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A Novel Method for Enhancing Strains’ Biodegradation of 4-Chloronitrobenzene
Accepted Manuscript Title: A Novel Method for Enhancing Strains’ Biodegradation of 4-Chloronitrobenzene Authors: Tian Li, Tian C. Zhang, Lin He PII: DOI: Reference:
S0168-1656(17)31690-5 https://doi.org/10.1016/j.jbiotec.2017.10.005 BIOTEC 8031
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
14-6-2017 4-10-2017 9-10-2017
Please cite this article as: Li, Tian, Zhang, Tian C., He, Lin, A Novel Method for Enhancing Strains’ Biodegradation of 4-Chloronitrobenzene.Journal of Biotechnology https://doi.org/10.1016/j.jbiotec.2017.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel Method for Enhancing Strains’
Biodegradation of
4-Chloronitrobenzene
Tian Lia, Tian C. Zhangb, Lin Hea a
Southwest University, Chongqing 400715, P. R. China Civil Engineering Department, University of Nebraska-Lincoln, Omaha, NE, USA
b
* Corresponding author: Pro. Lin He, Southwest University, Chongqing 400715, P. R. China.
Tel:
(86)-23-68251269; Fax: (86)-23-68251269；E-mail: [email protected]
Highlights：
A novel method for effectively enhancing strains’ 4-chloronitrobenzene biodegradation rate was established.
External nitrogen (not carbon) source could enhance the rate of 4-chloronitrobenzene biodegradation.
The organic reductant and the substrate had synergistic effect on 4-chloronitrobenzene biodegradation. The method has a good prospect to enhance biodegradation of other nitroaromatic compounds.
ABSTRACT: This paper introduces a novel approach to enhance the strains’ biodegradation of 4-chloronitrobenzene by utilizing the synergistic effect of the organic reductant mannitol and the substrate beef extraction. Our results demonstrate that 4-chloronitrobenzene could not be an available nitrogen source to support target strains’ growth, which induced the limited 4-chloronitrobenzene biodegradation. In addition, the organic reducing agent and substrate had a better synergistic effect than inorganic reducing agent and substrate to enhance the strains’ 4-chloronitrobenzene cometabolic biodegradation. Employing the synergistic effect of the optimal mixture (mannitol and beef extraction), the biodegradation rates of 50 mg L-1 4-chloronitrobenzene by seven of the ten target strains were enhanced up to 100% from
previous removals of no more than 19.1% after 7 days. Three of the strains could even completely degrade 100 mg L-1 4-chloronitrobenzene while five strains degraded over 91.4%. The
method
has
good
potential
to
enhance
bioremediation
of
various
4-Chloronitrobenzene-contaminated environments as mannitol and beef extraction are non-toxic to the environment.
Keywords: 4-chloronitrobenzene; cometabolic biodegradation; synergistic effect; organic reducing agent; nitrogen source; method
1. Introduction Microbial degradation of chemical compounds is an important route for removal of hazardous organic pollutants in the environment (Amado et al., 2015). However, the biodegradation process can be lengthy, especially for sudden, accidental toxic releases, because most native microorganisms have limited ability for biodegradation of toxic compounds. A common approach for screening highly efficient microorganisms is to collect samples from contaminated sites and then to enrich these microbes in the laboratory. However, such approach usually is quite time consuming, labor intensive, and costly. Thus, it would be beneficial to find a universal, simple approach that could effectively enhance the biodegradation ability of refractory organic pollutants. 4-chloronitrobenzene, as an important nitroaromatic compound, has been widely used (e.g., drug manufacture, herbicides, photographic chemicals, dyes, antioxidants) for many years (Xu et al., 2016). 4-Chloronitrobenzene causes methemoglobinemia and is potentially
carcinogenic and genotoxic to humans (Xu and Zhou, 2017). The nitro and chloride groups are the essential functional groups for 4-chloronitrobenzene’s antibiotic properties (Shukor et al., 2009). It was reported that not all microorganisms can reduce 4-chloronitrobenzene at a low redox potential, demonstrating its biodegradation being very difficult (Ma et al., 2007; Xiao et al., 2006). To date, several 4-Chloronitrobenzene-degrading microorganisms have been screened (Corbett and Corbett, 1981; Gvozdiak et al., 1983; Katsivela et al., 1999; Wu et al., 2006; Zhang et al., 2013; Zhen et al., 2006), but most of their biodegradation rates were not satisfying. Due to the strong electron affinity of the nitro or chloride groups of 4-chloronitrobenzene, increasing the electron transfer could enhance 4-chloronitrobenzene reduction (Guo et al., 2015; Xu et al., 2016); adding reducing agents could also increase the biodegradation rate of strains for nitroaromatic compounds (Klausen et al., 2003; Xu et al., 2011). However, there has been no report yet on adding organic reducing agents for enhancing 4-chloronitrobenzene biodegradation. On the other hand, co-metabolism has been used in the field for more than 20 years for bioremediation of recalcitrant contaminants through the introduction of substrates to stimulate biomass growth ( Ambrósio and Campos-Takaki, 2004). There have been reports of microbial co-metabolism of nitroaromatic compounds, but only one study determined the co-metabolic pathway of 4-chloronitrobenzene with the addition of succinate (Park et al., 1999). Our previous study has established a novel approach that could efficiently enhance strains’ nitrobenzene biodegradation rate by utilizing the synergistic effect of organic reductants and a substrate (Li et al., 2017). In the present study, we found that the strains’
4-chloronitrobenzene biodegradation efficiency was greatly enhanced with the synergistic effect of the organic reductant mannitol and the substrate beef extraction. We demonstrated that the method could have great potential in dealing with 4-chloronitrobenzene contaminated environments, and may also enhance strains’ biodegradation rate of other nitroaromatic compounds.
