. 61: 21-26. Drotar, A., Burton, A., Tavernier, J. E. and Fall, R. 1987. Widespread occurrence of bacterial thiol 8
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methyltransferases and the biogenic emission of methylated sulfur gases. Appl Environ Microbiol. 53: 1626-1631. Food and Agricultural Organization FAO 2013 FAO Statistical Yearbook 2013 FAOSTAT. Available at http://issuu.com/faooftheun/docs/syb2013issuu Graff, A. and Stubner, S. 2003. Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil. Syst Appl Microbiol. 26: 445-452. Hatakeyama, S. and Akimoto, H. 1983. Reactions of OH radicals with methanethiol, dimethyl sulfide, and dimethyl disulfide in air. J Phsy Chem. 87: 2387-2395. Higgins, M. J., Chen, Y. C., Yarosz, D. P., Murthy, S. N., Maas, N. A., Glindemann, D. and Novak, J. T. 2006. Cycling of volatile organic sulfur compounds in anaerobically digested biosolids and its implications for odors. Water Environ Res. 78: 243-252. Insam, H. and Seewald, M. 2010. Volatile organic compounds VOCs in soils. Bio Fert Soils. 46: 199-213. Kadota, H. and Ishida, Y. 1972. Production of volatile sulfur compounds by microorganisms. Annu Rev Microbiol. 26: 127-138. Lei, Q. and Boatright, W. L., 2001. Development of a new methanethiol quantification method using ethanethiol as an internal standard. J Agr Food Chem. 49: 3567-3572. Liu, Z., Yang, H., Huang, Z., Zhou, P. and Liu, S. J. 2002. Degradation of aniline by newly isolated, extremely aniline-tolerant Delftia sp. AN3. Appl Microbiol Biotechnol. 58: 679-682. Lomans, B. P., Van der Drift, C., Pol, A. and Op den Camp, H. J. M. 2002. Microbial cycling of volatile organic sulfur compounds. Cell Mol life Sci. 59: 575-588. Patil, N. K., Kundapur, R., Shouche, Y. S. and Karegoudar, T. B. 2006. Degradation of plasticizer di-n-butylphathalate by Delftia sp.TBKNP-05. Curr Microbiol. 52: 369-374. Qiao, W. C., Xi, S. Q., Zhang, J. H. and Yang, Z. 2000. The influence of physcio-chemical factors on the emission of volatile sulfur gases from soil. Environ Sci. (in Chinese). 01: 78-80. Rappert, S. and Müller, R. 2005. Odor compounds in waste gas emissions from agricultural operations and food industries. Waste Manage. 25: 887-907. Saari, A. and Martikainen, P. J. 2003. Dimethyl sulphoxide DMSO and dimethyl sulphide DMS as inhibitors of methane oxidation in forest soil. Soil Biol Biochem. 35: 383-389. Segal, W. and Starkey, R. L. 1969. Microbial decomposition of methionine and identity of the resulting sulfur products. J Bacteriol. 98: 908-913. Sreekumar, R., Al-Attabi, Z., Deeth, H. C. and Turner, M. S., 2009. Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine. Lett Appl Microbiol. 48: 777-782. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 28: 2731-2739. Tanaka, H., Esaki, N. and Soda, K. 1985. A versatile bacterial enzyme: L-methionine γ-lyase. Enzyme Microb Technol. 7: 530-537. Tang, M. Q. and You, M. S. 2012. Isolation, identification and characterization of a novel triazophos-degrading Bacillus sp. TAP-1. Microbio Res. 167: 299-305. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882 Ting, A. S. W., Mah, S. W. and Tee, C. S. 2011. Detection of potential volatile inhibitory compounds produced by endobacteria with biocontrol properties towards Fusarium oxysporum f. sp. cubense race 4. World J Microbiol Biotechnol. 27: 229-235. 9
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Watts, S. F. 2000. The mass budgets of carbonyl sulfide, dimethyl sulfide, carbon disulfide and hydrogen sulfide. Atmos Environ. 34: 761-779. Wen, A., Fegan, M., Hayward, C., Chakraborty, S. and Sly, L. I. 1999. Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans den Dooren de Jong 1926 and Tamaoka et al. 1987 gen. nov., comb. nov. Int J Syst Evol Micr. 49: 567-576. Yoshimura, M., Nakano, Y., Yamashita, Y., Oho, T., Saito, T. and Koga, T. 2000. Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis. Infect Immun. 68: 6912-6916. Zhao, J., Wu, X. B., Nie, C. P., Wu, T., Dai, W. H., Liu, H. and Yang, R, Y. 2012. Analysis of unculturable bacterial communities in tea orchard soils based on nested PCR-DGGE. World J Microbiol Biotechnol. 28: 1967-1979. Zhang, C. Z., Zhang, W. J. and Xu, J. 2013. Isolation and identification of methanethiol-utilizing bacterium CZ05 and its application in bio-trickling filter of biogas. Bioresource Technol. 150: 338-343.
ACKNOWLEDGEMENT
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This research was upported by the Natural Science Foundation of China (41273095, 41025012 and 41103067) TABLE I
The basic characteristics of the soil samples used for enrichment and isolation of MT-producing strains. Longitude
sites
and Latitude
Wuhu
E118o35′14′′,
pH
Total C -1
(g kg ) 5.2
17.9
Total N
Total S
M
Sampling
-1
-1
OM
a)
-1
Available P
Available K
-1
-1
Available S
(g kg )
(g kg )
(g kg )
(mg kg )
(mg kg )
(mg kg-1)
5.16
1.74
23.8
7.35
65.7
36.8
o
E117o44′50′′,
Chaohu
6.2
27.2
3.75
1.67
22.1
14.3
98.7
47.4
8.2
21.0
4.96
2.15
12.5
11.0
78.0
39.0
o
N 31 46′52′′ E117o35′04′′,
ce pt
Suzhou
ed
N 31 16′04′′
o
N 33 32′49′′
a) organic matter. TABLE II
Ac
Morphological, physiological and biochemical characteristics of the five MT-producing strains.