2. Materials and methods 2.1 Sampling and culturing strains The samples, representing eight distinct types of soil: sediment, active sludge, arable soils used for cultivation of corn, vegetable gardens, and river shore soils, etc., were obtained from different sites in China (Table 1). All samples were taken from the upper 5 cm of the site and stored in glass bottles at 4°C for no more than 2 days before processing. In order to isolate a certain amount of strains for 4-chloronitrobenzene degradation, aerobic cultivation was conducted in 250 mL Erlenmeyer flasks, each flask with one gram of each soil sample with 50 mL of lysogeny broth (LB) medium as a carbon and energy source, and 4-chloronitrobenzene (an initial concentration of 50 mg/L) as a co-substrate. The flasks were incubated on a rotary shaker (SPH-103B, SHIPING Temperature, Shanghai, China) at 150 rpm and at 30 °C for 7 days. Then, each culture was serially diluted in sterilized saline solution (NaCl 0.85%, wt/vol), and spread on a Petri dish with solid LB (SLB) and incubated at 28 °C for 4−5 days. Individual colonies grown on the SLB with distinct morphologies were picked and purified by sequentially culturing on the same medium. Culture purity was confirmed by microscopic examination (Alvarez et al., 2009). Finally, the pure colonies were transferred into mineral salt medium (MSM) (Yi et al., 2006), which contained 50 mg L-1
4-chloronitrobenzene, to investigate their ability to degrade 4-chloronitrobenzene under aerobic conditions. In this study, ten target strains were isolated (as per the aforementioned procedure) and used as candidates. To collect the selected strains, 50 mL of each of the cultural solutions were taken from the 250 mL flask, and strains were harvested by centrifugation (14438g at 4 ℃ for 10 min) and washed twice with 50 m mol L-1 potassium phosphate buffer (pH 7.5). Washed cells were re-suspended in 1 mL of MSM, which was ready as the strain for inoculation in the tests below. Fig. 1A and Fig. 1B show a phylogenetic tree of the 16S rDNA of the strains compared with the sequences available in the GenBank database. 2.2 Experimental design Batch experiments were conducted in 250 mL bottles filled with 50 mL reaction solution and placed in the aforementioned rotary shaker at 30°C and 150 rpm for a certain time period (e.g., 7 days). Five sets of the tests were designed as shown in Table 2 and conducted. All of the 50 mL reaction solutions contained 1) the strain re-suspended in 1 mL of MSM; and 2) 49 mL MSM, but some of them also contained additional reagents as below: Set 1: for testing the biodegradation of the strain for 4-Chloronitrobenzene and the results were used as a benchmark: the 50 mL reaction solution contained 1) and 2), plus 3) 4-chloronitrobenzene (with a final concentration = 50 mg L-1). Set 2: for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of additional substrates, such as a carbon source (i.e., 1% sucrose, 1% glucose) or a nitrogen source (i.e., 1% peptone) or both a carbon and nitrogen source (i.e., 1% beef extract): the 50 mL reaction solutions contained 1) to 3) as in Set 1, and 4) 1% of each of the additional substrates (%,
w/v). Set 3: for testing the biodegradation of the strains for 4-Chloronitrobenzene under the influence of each of the selected reductants [i.e., Fe2+, Fe2+/Fe (1:1 ratio), VC (= L-lactose), mannitol]: the 50 mL reaction solution contained 1) to 3) as in Set 1, and 5) 0.02% of each of the additional reductants (%, w/v). Set 4: for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of adding one of the 4 reductants and one substrate (beef extract): the 50 mL reaction solution contained 1) to 3) as in Set 1, and 5) 0.02% of each of the 4 reductants [i.e., Fe2+, Fe2+/Fe (1:1 ratio), VC, mannitol] (%, w/v) and 1% of beef Extract. Set 5: for testing the biodegradation of the strain for 4-chloronitrobenzene of a higher concentration and under the influence of additional substrate and reductant: the 50 mL reaction solution contained 1) and 2), plus 3) 4-chloronitrobenzene (with a final concentration = 100 mg L-1, and 5) 0.02% of each of the 2 reductants (VC, mannitol ) and 1% of beef extract. Control tests with no strains inoculation in the flask were conducted as well for all test sets. In all sets, the strains’ density (OD) and 4-Chloronitrobenzene concentrations were measured after 7 days; each experiment was replicated four times (n = 4). 2.3 Analytical methods 4-Chloronitrobenzene was measured using high-performance liquid chromatography (HPLC) (Agilent 1260, Wilmington, DE, USA) equipped with an Agilent Extend-C18 column (150 mm×4.6 mm). The analysis was performed with a flow of methanol/water (v/v, 7:3) at a rate of 1.0 mL min-1. Bacterial cell density was measured as OD600 and 4-chloronitrobenzene concentrations were determined by HPLC.
3. Results 3.1 Phylogenetic analysis of 4-Chloronitrobenzene-degrading strains The strains were isolated from different conditions and locations (Table1). Fig. 1A and Fig.1B show that the ten novel 4-chloronitrobenzene-degrading strains were Staphylococcus carnosus strain S2 (S.c S2), Bacillus amyloliquefaciens lihwang YX0 (B.a YX0), Bacillus subtilis lihwang strian YX3 (B.s YX3), Bacillus cereus lihwang Y10 (B.c Y10), Bacillus cereus lihwang YX2 (B.c YX2), Serratia marcescens LWJ1 (S.m LWJ1), Lysinibacillus fusiformis strain LWJ2 (L.f LWJ2), Stenotrophomonas maltophilia strain LWJ3 (S.m LWJ3), Xanthomonas retroflexus strain LWJ4 (X.r LWJ4) and Staphylococcus equorum strain LWJ5 (S.e LWJ5). In Fig. 1A, 5 4-chloronitrobenzene-degrading strains previously reported by others are also listed.
(Niu et al., 2009; Wu et al., 2006; Zhang et al.,
2013). Phylogenetic trees reveal the relationship of the fifteen strains, and all of them fall into the two phylums of firmicutes and proteobacteria. Fig. 1A shows a phylogenetic tree of proteobacteria grouped into 3 clusters. The first cluster of gammaproteobacterial class, pseudomonas putida ZWL73，S.m LWJ1，X.r LWJ4 and S.m LWJ3 falls into three orders (Pseudomonadales, Enterobacteriales，and Xanthomonadales), while the second cluster contains Acidovorax sp. LW1, comamonas testosteroni CNB-1 and burkholderia sp. CAN6, which fall into the class Betaproteobacteria, while the third cluster contains Sphingomonas sp. CNB3, falling into the gammaproteobacterial class. The seven strains in Fig. 1B exhibit low divergence in the 16S rRNA sequence. They are members of the Firmicutes, falling into the same Bacillales order. In this tree, the four strains B.c Y10, B.c YX2, B.a YX0 and B.s YX3 are of the Bacillus genus, while strains S.c S2 and
S.e LWJ5 are of the Staphylococcaceae genus, and strain L.f LWJ2 is of the Lysinibacillus genus. Thus, the seven target strains are closely related genetically, while the other three target strains (S.m LWJ1, X.r LWJ4 and S.m LWJ3) are not so closely related. 3.2 Effect of substrate on cometabolic biodegradation of 4-chloronitrobenzene by the strains As shown in Fig. 2A, the ten target strains’ 4-chloronitrobenzene-degrading ability was limited. 4-chloronitrobenzene removal was 7.1%, 7.6%, 18.9%, 8.4%,7.6%, 13.6%, 6.2%, 13.5%, 7.0% and 11.0% over the course of 7 days for the ten strains (B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3), respectively (Fig. 2A). The OD values of the strains exhibited a similar tendency as 4-chloronitrobenzene removal with the highest OD values of strain LWJ1 being 0.061 and the lowest OD values of strain LWJ2 being 0.031 (Fig. 2B), which indicates that 4-chloronitrobenzene could hardly support the target strains’ growth, possibly one major reason that the strains had a limited degradation of 4-chloronitrobenzene. One option to effectively enhance the strains’ 4-chloronitrobenzene-degrading capability is to increase the biomass of microorganisms. Thus, we firstly analyzed how the additional carbon source (i.e., glucose and sucrose) would impact the ten target strains’ 4-chloronitrobenzene degradation rate and the OD values of the strains. Fig. 2B shows that each of the additional carbon sources has little impact on strains’ growth as compared with the basic culture (MSM), which led to almost no variation in the 4-chloronitrobenzene removal of the ten target strains (Fig. 2A). With glucose addition, the OD values of the ten strains ranged from 0.031 to 0.094, and the highest degradation rate of strain LWJ3 was just
16.2%, while the degradation rates of the other nine target strains appeared to be inhibited and ranged from 4.4% to 12.4%. With the additional sucrose, 4-chloronitrobenzene removal was slightly higher than those with the additional glucose, ranging from 14.1% to 27.6%, but the OD values showed a similar tendency, ranging from 0.075 to 0.146. This demonstrates that the additional carbon source could not effectively enhance the strains’ growth, resulting in the limited increase in 4-chloronitrobenzene-degradation rates of the ten strains. Then,
to assess the effect of
an
additional
nitrogen
source
on
the
strains’
4-chloronitrobenzene-degradation and their growth, 4-chloronitrobenzene removals were observed with the ten strains in the presence of 50 mg L-1 of 4-chloronitrobenzene and peptone. The OD values of the strains (B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3) were 1.24, 1.02, 1.46, 1.35, 1.17, 1.24, 1.78, 1.24, 1.39 and 1.12, respectively (Fig. 2B). At the same time, 4-chloronitrobenzene removal was found to increase to 47.7%, 45.0%, 62.9%, 44.0%, 45.9%, 64.8%, 53.4%, 55.8%, 57.8% and 52.7% for the strains of B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 or B.sYX3, respectively (Fig. 2A). This result indicates that the additional peptone source could support the ten strains’ growth, causing their 4-chloronitrobenzene-degradation rates to increase. Finally, we investigated how a primary substrate as an additional carbon and nitrogen source affects the strains’ 4-chloronitrobenzene degradation and their growth. With beef extract, the OD values of the ten strains were over 1.24, and all OD values were almost ten times those without beef extract and peptone addition (Fig. 2B). The 4-chloronitrobenzene removals of strains were increased, with six strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m
LWJ3, X.r LWJ4 and B.c YX2) having the 4-chloronitrobenzene removal over 60%. The lowest degrading rate of strain B.c Y10 was 43.1% and the rates of other three strains (Sc. S2, S.e LWJ5 and B.s YX3) were 52.0%, 52.6% and 54.2% (Fig. 2A). Fig. 2A clearly shows that the additional peptone or beef extract had a greater effect on the ten target strains’ biodegradation rates than additional glucose or sucrose. Taking into account all of these results, beef extract was screened for the optimal primary substrate in our following experiments. 3.3 Effects of reducing agent on biodegradation of 4-chloronitrobenzene by the strains According
to
previous
reports,
addition of
reducing
agents would
enhance
the biodegradation of nitroaromatic compounds (Li et al., 2017; Singh et al., 2014). Fig. 3A and Fig. 3B show that the 4-chloronitrobenzene removal of strains B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3 were increased to 19.7%, 25.0%, 24.7%, 22.8%, 13.7%, 23.1%, 34.2%, 21.5%, 24.4% and 23.6%, respectively, while their OD values did not vary significantly. In addition, Fig. 3B shows that, compared with the results from the MSM culture, the OD values had only limited enhancement with the addition of the other three reducing agents (Fe2+, Fe2+/Fe, and VC). Nevertheless, Fig. 3A shows that 4-chloronitrobenzene removal of some strains was increased with the addition of Fe2+, Fe2+/Fe, and VC. With the addition of Fe2+, the ten strains’ removal of 4-chloronitrobenzene increased to 9.2–31.6% as compared with 6.2–18.9% without Fe2+. When Fe2+/Fe (1:1) was added, all ten target strains’ 4-chloronitrobenzene degradation increased to 9.9–35.7% even though the corresponding OD values showed limited increase only, demonstrating that the addition of iron had limited