Characteristics Colony characteristics
Shape of cells Oxygen Gram staining Oxidase Catalase Starch hydrolysis Glucose fermentation
WH-R1 a)
WH-R2
WH-R3
CHZG-R4
SZLB-W3
Pale yellow Opaque Smooth Moist Rod +w + + + + +
Pale yellow Opaque Smooth Moist Rod +w + + + + +
Pale yellow Opaque Smooth Moist Rod +w + + + + +
White Transluscent Smooth Moist Rod +w – + + – –
Yellow Transluscent Smooth Moist Coccus +w – – + + +
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Nitrate reduction a)
–
–
–
+
+, positive reaction; –, negative reaction; w, weak positive.
TABLE III MT productions of the five MT-producing strains in 5 mL culture with OD600 nm of 1.0 incubated at 30˚C and pH 7.0 for 4 h.
MT Production (μg)
Accession No.
a)
Sulfur conversion (%)
b)
KJ492944
0.30 ± 0.02 b
Bacillus sp. WH-R2
KJ492945
0.26 ± 0.01 b
0.28
Bacillus sp. WH-R3
KJ492943
0.22 ± 0.02 b
0.23
Arthrobacter sp. SZLB-W3 Delftia sp. CHZG-R4
KJ492947 KJ492946
0.05 ± 0.002 b 21.0±0.44 a
0.05 21.8
a)
Mean ± SD of three replicates.
b)
Means followed by the same letter are not significantly different at P < 0.05.
0.31
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Bacillus sp. WH-R1
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Strains
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Bacillus megaterium IAM 13418 T (D16273) 98
Bacillus aryabhattai B8W22 T (EF114313) WH-R2 (KJ 492945)
97
99
WH-R3 (KJ492943) WH-R1 (KJ492944)
100
Bacillus flexus IFO 15715T (AB021185) 100
M
Bacillus paraflexus RC2 T (FN999943)
Bacillus koreensis BR030 T (AY667496) Arthrobacter gyeryongensis DCY72 T (JX141781)
98
ed
Arthrobacter ramosus CCM 1646 T (AM039435)
100
SZLB-W3 (KJ492947) Arthrobacter nitroguajacolicus G2-1 T (AJ512504)
84
ce pt
99
Arthrobacter aurescens DSM 20116 T (X83405)
Xenophilus aerolatus 5516S-2 T (EF660342) Delftia litopenaei wsw-7 T (GU721027)
100
Delftia acidovorans ACM 489 T (AF078774)
100
65
Ac
0.02
Delftia tsuruhatensis T7 T (AB075017)
99 CHZG-R4 (KJ492946) 60 Delftia lacustris DSM 21246 T (EU888308)
Fig. 1 Phylogenetic tree generated by the neighbor-joining method on the basis of partial 16S rRNA sequences showing the phylogenetic relationships between MT-producing bacteria and the blast sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at major branching points.
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40
40
MT production (μg)
A
a
30 b 20
c d
10
a
B
MT production (μg)
a
e
b
30
c d
20
e
de
10
f
f
f
0
0 20
25
30
35
40
45
4
50
5
6
7
o
8
9
10 11
pH
Temperature ( C)
t
Fig. 2 Effects of temperature (A) and pH (B) on MT production by Delftia sp. CHZG-R4 in 5 mL culture with OD600 nm of 1.0
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incubated for 4 h. Error bars, mean ± SD of three replicates. Bar with different letters are significantly different at 0. 05 level according to a LSD test.
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Initial pH Incubated after 4 hours
10
pH
8
4 2 2
3
4 5 Samples
6
7
8
ed
1
M
6
30 20 10 0
c
bc
a
a
40
b
Ac
MT production (μg)
A
trol cose Starch rcose CMC Con Glu Su
MT production (μg)
40
ce pt
Fig. 3 pH changes of Delftia sp. CHZG-R4 suspension before and after incubation at 35˚C for 4 h.
a B
30
b
a
c
20 10 0
d
trol lfate Urea nitrate ptone Con nia su Pe o sium s m a t Am Po
Fig. 4 Effects of carbon sources (A) and nitrogen sources (B) on MT production by Delftia sp. CHZG-R4 in 5 mL culture with OD600 nm of 1.0 incubated at 35˚C and pH 6.0 for 4 h. Error bars, mean ± SD of three replicates. CMC, carboxymethylcellulose sodium. Bar with different letters are significantly different at 0.05 level according to a LSD test.
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Title: Isolation and characterization of methanethiol-producing bacteria from agricultural soils
Author: LIU Hui, SHI Cheng-Fei, WU Ting, JIA Qi-Na, ZHAO Juan, WANG Xin-Ming
PII: DOI: Reference:
S1002-0160(17)60411-9 10.1016/S1002-0160(17)60411-9 NA
To appear in:
Received date: Revised date: Accepted date:
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Please cite this article as: LIU Hui, SHI Cheng-Fei, WU Ting, JIA Qi-Na, ZHAO Juan, WANG Xin-Ming, Isolation and characterization of methanethiol-producing bacteria from agricultural soils, Pedosphere (2017), 10.1016/S1002-0160(17)60411-9.