effect on strains’ growth. When VC was added, the OD values of the ten target strains ranged from 0.034 to 0.063, showing little variation. But the ten strains could degrade 4-chloronitrobenzene more effectively with the biodegradation efficiency of strains B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3 being 14.6%, 9.6%, 17.6%, 11.2%, 10.1%, 14.3%, 8.2%, 29.9%, 19.7% and 18.4%, respectively. 3.4
Synergistic
effect
of
the
organic
reductant
and
the
substrate
on
4-chloronitrobenzene biodegradation Our previous study found that the rates of strains’ nitrobenzene biodegradation were increased by utilizing the synergistic effect of organic reductants and a substrate. (Li et al., 2017). In the present study, the addition of organic reductant and substrate was tested to see if it has synergistic effect on 4-chloronitrobenzene degradation. Fig. 4 shows that with Fe2+ and beef extract, 4-chloronitrobenzene was completely removed by strain S2 and were 84.7%, 83.2%, 89.8%, 76.8%, 84.6%, 64.7%, 80%, 62.2%, and 82.5% for strains of B.a YX0 , S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c Y10, B.c YX2 and B.sYX3, respectively. With the addition of Fe2+/Fe (1:1) and beef extract, the degradation efficiencies were more effectively enhanced than those with the addition of both Fe2+ and beef extract (except that for B.c Y10), with 86.3%, 100%, 95.9%, 90.7, 78.4%, 96.8%, 73.9%, 92.7% and 87.5% for strains B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2 and B.sYX3, respectively. When VC and beef extract were added, 4-chloronitrobenzene could be completely removed by five strains (S.c S2, X.r LWJ4, S.e LWJ5, B.c YX2, B.s YX3) in 7 days, while
strains B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3 and B.c Y10 had a removal of
90.2%,
88.7%, 82.6%, 70% and 82%, respectively. Even with a higher concentration of 4-chloronitrobenzene (100 mgL-1), 100% was removed by strain S.c S2, and the other strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.s YX3) had a removal of 77.8%, 81.7%, 72.6%, 51.3%, 86.3%,98.4%, 99.0%, 66.3% and 85.0%, respectively (Fig. 5). With mannitol and beef extract, all the strains’ degrading abilities were efficiently enhanced, with the degradation of strains S.c S2, S.m LWJ1, L.f LWJ2, X.r LWJ4, S.e LWJ5, B.c YX2 and B.c Y10 reaching up to 100% after 7 days, and none of the other removals were less than 90%. When with 4-chloronitrobenzene of 100 mgL-1, the removal of strains S.c S2, X.r LWJ4 and B.c YX2 was 100% after 7 days, while the removal of the other seven strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3, S.e LWJ5, B.c Y10 and B.s YX3 ) was 92.5%, 97.3%, 94.6%, 91.4%, 95.2%, 79.8% and 88.1%, respectively. With the addition of four different mixtures (Fe2+/Fe + beef extract, Fe2+ + beef extract, VC + beef extract, or mannitol + beef extract ), the strains’ 4-chloronitrobenzene removal showed a similar tendency, i.e., a reductant and a substrate working better together than alone. However, Fig. 4 and Fig. 5 demonstrate that mixtures of organic reductant and substrate have a better synergistic effect on 4-chloronitrobenzene degradation than inorganic reductant and substrate. Since the synergistic effect of mannitol and beef extract was the most efficient on enhancing the strains’ 4-chloronitrobenzene biodegradation, we recommend the optimal additive of mannitol and beef extract.
4. Discussion
In our study, ten novel 4-chloronitrobenzene degrading bacteria were screened, with six of them falling into Bacillales order, which were previously reported to degrade nitrobenzene (Hu et al., 2008; Li et al., 2014; Wang et al., 2012; Zhao et al., 2011; Zheng et al., 2008). The microbial cometabolism, i.e. transformation of a non-growth substrate, namely co-substrate, in the obligate presence of a growth substrate by growing cells, or by resting cells in the absence of a growth substrate, is a significant part of the total biodegradative activity toward xenobiotic compounds (Nava et al., 2007). Hence, 4-chloronitrobenzene was biodegraded via cometabolic biodegradation pathway. As shown in Fig. 2A and Fig. 2B, the addition of a nitrogen source enhanced the strains’ removal of 4-chloronitrobenzene while increasing the strains’ biomass. In contrast, the addition of a carbon source had little effect on the strains’ removal of 4-chloronitrobenzene without increasing the strains’ biomass. These results indicate that nitrogen from 4-chloronitrobenzene was utilized by the strains to generate energy to drive metabolic reactions, not for the production of new biomass, while carbon from 4-chloronitrobenzene was utilized by the strains for the production of new biomass. Similar results were reported in previous studies. Bacillus licheniformis strainYX2 could not utilize nitrobenzene as the sole carbon and nitrogen source to grow. The addition of a nitrogen source (peptone) stimulated the bioreduction of nitrobenzene, whereas the addition of a carbon source had little effect (Li et al., 2014). On the other hand, M. odoratimimus strainYX6 and K. ornithinolytica strain NB1 could utilize nitrobenzene as the sole carbon and nitrogen source to grow, and the addition of carbon (glucose ) stimulated the bioreduction of nitrobenzene (Li et al., 2013; Wang et al., 2012). These reports demonstrate that whether the
bacteria could utilize nitroaromatic compound as a sole carbon and nitrogen source may decide the effectiveness of adding carbon source or nitrogen source to enhance strain’s biodegradation of nitroaromatic compounds. With the nitro and chloride groups, 4-chloronitrobenzene is both a chlorinated organic compound and a nitroaromatic compound. The removal of chlorinated organic compound by a microorganism is often limited because of a lack of electron donors (Aulenta et al., 2006; Bradley, 2003). Besides, oxygenases, which play a crucial role in the aerobic degradation of all aromatic compounds, cannot act on electron-deficient compounds, such as chlorinated organic compounds and nitroaromatic compounds (Arora et al., 2009). Under aerobic conditions, the reductive pathway of 4-chloronitrobenzene microbial degradation is initiated (Arora et al., 2012). In previous studies, 4-chloronitrobenzene was converted to an unstable intermediate 1-hydroxylamino-4-chlorobenzene by strain LW1 and strain CNB-1 (Katsivela et al., 1999; Wu et al., 2006); and 4-chloronitrobenzene could be transformed by Pseudomonas
sp.