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doi:10.1016/S1002-0160(17)60411-9
Isolation and characterization of methanethiol-producing bacteria from agricultural soils LIU Hui1, SHI Cheng-Fei1, WU Ting1, 2, JIA Qi-Na1, ZHAO Juan1, WANG Xin-Ming2, 1
College of Environmental Science and Engineering, Anhui Normal University, Wuhu 241000, China State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China
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ABSTRACT Methanethiol-producing bacteria (MPB) were enriched and isolated from agricultural soils in a modified basal medium (MBM) containing methionine (Met) as the sole carbon sources. The isolates were identified as Bacillus sp. WH-R1, WH-R2 and WH-R3, Arthrobacter sp. SZLB-W3 and Delftia sp. CHZG-R4 based on cell morphology, physiological and biochemical characteristics and 16S rRNA sequence analysis. Delftia sp. CHZG-R4 was reported as a novel strain to produce methanethiol (MT) using Met as precursor, and had the most active MT-producing potential, with the production of 21.8 μg at 30 ˚C and pH 7.0. Its optimal MT production occurred at 35 ˚C and pH 6.0, with 51.3% of sulfur content in Met converting into MT. Under these conditions, its MT production was changed by the supply of both carbon and nitrogen sources. The addition of 2 g L-1 starch and 2 g L-1 sucrose, 2 g L-1 urea and 2 g L-1 potassium nitrate into the MBM promoted above 10% of the MT production, while the provision of 2 g L-1 ammonia sulfate and 2 g L-1 peptone decreased 16% and 87% of the MT production, respectively. This is the first study to report Delftia sp. CHZG-R4 capable of producing MT, which might provide useful information for the microbial mechanism of MT production from agricultural soils and also contribute to the limited knowledge about the function of Delftia sp. CHZG-R4. Key words: agricultural soils, Delftia sp., methanethiol (MT), Methanethiol-producing bacteria (MPB), methionine (Met) INTRODUCTION Volatile organic sulfur compounds (VOSCs) produced from terrestrial soils can have important effects on atmospheric chemistry and ecosystem-level processes. In the atmosphere, they play an important role in acid precipitation, cloud formation, and global warming and sulfur budget (Andreae and Crutzen, 1997; Charlson et al., 1987; Cox and Sheppard, 1980; Watts, 2000). Methanethiol (MT) is one important member of the VOSC group. As a very reactive gas in the atmosphere, it is readily oxidized to methanesulfonic acid and SO2, and subsequently gives a rise to acid rain (Hatakeyama and Aklmoto, 1983). As an important intermediate, it can be further transformed to dimethyl sulfide (DMS) and thus contribute to the formation of
Corresponding author. E-mail address: [email protected] (T. Wu); [email protected] (X. Wang)
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cloud-condensation nuclei (CCN) and the change of global climate (Drotar et al., 1987; Bentley and Chasteen, 2004). There is also evidence that MT together with other VOSCs can influence biogeochemical processes within soils, altering the rates of carbon cycling by serving as substrates for microbial metabolism, inhibiting microbial processes associated with the nitrogen cycle, and either stimulating or inhibiting the growth of specific microbial taxa, and thus influence the growth of plants (Bremner and Bundy, 1974; Insam and Seewald, 2010; Saari and Martikainen, 2003). As one major component of the VOSC mixtures evolved from soils, MT originates mainly from the metabolism of sulfur-containing amino acids such as cysteine and methionine (Met) by microorganisms (Higgins et al., 2006). Met can be firstly deaminated and then demethiolated to produce MT by methionine γ-lyase catalysis in microorganisms (Bonnarme et al., 2000; Kadota and Ishida, 1972; Lomans et al., 2002; Segal and Starkey, 1969; Tanaka et al., 1985). A great variety of microorganisms have been isolated and identified to involve in this reaction in food industry, biosolid degradation, and freshwater and saline water ecosystem, including Achrobacter sp., Aspergillus sp., Aspergillus oryzae, Fusarium culmorum, Streptomyces griseus, Clostridium sp., Escherichia coil, Pseudomonas sp., Pseudomonas putifaciens, Pseudomonas fluorescens, Pseudomonas perolens and so on (Bonnarme et al., 2000; Kadota and Ishida, 1972; Rappert and Müller, 2005; Segal and Starkey, 1969). The area of agricultural fields worldwide is about 4912 million hectares, accounting for about 37.4% of the global land area (FAO, 2013). However, only a few bacteria have been reported to produce MT in agricultural soils, which include Parasporobacterium paucivorans, Holophaga foetida, Sporobacterium olearium and Rhodobacter capsulatus (Rappert and Müller, 2005 and its referencein). It would be of great importance to find more MT-producing bacterial species to provide useful information for understanding the microbial mechanism of MT production from agricultural soils. The aim of this study was to isolate, identify and characterize bacteria that can use Met as the sole carbon sources to produce MT in agricultural soils. The influence of several factors including temperature, pH, and carbon and nitrogen sources on MT production were also investigated. MATERIALS AND METHODS Media Enrichment, isolation and cultivation were carried out with the following medium. The modified basal medium (MBM) was prepared according to the literature (de Souza and Yoch, 1995) with minor modifications: KH2PO4, 0.6 g L-1; K2HPO4, 0.9 g L-1; MgSO4·7H2O, 0.2 g L-1; CaCl2·2H2O, 0.075 g L-1; Fe-EDTA, 0.0295 g L-1; MnSO4, 0.1599 g L-1; H3BO4, 0.28 g L-1; CuSO4·5H2O, 0.0079 g L-1; ZnSO4·7H2O, 0.024 g L-1; NaMoO4·2H2O, 0.126 g L-1; (NH4)2SO4, 0.66 g L-1; NaCl, 1.17 g L-1; yeast extract, 0.05 g L-1; methionine (Met), 0.0597 g L-1 (0.4 mmol L-1); vitamin B12, 2 μg L-1; p-aminobenzoic acid, 10 μg L-1; riboflavin, 10 μg L-1; vitamin B6, 10 μg L-1; niacin, 10 μg L-1; vitamin C, 10 μg L-1; thiamine, 10 μg L-1; and D-(+)-biotin, 10 μg L-1. The final pH was adjusted to 7.0. Agar (20 g L-1) was added into the liquid media to prepare solid medium plates. Nutrient Broth (NB) medium contained beef extract (3.0 g L-1), NaCl (5.0 g L-1) and peptone (10.0 g L-1) with pH 7.2-7.4. Enrichment, isolation and screening of MPB Soil samples used for enrichment and isolation of MT-producing strains were collected from two typical paddy rice fields in Wuhu and Chaohu and one typical wheat field in Suzhou from Anhui province in China. The basic characteristics of the soil samples were provided in Table I. Two grams of the soil were added to 50 mL MBM. The enrichment culture was incubated in a rotary shaker at 200 r min-1 at 30˚C for about 4 days. After shaking, dilutions of the sequential enrichment were spread onto the solidified agar MBM plates, and incubated at 30˚C. Then, colonies with distinct morphologies were picked and purified by the streaking method. The purified isolates were used for screening MPB. 3