strain
CBS3
to
4-chloroaniline,
N-acetyl-4-chloroaniline,
and
4-chloronitrosobenzene at low rates without any further degradation (Schackmann and Müller, 1991). Since oxidation and reduction are the processes of losing and gaining of electrons, the ion-exchange effect has been recognized as the main mechanism for remediation technology (Zeng et al., 2017). In addition, the addition of electron donors (e.g., glucose) could accelerate reduction of nitroaromatic antibiotic chloramphenicol, which has the same groups (chloro and nitro) as 4-Chloronitrobenzene (Liang et al., 2013). Fig. 3A shows that 4-chloronitrobenzene removal of the eight strains with an inorganic reducing agent were higher than those with an organic reducing agent, while the other two
strains had opposite results. The individual additional inorganic reducing agent can have a better effect on strains’ biodegradation of nitroaromatic comounds than the individual additional organic reducing agent, which may be a major reason why there is such limited research about the utilization of an organic reducing agent to reduce nitroaromatic compounds. In our experiments, the additional reducing agents could enhance the ten strains’ 4-chloronitrobenzene removal rates, but they had little impact on the strains’ growth at same time.
The
results
demonstrate
that
reducing
agents
can
enhance
the
strains’
4-chloronitrobenzene degrading ability. The iron (Fe2+ or Fe2+/Fe) as electron donors could enhance 4-chloronitrobenzene bioremediation, and the growth of the electron-donating power could contribute to increasing the strains’ 4-chloronitrobenzene bioremediation, which is consistent with the reports about catalytic reductants contributing to the nitroaromatic compound degradation (An et al., 2012; Luan et al., 2010; Salter-Blanc et al., 2015; Zeng et al., 2013). The ten target strains belong to eight different families, but the addition of reducing agents had similar effects on their growth and 4-chloronitrobenzene degradation, demonstrating that adding a reducing agent may generally promote the strains’ 4-Chloronitrobenzene-degrading ability. The additional reductant (mannitol) as electron donors catalyzed 4-chloronitrobenzene biodegradation without supporting the strains’ growth, and it didn’t utilize cometabolic method to enhance the 4-chloronitrobenzene biodegradation rate. The addition of beef extraction as the obligatory substrate to enhance the biomass of the strains induced enhancement of the 4-chloronitrobenzene cometabolic biodegradation rate. Therefore, the addition of mannitol and beef extract had synergistic effect on 4-chloronitrobenzene
degradation. Employing the new method, the rates of 50 mgL-1 4-Chloronitrobenzene biodegradation of seven target strains (S.c S2, S.m LWJ1, L.f LWJ2, X.r LWJ4, S.e LWJ5, B.c YX2 and B.c Y10) were enhanced from 7.6%, 18.9%, 8.4%, 13.6%, 6.2%, 13.5% and 7.0% in MSM up to 100%, respectively. In addition, three strains (S.c S2, X.r LWJ4 and B.c YX2) could completely remove 100 mgL-1 4-chloronitrobenzene, while the removal of five strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3, S.e LWJ5) ranged from 90% to 100%. Therefore, as long as a strain can degrade 4-chloronitrobenzene without using 4-chloronitrobenzene as its sole carbon and nitrogen source to support growth, this method of utilizing the synergistic effect of mannitol and beef extract to increase its degradation rate is effective. Because the ten strains were isolated from various environments, this method has the potential to deal with different 4-chloronitrobenzene-contaminated environments and could deal with wastewater with high 4-chloronitrobenzene concentrations. What’s more, the addition of mannitol and beef extract is non-toxic to the environment. Thus, the method has a good prospect to enhance remediation in nitroaromatic compounds contaminated environments. In addition, results of this study provide a clue for development of novel approaches to enrichment and culturing of microbes with enhanced biodegradation ability of refractory organic pollutants.
Acknowledgements We are grateful for the support of the National Key Research and Development Program of China (No：2017YFD 0200301).
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Xiao, Y., Wu, J., Liu, H., Wang, S., Liu, S., Zhou, N., (2006) Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonas putida ZWL73. Applied Microbiology & Biotechnology 73, 166-171. Xu, X., Lin, H., Zhu, L., Yang, Y., Feng, J., (2011) Enhanced biodegradation of 2-chloronitro-benzene using a coupled zero-valent iron column and sequencing batch reactor system. Journal of Chemical Technology & Biotechnology 86, 993-1000. Xu, X., Shao, J., Li, M., Gao, K., Jin, J., Zhu, L., (2016) Reductive Transformation of p-chloronitrobenzene in the upflow anaerobic sludge blanket reactor coupled with microbial electrolysis cell: performance and microbial community. Bioresource Technology 218, 1037-1045. Xu, Y., Zhou, N., (2017) Microbial remediation of aromatics-contaminated soil. Frontiers of Environmental Science & Engineering 11, 1-9. Yi, S., Zhuang, W., Wu, B., Tay, S.T., Tay, J.H., (2006) Biodegradation of p-nitrophenol by aerobic granules in a sequencing batch reactor. Environmental Science & Technology 40, 2396-2401. Zeng, G., Wan, J., Huang, D., Hu, L., Huang, C., Cheng, M., Xue, W., Gong, X., Wang, R., Jiang, D., (2017) Precipitation, adsorption and rhizosphere effect: The mechanisms for Phosphate-induced Pb immobilization in soils-A review. Journal of Hazardous Materials 339, 354-367.