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MPB were screened similarly to the method developed by previous studies (Drotar et al., 1987; Yoshimura et al., 2000; Tang and You, 2012). Briefly, the isolates were inoculated on NB medium and cultured on a rotary shaker at 200 r min-1 at 30˚C for about 20 h when they get to the late log phase with the optical density value at 600 nm (OD600 nm) of about 1.0 (approximately 108 CFU mL-1). After incubation, 5mL of cultures were harvested by centrifugation (TDZ5-WS centrifuge, Changsha centrifuge Co. Ltd., China) at 5000 r min-1 for 20 min, washed for triplicate and re-suspended with 5mL MBM not containing Met. Then, the suspension was transferred into a 22 mL headspace vial (PerkinElmer, USA), and 20 μL of 0.1 mol L-1 Met was added. The final pH was 7.0. The vials were sealed with a silicone/polytetrafluoroethylene (PTFE) septum and incubated at 30˚C for 4 h, and then the headspace gases were used to measure MT. Uninoculated solution of MBM containing Met was served as the negative control. The isolates producing MT were considered as potential MPB strains. The screened strains were preserved in 25% glycerol at -70˚C. Identification of MT-producing bacteria All the potential MPB strains were identified by morphology and physio-biochemistry, and 16S rRNA gene analyses. The cell morphology was observed under a microscopy (Olympus BX51, Japan). The physiological and biochemical characteristics were determined according to Bergey’s Manual of Determinative Bacteriology (8th Ed). The 16S rRNA gene was amplified by polymerase chain reaction (PCR) according to the method described by Zhao et al. (2012). Briefly, genomic DNA was extracted from a pure colony using Genomic DNA Extraction Kit for Bacteria (Sangon Biotech, Shanghai, China) as described in the manufacturer’s instructions. Then, the bacterial 16SrRNA was amplified by PCR with primers 5’-AGAGTTTGATCMTGGCTCAG-3’ (E.coli bases 8-27) and 5’-ACGGTTACCTTGTTACGACTT-3’ (E.coli bases 1,487-1,507). A 30 μL PCR mixture contained 2μL of dNTP at 2.5 μM, 5 μL of 10 × Taq buffer, 1 μL of 2.5 U Taq DNA polymerase, 1μL of each primer, 19 μL of sterile water and 1μL of template DNA. The PCR was performed in a PTC-200 Thermocycler (MJ Research Inc, Watertown, MA, USA). The PCR was programed as follows: an initial denaturation step at 94˚C for 5 min, and 30 cycles of denaturation at 94˚C for 45 s, primer annealing at 55˚C for 45 s and elongation at 72˚C for 1.5 min were performed, which was followed by a final elongation step at 72˚C for 10 min. The PCR product was subjected to electrophoresis on 1.5% (w/v) agarose gel with ethidium bromide under U.V. light, and then were excised and purified by AxyPrepTM PCR Cleanup Kit. (AXYGEN Biotech, Hangzhou, China) as recommended by the manufacturer. The PCR product was commercially sequenced by Sangon Biotech (Shanghai, China) Co., Ltd.. The sequences were obtained from the GenBank database on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) and EzBioCloud (http://www.ezbiocloud.net/) using the BLAST search program. Alignment of 16S rRNA gene sequences from GenBank database was performed using ClustalX 1.8.3 with default settings (Thompson et al., 1997). Phylogenesis was analyzed by MEGA (version 5.05). Distances were calculated using the Kimura two-parameter distance model. An unrooted tree was built by the neighbor-joining method. The dataset was boot-strapped 1000 times (Tamura et al., 2011). Determination of MT-producing ability and impact factors The five fastest-growing MPB strains were selected to determine the MT-producing capability. Strains were cultured in NB media to late log phase with OD600 nm of about 1.0 and cell concentration of approximately 108 CFU mL-1. Cells were harvested, washed, re-suspended and transferred into a 22 mL headspace vial containing Met, and then incubated and measured MT production in the same way as aforementioned. One MPB strain, named CHZG-R4, was used to study the influence of incubation conditions on MT 4
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production. The effects of different incubation temperatures (20˚C, 25˚C, 30˚C, 35˚C, 40˚C, 45˚C and 50˚C, at pH 6.0), pH values (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, at 35˚C) on the MT production were studied. All these experiments were performed in triplicate. Since some bacteria required other organic compounds to support their growth and/or provide energy when they decomposed Met (Segal and Starkey, 1969; Sreekumar et al., 2009), the effects of different carbon and nitrogen sources on MT production were investigated under optimal condition (35˚C, pH 6.0). The effects of carbon and nitrogen sources, respectively, were studied by adding different carbon sources (control without addition, 2 g L-1 glucose, 2 g L-1 starch, 2 g L-1 sucrose and 2 g L-1 carboxymethylcellulose sodium (CMC)) and nitrogen-containing materials (control without addition, 2 g L-1 ammonia sulfate, 2 g L-1 urea, 2 g L-1 potassium nitrate and 2 g L-1 peptone) into cell suspension in headspace vial, and then incubated and measured MT production as described above. All these experiments were performed in triplicate. MT Analysis MT was analyzed by a TurboMatrix 16 headspace (PerkinElmer, USA) coupled with a Shimazu QP2010 gas chromatography-mass selective detector (GC-MSD, Shimazu, Japan). Details about sample analysis, standard preparation and calibration were similar to those presented previously (Lei and Boatright, 2001; Zhang et al., 2013). Briefly, MT in the headspace gas was injected using a TurboMatrix headspace. 5 μL of ethanethiol (100 μg mL-1) was added to each vial as internal standard before analysis. The headspace injector conditions were vial pressure of 19 psi, oven temperature of 80˚C, loop temperature of 90˚C, transfer line temperature of 100˚C, vial equilibration for 15 min, vial pressurization for 1 min, loop fill for 0.15 min, loop equilibration for 0.5 min, sample injection for 0.04 min, and GC cycle for 30 min. After headspace, MT was transferred into the GC-MS system for analysis. The mixture was firstly separated by a HP-1 capillary column (30 m × 0.25 mm i.d. × 0.25 μm film, Agilent Technologies, USA) with helium as carrier gas. The GC oven temperature was programmed to be initially at 30˚C, holding for 3 min, increasing to 250˚C at 20˚C min-1 and holding for 10 min. The MSD was used in selected ion monitoring (SIM) mode with the target ion of m/z 47 and 48 for MT and m/z 62 and 47 for ethanethiol, and the ionization method was electron impacting (EI). MT was identified based on its retention time, and quantified by multi-point internal calibration method. To prepare calibration curves, standard MT liquid in propanediol (10% w/w, Shanghai Xiangjie Perfumery Plant, China) was diluted with MBM not containing Met to 0 (only MBM), 10, 100, 500, 1000, 2000 and 5000 ng mL-1. The diluted MT standards were analyzed in the same way as the MPB samples. Good dose-response correlation (R > 0.99) was found in the range 0-5000 ng mL-1. The analytical system was challenged daily with a one-point (typically 500 ng mL-1) calibration before running MPB samples. If the response was beyond ± 10% of the initial calibration curve, recalibration was performed. The method detection limits (MDLs) for MT was 2 ng mL-1 with a sample volume of 5 mL. The relative standard deviations (RSDs) were less than 6% after 10 replicate analysis of a standard (500 ng mL-1) in 10 consecutive days. Data analysis All date reported were means of at least three replicates and were analyzed by one-way analysis of variance (ANOVA) with a LSD test. All statistical significance was accepted at p < 0.05. RESULTS AND DISCUSSION Isolation and identification of MPB A number of colonies with MT-producing potential were successfully enriched and isolated from agricultural soils in the MBM with Met as sole carbon source. Among them, five fastest-growing strains were selected for further study, and they were designated WH-R1, WH-R2, WH-R3, SZLB-W3 and 5
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CHZG-R4 (Table II), respectively. The strains of WH-R1, WH-R2 and WH-R3 showed pale yellow, opaque colonies, whereas CHZG-R4 and SZLB-W3 showed white, translucent and yellow, translucent colony, respectively. All the colonies were smooth and moist. SZLB-W5 was coccus-shaped, while the other four strains were rod-shaped. Both CHZG-R4 and SZLB-W5 were gram-negative bacteria, while WH-R1, WH-R2 and WH-R3 were gram-positive. Detail morphological and physio-biochemical characteristics were listed in Table II. The 16S rRNA gene sequences of WH-R1, WH-R2 and WH-R3 exhibited 99% similarity to Bacillus sp., while SZLB-W3 and CHZG-R4 had 100% and 99% similarity to Arthrobacter sp. and Delftia sp., respectively. The results were consistent with the physiologic and biochemical characteristics of these strains (Table II). Therefore, these strains of WH-R1, WH-R2, WH-R3, SZLB-W3 and CHZG-R4 were named Bacillus sp. WH-R1, WH-R2 and WH-R3, Arthrobacter sp. SZLB-W3 and Delftia sp. CHZG-R4, respectively. The sequences of five representative strains were deposited in the GenBank nucleotide sequence date library under the following accession numbers: KJ492943 (WH-R3, Bacillus sp.), KJ492944 (WH-R1, Bacillus sp.), KJ492945 (WH-R2, Bacillus sp.), KJ492946 (CHZG-R4, Delftia sp.), KJ492947 (SZLB-W3, Arthrobacter sp.). The identification of the five selected strains based on 16S rRNA sequence and their phylogenetic tree were presented in Fig. 1. Pure cultures of various bacteria, actinomycetes and fungi can produce MT ( Buzzini et al., 2002; Kadota and Ishida, 1972; Segal and Starkey, 1969; Sreekumar et al., 2009). The genera Bacillus and Arthrobacter have been reported to convert Met into MT in several previous studies (Bonnarme et al., 2000; Burra et al., 2010; Rappert and Müller, 2005; Ting et al., 2010). However, to the best of our knowledge, the genus Delftia was found to produce MT from Met for the first time, although it was isolated from various environment (Wen et al., 1999) and had a capability to oxidize thiosulfate to sulfate (Graff and Stubner, 2003), degrade aniline into TCA-cycle intermediates (Liu et al., 2002) and decompose di-n-butylphthalate into intermediates of monobutylphthalate, phthalate and protocatechuate (Patil et al., 2006). Ability to produce MT of different strains As shown in Table III, all the five strains had the high MT-producing ability, with the production ranging from 0.07 to 21.0 μg. Substantial differences in MT-producing ability existed between Delftia sp. CHZG-R4 and other four strains (p < 0.05). Delftia sp. CHZG-R4 strain had the highest MT-producing capability at a rate of 21.0 μg for 4 h incubation at 30˚C and pH 7.0, which was 2-3 orders of magnitude greater than those of other four strains. There was no significant difference among Bacillus sp. WH-R1, WH-R2, WH-R3 and Arthrobacter sp. SZLB-W3, although the MT-forming rate of Arthrobacter sp. SZLB-W3 strain was one order of magnitude lower than those of three Bacillus sp. SZLB-W3 strains. The results were consistent with the fact that 21.8% of sulfur content added as Met was converted into MT by Arthrobacter sp. SZLB-W3, while less than 1% of sulfur was transferred into MT by other four strains. Differences in MT production levels of five strains are likely attributed to differences in the presence or activity of amino acid transporters and/or catabolic enzymes (Sreekumar et al., 2009). Many literatures have already studied the MT-producing ability of various microorganisms. For example, Drotar et al. (1987) reported that 40 strains of heterotrophic bacteria utilized ammonium sulfide as Met-breakdown inducer to formed MT at a rate ranging from 0-300 pmol min-1 per mL of headspace per 1010 cells. Buzzini et al. (2005) investigated the VOSC-producing capability of 37 basidiomycetous yeasts grown on media containing L-cysteine or L-Met as sole nitrogen sources, and found that only 10 strains were able to grow on media containing L-Met and produce MT at a rate of 0.01-0.12 mg L-1 culture. However, the MT-producing capabilities of microbes in these studies cannot be compared with those in our study because of the difference in the culture conditions of strains and the analysis methods of MT. It is further noted that the MT-producing ability of MPB strains may be not the same at different growth phase 6