Zeng, T., Zhang, X., Niu, H., Ma, Y., Li, W, Cai, Y., (2013) In situ growth of gold nanoparticles onto polydopamine-encapsulated magnetic microspheres for catalytic reduction of nitrobenzene. Applied Catalysis B Environmental 134-135, 26-33. Zhang, L., Wang, X., Jiao, Y., Chen, X., Zhou, L., Guo, K., Ge, F., Wu, J., (2013) Biodegradation of 4-chloronitrobenzene by biochemical cooperation between Sphingomonas sp. strain CNB3 and Burkholderia sp. strain CAN6 isolated from activated sludge. Chemosphere 91, 1243-1249. Zhao, D., Liu, C., Zhang, Y., Liu, Q., (2011) Biodegradation of nitrobenzene by aerobic granular sludge in a sequencing batch reactor (SBR). Desalination 281, 17-22. Zhen, D., Liu, H., Wang, S., Zhang, J., Zhao, F., Zhou, N., (2006) Plasmid-mediated degradation of 4-chloronitrobenzene by newly isolated Pseudomonas putida strain ZWL73. Applied Microbiology & Biotechnology 72, 797-803.
Zheng, C., Zhou, J., Wang, J., Qu, B., (2008) Isolation and characterization of a nitrobenzene degrading yeast strain from activated sludge. Journal of Hazardous Materials 160, 194-199.
Figure Captions Fig. 1. Neighbour-joining dendrogram of 16S rDNA sequences of 4-Chloronitrobenzene -degrading strains. Bootstrap values (in percentage of 1000 replicates) and DNA database accession numbers are indicated. Fig. 1
Proteobacteria
Pseudomonas putida ZWL73
89
Serratia marcescens LWJ1
73
Xanthomonas retroflexus LWJ4
100
Stenotrophomonas maltophili LWJ3 Burkholderia sp. CAN6 Acidovorax sp. LW1
99
Comamonas testosteroni CNB-1
98
Sphingomonas sp. CNB3
A
0.1
Firmicutes 10
Bacillus amyloliquefaciens YX0
0
6
Bacillus subtilis YX3 Staphylococcus carnosus S2
1 10
Staphylococcus equorum LWJ5
0 Bacillus cereus Y10 10 0
0.01
Bacillus cereus YX2 Lysinibacillus fusiformis LWJ2
B
Fig. 2. Effect of substrate (
MSM,
glucose,
sucrose,
peptone and
beef extration ) on the
% 4-Chloronitrobenzene removal
degradation of 4-Chloronitrobenzene and OD600 by the ten target strains. (Set 1 and Set 2) Fig. 2
A
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2 S.m LWJ3
X.r LWJ4
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Strain
OD600
B
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2 S.m LWJ3
Strain
X.r LWJ4
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Fig. 3. Effect of reductant (
MSM,
Fe2+,
Fe2+/Fe,
Vc,
mannitol) on the biodegradation of 50 mg
L-1 nitrobenzene and OD60 by the ten target strains. (Set 1 and Set 3)
% 4-Chloronitrobenzene removal
Fig. 3
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2 S.m LWJ3
X.r LWJ4 S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
B.c Y10
B.s YX3
OD600
Strain
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2
S.m LWJ3 X.r LWJ4
Strain
S.e LWJ5
B.c YX2
Fig. 4. Synergistic effect of reductant and substrate ( extract,
Vc and beef extract,
MSM,
Fe2+ and beef extract,
Fe2+/Fe and beef
Mannitol and beef extract) on the degradation of 50 mgL-1
4-Chloronitrobenzene by the ten strains. (Set 4)
% 4-Chloronitrobenzene removal
Fig. 4
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2
S.m LWJ3 X.r LWJ4
Strain
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Fig. 5. Synergistic effect of reductant and substrate (
Vc and beef extract,
Mannitol and beef extract) on
-1
the degradation of 100 mgL 4-Chloronitrobenzene by the ten strains. (Set 5)
%4-Chloronitrobenzene removal
Fig. 5
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2
S.m LWJ3 X.r LWJ4
Strain
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Table. 1. Identification and comparison of bacterial isolates from different locations Table 1.