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and that microorganism species generally have the maximum metabolic activity at late logarithmic phase. So we will determine the growth curve of each analyzed bacteria species as well as their MT-producing ability at different growth phase in the future work. Effect of temperature and pH on MT production As a novel and most active MT-producing strain, Delftia sp. CHZG-R4 may be important for sulfur cycle in agricultural soil, so it was selected for further study. The effects of temperature and pH on MT production were investigated with Met as sole carbon source. As shown in Fig. 2, suitable temperature and pH favored the MT production of Delftia sp. CHZG-R4. The highest MT production of Delftia sp. CHZG-R4 was measured at 35˚C, with a rate of 32.8 μg, followed by 40˚C (31.0 μg). The production of MT was still significantly increased from 25˚C to 30˚C, but was seriously inhibited at 20˚C and above 45˚C. Quite similar, Patil et al. (2006) also found Delftia sp. growth occurred on mineral salts medium supplemented with di-n-butylphthalate between 20˚C and 40˚C, with optimum growth at 34˚C. The optimal pH for MT production of Delftia sp. CHZG-R4 was 6.0 (32.8 μg), with a wide stable range from 5.0 to 10.0. When pH was 4.0 and 11.0, the production of MT decreased sharply to 0.07 and 0.03 μg, respectively. This result was inconsistent with previous studies, in which Delftia sp. growth occurred at a narrow range of 6.0-8.0, with optimum growth at pH 7.0 (Liu et al., 2002; Patil et al., 2006). This could be explained by the change of the suspension pH in the culture process in the vials. The initial pH values above 6.0 decreased at the end of culture, while those below 6.0 increased (Fig. 3). Particularly, the suspensions at initial pH 5.0-10.0 finally get to approximately 6.0-8.0 during the incubation. The Delftia sp. CHZG-R4 strain could simultaneously metabolize Met to α-ketobutyrate and ammonia (Lomans et al., 2002), which had a neutralization and made this strain thrive in such a wide pH range. It’s worth noting that MT production by Delftia sp. CHZG-R4 at its optimal temperature (35˚C) and pH (6.0) accounted for 51.3% of sulfur content added as Met, supporting that the genera Delftia bacteria had the high MT-producing capability. Effect of carbon and nitrogen source on MT production The effects of different carbon sources on the MT production of Delftia sp. CHZG-R4 strain were shown in Fig. 4A. Even though the strain CHZG-R4 grew well in MBM with Met as the sole carbon source, the addition of starch, sucrose and CMC into culture medium had a positive influence on MT production. Sucrose was the strongest in promoting the MT production, followed by starch and CMC. When sucrose, starch and CMC were added, the MT productions get to 36.1 , 35.0 and 32.8 μg MT after 4 h incubation at 35˚C and pH 6.0, respectively, 1.17 , 1.13 and 1.05 times those in the control treatment (31.0 μg). For glucose, its supply only slightly enhanced the MT production, with a value of 32.4 μg. The increasing of production might be attributed to the reason that the available organic carbon sources could promote the growth and/or activity of Delftia sp. CHZG-R4 strain and thus enhance its uptake of Met, so that more Met were converted into MT. Quite similar, Arfi et al. (2003) also found the addition of glycerol and glucose significantly increased the production of volatile sulfur compounds from L-Met by Geotrichum candidum, because the carbon sources had a dramatic effect on the activity of aminotransferase, a key enzyme in L-Met catabolism. Fig. 4B showed the significant differences of MT production between control treatment (without other nitrogen source addition) and each experiment treatment (with other nitrogen source addition). The addition of urea and potassium nitrate obviously promoted the MT production, while the addition of ammonia sulfate and peptone notably decreased the MT production (p < 0.05). The MT productions in the treatments with the addition of urea and potassium nitrate were 34.5 and 33.7 μg, respectively, after 4 h incubation at 35˚C and pH 6.0, which were 1.11 and 1.10 times those in the control treatment (31.0 μg). One explanation for the enhancement of production was that the soluble organic nitrogen sources could promote the growth and/or 7
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activity of Delftia sp. CHZG-R4 strain and thus increase its consumption of Met, so that more MT were produced. However, when ammonia sulfate and peptone was added into the MBM, the MT productions were 26.0 and 4.01 μg, respectively, that is, 0.84 and 0.13 times those in the control treatment. This result could account for the reason that Delftia sp. CHZG-R4 strain prefer to ammonia sulfate and peptone as nitrogen sources or that the growth and/or activity of Delftia sp. CHZG-R4 strain was suppressed by ammonia sulfate and peptone, so that transformation of Met into MT by this strain was inhibited. Qiao et al. (2000) also found that the addition of ammonia nitrogen decreased the production of VOSCs from rice paddy soils. CONCLUSIONS Five MT-producing strains of WH-R1, WH-R2, WH-R3, SZLB-W3 and CHZG-R4 were isolated and characterized, utilizing Met as the sole carbon source for growth. WH-R1, WH-R2 and WH-R3 belong to the genus Bacillus, and SZLB-W3 and CHZG-R4 were identified as Arthrobacter sp. SZLB-W3 and Delftia sp. CHZG-R4, respectively. The strain of Delftia sp. CHZG-R4 was reported to produce MT for the first time, and had the highest capability in producing MT among five strains. Its optimal MT production appeared at 35 °C and pH 6.0. Under these conditions, Delftia sp. CHZG-R4 transferred 51.3% of sulfur content in Met into MT, and its MT production was increased above 10% by the addition of 2 g L-1 starch and 2 g L-1 sucrose, 2 g L-1 urea and 2 g L-1 potassium nitrate after 4 h incubation, but decreased 16% and 87% by the provision of 2 g L-1 ammonia sulfate and 2 g L-1 peptone, respectively. The results might provide useful information for the development of microbial resources and the microbial mechanism of MT production from agricultural soils. ACKNOWLEDGEMENTS REFERENCES Andreae, M. O. and Crutzen, P. J. 1997. Atmospheric aerosols: biogeochemical source and roles in atmospheric chemistry. Science. 276: 1052-1058. Arfi, K., Tâche, R., Spinnler, H. E. and Bonnarme, P. 2003. Dual influence of the carbon source and L-methionine on the synthesis of sulphur compounds in the cheese-ripening yeast Geotrichum candidum. Appl Microbiol Biotechnol. 61: 359-365. Bentley, R. and Chasteen, T. G. 2004. Environmental VOSCs-formation and degradation of dimethyl sulfide, methanethiol and related materials. Chemosphere. 55: 291-317. Bonnarme, P., Psoni, L. and Spinnler, H. E. 2000. Diversity of L-methionine catabolism pathways in cheese-ripening bacteria. Appl Environ Microbiol. 66: 5514-5517. Bremner, J. M. and Bundy, L. G. 1974. Inhibition of nitrification in soils by volatile sulfur compounds. Soil Biol Biochem. 6: 161-165. Burra, R., Pradenas, G. A., Montes, R. A., Vásquez, C. C. and Chasteen, T. G. 2010. Production of dimethyl triselenide and dimethyl diselenenyl sulfide in the headspace of metalloid-resistant Bacillus species grown in the presence of selenium oxyanions. Anal Biochem. 396: 217-222. Buzzini, P., Romano, S., Turchetti, B., Vaughan, A., Pagnoni, U. M. and Davoli, P. 2005. Production of volatile organic sulfur compounds VOSCs by basidiomycetous yeasts. FEMS Yeast Res. 5: 379-385. Charlson, R. J., Lovelock, J. E., Andreae, M.O. and Warren, S. G. 1987. Oceanic phytoplankton, atmospheric sulphur, cloud albedo and climate. Nature. 326: 655-661. Cox, R. A.and Sheppard, D. 1980. Reactions of OH radicals with gaseous sulphur compounds. Nature. 284: 330-331. de Souza, M. P. and Yoch, D. C. 1995. Purification and characterization of dimethylsulfoniumpropionate lyase from an Alcaligenes-like dimethyl sulfide-producing marine isolate. Appl Environ Microbiol. 61: 21-26. Drotar, A., Burton, A., Tavernier, J. E. and Fall, R. 1987. Widespread occurrence of bacterial thiol 8
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methyltransferases and the biogenic emission of methylated sulfur gases. Appl Environ Microbiol. 53: 1626-1631. Food and Agricultural Organization FAO 2013 FAO Statistical Yearbook 2013 FAOSTAT. Available at http://issuu.com/faooftheun/docs/syb2013issuu Graff, A. and Stubner, S. 2003. Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil. Syst Appl Microbiol. 26: 445-452. Hatakeyama, S. and Akimoto, H. 1983. Reactions of OH radicals with methanethiol, dimethyl sulfide, and dimethyl disulfide in air. J Phsy Chem. 87: 2387-2395. Higgins, M. J., Chen, Y. C., Yarosz, D. P., Murthy, S. N., Maas, N. A., Glindemann, D. and Novak, J. T. 2006. Cycling of volatile organic sulfur compounds in anaerobically digested biosolids and its implications for odors. Water Environ Res. 78: 243-252. Insam, H. and Seewald, M. 2010. Volatile organic compounds VOCs in soils. Bio Fert Soils. 46: 199-213. Kadota, H. and Ishida, Y. 1972. Production of volatile sulfur compounds by microorganisms. Annu Rev Microbiol. 26: 127-138. Lei, Q. and Boatright, W. L., 2001. Development of a new methanethiol quantification method using ethanethiol as an internal standard. J Agr Food Chem. 49: 3567-3572. Liu, Z., Yang, H., Huang, Z., Zhou, P. and Liu, S. J. 2002. Degradation of aniline by newly isolated, extremely aniline-tolerant Delftia sp. AN3. Appl Microbiol Biotechnol. 58: 679-682. Lomans, B. P., Van der Drift, C., Pol, A. and Op den Camp, H. J. M. 2002. Microbial cycling of volatile organic sulfur compounds. Cell Mol life Sci. 59: 575-588. Patil, N. K., Kundapur, R., Shouche, Y. S. and Karegoudar, T. B. 2006. Degradation of plasticizer di-n-butylphathalate by Delftia sp.TBKNP-05. Curr Microbiol. 52: 369-374. Qiao, W. C., Xi, S. Q., Zhang, J. H. and Yang, Z. 2000. The influence of physcio-chemical factors on the emission of volatile sulfur gases from soil. Environ Sci. (in Chinese). 01: 78-80. Rappert, S. and Müller, R. 2005. Odor compounds in waste gas emissions from agricultural operations and food industries. Waste Manage. 25: 887-907. Saari, A. and Martikainen, P. J. 2003. Dimethyl sulphoxide DMSO and dimethyl sulphide DMS as inhibitors of methane oxidation in forest soil. Soil Biol Biochem. 35: 383-389. Segal, W. and Starkey, R. L. 1969. Microbial decomposition of methionine and identity of the resulting sulfur products. J Bacteriol. 98: 908-913. Sreekumar, R., Al-Attabi, Z., Deeth, H. C. and Turner, M. S., 2009. Volatile sulfur compounds produced by probiotic bacteria in the presence of cysteine or methionine. Lett Appl Microbiol. 48: 777-782. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 28: 2731-2739. Tanaka, H., Esaki, N. and Soda, K. 1985. A versatile bacterial enzyme: L-methionine γ-lyase. Enzyme Microb Technol. 7: 530-537. Tang, M. Q. and You, M. S. 2012. Isolation, identification and characterization of a novel triazophos-degrading Bacillus sp. TAP-1. Microbio Res. 167: 299-305. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882 Ting, A. S. W., Mah, S. W. and Tee, C. S. 2011. Detection of potential volatile inhibitory compounds produced by endobacteria with biocontrol properties towards Fusarium oxysporum f. sp. cubense race 4. World J Microbiol Biotechnol. 27: 229-235. 9
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Watts, S. F. 2000. The mass budgets of carbonyl sulfide, dimethyl sulfide, carbon disulfide and hydrogen sulfide. Atmos Environ. 34: 761-779. Wen, A., Fegan, M., Hayward, C., Chakraborty, S. and Sly, L. I. 1999. Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans den Dooren de Jong 1926 and Tamaoka et al. 1987 gen. nov., comb. nov. Int J Syst Evol Micr. 49: 567-576. Yoshimura, M., Nakano, Y., Yamashita, Y., Oho, T., Saito, T. and Koga, T. 2000. Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis. Infect Immun. 68: 6912-6916. Zhao, J., Wu, X. B., Nie, C. P., Wu, T., Dai, W. H., Liu, H. and Yang, R, Y. 2012. Analysis of unculturable bacterial communities in tea orchard soils based on nested PCR-DGGE. World J Microbiol Biotechnol. 28: 1967-1979. Zhang, C. Z., Zhang, W. J. and Xu, J. 2013. Isolation and identification of methanethiol-utilizing bacterium CZ05 and its application in bio-trickling filter of biogas. Bioresource Technol. 150: 338-343.