Identification and comparison of bacterial isolates from different locations Isolate
Staphylococcus carnosus strain S2 （S.c S2） Bacillus amyloliquefaciens lihwang YX0（B.a YX0） Bacillus subtilis lihwang strian YX3 ( B.s YX3) Bacillus cereus lihwang Y10 ( B.c Y10) Bacillus cereus lihwang YX2 (B.c YX2) Serratia marcescens LWJ1 (S.m LWJ1 ) Lysinibacillus fusiformis strain LWJ2 (L.f LWJ2) Stenotrophomonas maltophilia strain LWJ3 (S.m LWJ3) Xanthomonas retroflexus strain LWJ4 (X.r LWJ4) Staphylococcus equorum strain LWJ5 (S.e LWJ5)
GenBank number KT932959
Reference strain
Bacterial taxonomy
% identity 99
Isolation source
Staphylococcus carnosus subsp. carnosus TM300 Bacillus amyloliquefaciens LFB112
Firmicutes; Bacilli; Bacillales; Staphylococcaceae ; Staphylococcus; Staphylococcus carnosus Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus amyloliquefaciens Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus subtilis Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus;Bacillus cereus
99
KM093861
Bacillus subtilis strain HDYM-28 Bacillus cereus F837/76
99
The active sludge of Henan The cornfield of Chongqing
KM093863
Bacillus cereus strain MT6
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus cereus
99
The vegetable garden of Chongqing
KT821502
Serratia marcescens WW4
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Serratia; Serratia marcescens
99
Jialingjiang river shore soil
KT861858
Lysinibacillus fusiformis strain S-1
99
The farm soil of Wuhan
KT932956
Stenotrophomonas maltophilia
Firmicutes; Bacilli; Bacillales; Bacillaceae; Lysinibacillus Lysinibacillus fusiformis Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Stenotrophomonas; Stenotrophomonas maltophilia
99
The Soil in orange grove
KT932957
Xanthomonas retroflexus
Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Xanthomonas; Xanthomonas retroflexus
99
The pool of Chongqing
KT932958
Staphylococcus equorum strain CTSP8
Firmicutes; Bacilli; Bacillales; Staphylococcaceae; Staphylococcus; Staphylococcus equorum
99
The meadow soil of Chongqing
KM403448 KM403447
99
The vegetable garden of Chongqing The farm soil of Chongqing
Table. 2. Experimental sets and conditions Table 2. Experimental sets and conditions Substrate
Reductant
4-Chloronitrobenzene
Experimental purpose
Set 1
-
-
+
for testing the biodegradation of the strain for 4-chloronitrobenzene and the results were used as a benchmark
Set 2
+
-
+
for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of additional substrates
Set 3
-
+
+
for testing the biodegradation of the strains for 4-chloronitrobenzene under the influence of each of the selected reductants
Set 4
+
+
+
for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of adding one of the 4 reductants and one substrate (beef extraction)
Set 5
+
+
+
for testing the biodegradation of the strain for 4-chloronitrobenzene of a higher concentration and under the influence of additional substrate and reductant
Note: All of the 50 mL reaction solutions contained 1) the strain re-suspended in 1 mL of MSM; and 2) 49 mL mineral salt medium (MSM). + represents the additional substance; － represents without the additional substance. In Set 2, substrate represents 1% sucrose, or 1% glucose, or 1% peptone or 1% beef extraction (W/W); in Set 3, reductant represents 0.02% Fe2+, or 0.02% Fe2+/Fe (1:1 ratio), or 0.02% VC (= L-lactose) or 0.02% mannitol (W/W); In set 4, substrate represents 1% beef extraction (W/W), and reductant represents 0.02% of each of the 4 reductants [i.e., Fe2+, Fe2+/Fe (1:1 ratio), VC, mannitol]; in Set 5, substrate represents 1% beef extraction (W/W), and reductant represents 0.02% of VC or mannitol (W/W). The concentration of 4-Chloronitrobenzene was 50 mg L-1 for Sets 1-4, and 100 mg L-1 for Set 5.
S0168-1656(17)31690-5 https://doi.org/10.1016/j.jbiotec.2017.10.005 BIOTEC 8031
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
14-6-2017 4-10-2017 9-10-2017
Please cite this article as: Li, Tian, Zhang, Tian C., He, Lin, A Novel Method for Enhancing Strains’ Biodegradation of 4-Chloronitrobenzene.Journal of Biotechnology https://doi.org/10.1016/j.jbiotec.2017.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel Method for Enhancing Strains’
Biodegradation of
4-Chloronitrobenzene
Tian Lia, Tian C. Zhangb, Lin Hea a
Southwest University, Chongqing 400715, P. R. China Civil Engineering Department, University of Nebraska-Lincoln, Omaha, NE, USA
b
* Corresponding author: Pro. Lin He, Southwest University, Chongqing 400715, P. R. China.
Tel:
(86)-23-68251269; Fax: (86)-23-68251269；E-mail: [email protected]
Highlights：
A novel method for effectively enhancing strains’ 4-chloronitrobenzene biodegradation rate was established.
External nitrogen (not carbon) source could enhance the rate of 4-chloronitrobenzene biodegradation.
The organic reductant and the substrate had synergistic effect on 4-chloronitrobenzene biodegradation. The method has a good prospect to enhance biodegradation of other nitroaromatic compounds.
ABSTRACT: This paper introduces a novel approach to enhance the strains’ biodegradation of 4-chloronitrobenzene by utilizing the synergistic effect of the organic reductant mannitol and the substrate beef extraction. Our results demonstrate that 4-chloronitrobenzene could not be an available nitrogen source to support target strains’ growth, which induced the limited 4-chloronitrobenzene biodegradation. In addition, the organic reducing agent and substrate had a better synergistic effect than inorganic reducing agent and substrate to enhance the strains’ 4-chloronitrobenzene cometabolic biodegradation. Employing the synergistic effect of the optimal mixture (mannitol and beef extraction), the biodegradation rates of 50 mg L-1 4-chloronitrobenzene by seven of the ten target strains were enhanced up to 100% from
previous removals of no more than 19.1% after 7 days. Three of the strains could even completely degrade 100 mg L-1 4-chloronitrobenzene while five strains degraded over 91.4%. The
method
has
good
potential
to
enhance
bioremediation
of
various
4-Chloronitrobenzene-contaminated environments as mannitol and beef extraction are non-toxic to the environment.