ACKNOWLEDGEMENT
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This research was upported by the Natural Science Foundation of China (41273095, 41025012 and 41103067) TABLE I
The basic characteristics of the soil samples used for enrichment and isolation of MT-producing strains. Longitude
sites
and Latitude
Wuhu
E118o35′14′′,
pH
Total C -1
(g kg ) 5.2
17.9
Total N
Total S
M
Sampling
-1
-1
OM
a)
-1
Available P
Available K
-1
-1
Available S
(g kg )
(g kg )
(g kg )
(mg kg )
(mg kg )
(mg kg-1)
5.16
1.74
23.8
7.35
65.7
36.8
o
E117o44′50′′,
Chaohu
6.2
27.2
3.75
1.67
22.1
14.3
98.7
47.4
8.2
21.0
4.96
2.15
12.5
11.0
78.0
39.0
o
N 31 46′52′′ E117o35′04′′,
ce pt
Suzhou
ed
N 31 16′04′′
o
N 33 32′49′′
a) organic matter. TABLE II
Ac
Morphological, physiological and biochemical characteristics of the five MT-producing strains.
Characteristics Colony characteristics
Shape of cells Oxygen Gram staining Oxidase Catalase Starch hydrolysis Glucose fermentation
WH-R1 a)
WH-R2
WH-R3
CHZG-R4
SZLB-W3
Pale yellow Opaque Smooth Moist Rod +w + + + + +
Pale yellow Opaque Smooth Moist Rod +w + + + + +
Pale yellow Opaque Smooth Moist Rod +w + + + + +
White Transluscent Smooth Moist Rod +w – + + – –
Yellow Transluscent Smooth Moist Coccus +w – – + + +
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Nitrate reduction a)
–
–
–
+
+, positive reaction; –, negative reaction; w, weak positive.
TABLE III MT productions of the five MT-producing strains in 5 mL culture with OD600 nm of 1.0 incubated at 30˚C and pH 7.0 for 4 h.
MT Production (μg)
Accession No.
a)
Sulfur conversion (%)
b)
KJ492944
0.30 ± 0.02 b
Bacillus sp. WH-R2
KJ492945
0.26 ± 0.01 b
0.28
Bacillus sp. WH-R3
KJ492943
0.22 ± 0.02 b
0.23
Arthrobacter sp. SZLB-W3 Delftia sp. CHZG-R4
KJ492947 KJ492946
0.05 ± 0.002 b 21.0±0.44 a
0.05 21.8
a)
Mean ± SD of three replicates.
b)
Means followed by the same letter are not significantly different at P < 0.05.
0.31
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Bacillus sp. WH-R1
t
Strains
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Bacillus megaterium IAM 13418 T (D16273) 98
Bacillus aryabhattai B8W22 T (EF114313) WH-R2 (KJ 492945)
97
99
WH-R3 (KJ492943) WH-R1 (KJ492944)
100
Bacillus flexus IFO 15715T (AB021185) 100
M
Bacillus paraflexus RC2 T (FN999943)
Bacillus koreensis BR030 T (AY667496) Arthrobacter gyeryongensis DCY72 T (JX141781)
98
ed
Arthrobacter ramosus CCM 1646 T (AM039435)
100
SZLB-W3 (KJ492947) Arthrobacter nitroguajacolicus G2-1 T (AJ512504)
84
ce pt
99
Arthrobacter aurescens DSM 20116 T (X83405)
Xenophilus aerolatus 5516S-2 T (EF660342) Delftia litopenaei wsw-7 T (GU721027)
100
Delftia acidovorans ACM 489 T (AF078774)
100
65
Ac
0.02
Delftia tsuruhatensis T7 T (AB075017)
99 CHZG-R4 (KJ492946) 60 Delftia lacustris DSM 21246 T (EU888308)
Fig. 1 Phylogenetic tree generated by the neighbor-joining method on the basis of partial 16S rRNA sequences showing the phylogenetic relationships between MT-producing bacteria and the blast sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at major branching points.
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40
40
MT production (μg)
A
a
30 b 20
c d
10
a
B
MT production (μg)
a
e
b
30
c d
20
e
de
10
f
f
f
0
0 20
25
30
35
40
45
4
50
5
6
7
o
8
9
10 11
pH
Temperature ( C)
t
Fig. 2 Effects of temperature (A) and pH (B) on MT production by Delftia sp. CHZG-R4 in 5 mL culture with OD600 nm of 1.0
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incubated for 4 h. Error bars, mean ± SD of three replicates. Bar with different letters are significantly different at 0. 05 level according to a LSD test.
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Initial pH Incubated after 4 hours
10
pH
8
4 2 2
3
4 5 Samples
6
7
8
ed
1
M
6
30 20 10 0
c
bc
a
a
40
b
Ac
MT production (μg)
A
trol cose Starch rcose CMC Con Glu Su
MT production (μg)
40
ce pt
Fig. 3 pH changes of Delftia sp. CHZG-R4 suspension before and after incubation at 35˚C for 4 h.
a B
30
b
a
c
20 10 0
d
trol lfate Urea nitrate ptone Con nia su Pe o sium s m a t Am Po
Fig. 4 Effects of carbon sources (A) and nitrogen sources (B) on MT production by Delftia sp. CHZG-R4 in 5 mL culture with OD600 nm of 1.0 incubated at 35˚C and pH 6.0 for 4 h. Error bars, mean ± SD of three replicates. CMC, carboxymethylcellulose sodium. Bar with different letters are significantly different at 0.05 level according to a LSD test.
12