Keywords: 4-chloronitrobenzene; cometabolic biodegradation; synergistic effect; organic reducing agent; nitrogen source; method
1. Introduction Microbial degradation of chemical compounds is an important route for removal of hazardous organic pollutants in the environment (Amado et al., 2015). However, the biodegradation process can be lengthy, especially for sudden, accidental toxic releases, because most native microorganisms have limited ability for biodegradation of toxic compounds. A common approach for screening highly efficient microorganisms is to collect samples from contaminated sites and then to enrich these microbes in the laboratory. However, such approach usually is quite time consuming, labor intensive, and costly. Thus, it would be beneficial to find a universal, simple approach that could effectively enhance the biodegradation ability of refractory organic pollutants. 4-chloronitrobenzene, as an important nitroaromatic compound, has been widely used (e.g., drug manufacture, herbicides, photographic chemicals, dyes, antioxidants) for many years (Xu et al., 2016). 4-Chloronitrobenzene causes methemoglobinemia and is potentially
carcinogenic and genotoxic to humans (Xu and Zhou, 2017). The nitro and chloride groups are the essential functional groups for 4-chloronitrobenzene’s antibiotic properties (Shukor et al., 2009). It was reported that not all microorganisms can reduce 4-chloronitrobenzene at a low redox potential, demonstrating its biodegradation being very difficult (Ma et al., 2007; Xiao et al., 2006). To date, several 4-Chloronitrobenzene-degrading microorganisms have been screened (Corbett and Corbett, 1981; Gvozdiak et al., 1983; Katsivela et al., 1999; Wu et al., 2006; Zhang et al., 2013; Zhen et al., 2006), but most of their biodegradation rates were not satisfying. Due to the strong electron affinity of the nitro or chloride groups of 4-chloronitrobenzene, increasing the electron transfer could enhance 4-chloronitrobenzene reduction (Guo et al., 2015; Xu et al., 2016); adding reducing agents could also increase the biodegradation rate of strains for nitroaromatic compounds (Klausen et al., 2003; Xu et al., 2011). However, there has been no report yet on adding organic reducing agents for enhancing 4-chloronitrobenzene biodegradation. On the other hand, co-metabolism has been used in the field for more than 20 years for bioremediation of recalcitrant contaminants through the introduction of substrates to stimulate biomass growth ( Ambrósio and Campos-Takaki, 2004). There have been reports of microbial co-metabolism of nitroaromatic compounds, but only one study determined the co-metabolic pathway of 4-chloronitrobenzene with the addition of succinate (Park et al., 1999). Our previous study has established a novel approach that could efficiently enhance strains’ nitrobenzene biodegradation rate by utilizing the synergistic effect of organic reductants and a substrate (Li et al., 2017). In the present study, we found that the strains’
4-chloronitrobenzene biodegradation efficiency was greatly enhanced with the synergistic effect of the organic reductant mannitol and the substrate beef extraction. We demonstrated that the method could have great potential in dealing with 4-chloronitrobenzene contaminated environments, and may also enhance strains’ biodegradation rate of other nitroaromatic compounds.
2. Materials and methods 2.1 Sampling and culturing strains The samples, representing eight distinct types of soil: sediment, active sludge, arable soils used for cultivation of corn, vegetable gardens, and river shore soils, etc., were obtained from different sites in China (Table 1). All samples were taken from the upper 5 cm of the site and stored in glass bottles at 4°C for no more than 2 days before processing. In order to isolate a certain amount of strains for 4-chloronitrobenzene degradation, aerobic cultivation was conducted in 250 mL Erlenmeyer flasks, each flask with one gram of each soil sample with 50 mL of lysogeny broth (LB) medium as a carbon and energy source, and 4-chloronitrobenzene (an initial concentration of 50 mg/L) as a co-substrate. The flasks were incubated on a rotary shaker (SPH-103B, SHIPING Temperature, Shanghai, China) at 150 rpm and at 30 °C for 7 days. Then, each culture was serially diluted in sterilized saline solution (NaCl 0.85%, wt/vol), and spread on a Petri dish with solid LB (SLB) and incubated at 28 °C for 4−5 days. Individual colonies grown on the SLB with distinct morphologies were picked and purified by sequentially culturing on the same medium. Culture purity was confirmed by microscopic examination (Alvarez et al., 2009). Finally, the pure colonies were transferred into mineral salt medium (MSM) (Yi et al., 2006), which contained 50 mg L-1
4-chloronitrobenzene, to investigate their ability to degrade 4-chloronitrobenzene under aerobic conditions. In this study, ten target strains were isolated (as per the aforementioned procedure) and used as candidates. To collect the selected strains, 50 mL of each of the cultural solutions were taken from the 250 mL flask, and strains were harvested by centrifugation (14438g at 4 ℃ for 10 min) and washed twice with 50 m mol L-1 potassium phosphate buffer (pH 7.5). Washed cells were re-suspended in 1 mL of MSM, which was ready as the strain for inoculation in the tests below. Fig. 1A and Fig. 1B show a phylogenetic tree of the 16S rDNA of the strains compared with the sequences available in the GenBank database. 2.2 Experimental design Batch experiments were conducted in 250 mL bottles filled with 50 mL reaction solution and placed in the aforementioned rotary shaker at 30°C and 150 rpm for a certain time period (e.g., 7 days). Five sets of the tests were designed as shown in Table 2 and conducted. All of the 50 mL reaction solutions contained 1) the strain re-suspended in 1 mL of MSM; and 2) 49 mL MSM, but some of them also contained additional reagents as below: Set 1: for testing the biodegradation of the strain for 4-Chloronitrobenzene and the results were used as a benchmark: the 50 mL reaction solution contained 1) and 2), plus 3) 4-chloronitrobenzene (with a final concentration = 50 mg L-1). Set 2: for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of additional substrates, such as a carbon source (i.e., 1% sucrose, 1% glucose) or a nitrogen source (i.e., 1% peptone) or both a carbon and nitrogen source (i.e., 1% beef extract): the 50 mL reaction solutions contained 1) to 3) as in Set 1, and 4) 1% of each of the additional substrates (%,
w/v). Set 3: for testing the biodegradation of the strains for 4-Chloronitrobenzene under the influence of each of the selected reductants [i.e., Fe2+, Fe2+/Fe (1:1 ratio), VC (= L-lactose), mannitol]: the 50 mL reaction solution contained 1) to 3) as in Set 1, and 5) 0.02% of each of the additional reductants (%, w/v). Set 4: for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of adding one of the 4 reductants and one substrate (beef extract): the 50 mL reaction solution contained 1) to 3) as in Set 1, and 5) 0.02% of each of the 4 reductants [i.e., Fe2+, Fe2+/Fe (1:1 ratio), VC, mannitol] (%, w/v) and 1% of beef Extract. Set 5: for testing the biodegradation of the strain for 4-chloronitrobenzene of a higher concentration and under the influence of additional substrate and reductant: the 50 mL reaction solution contained 1) and 2), plus 3) 4-chloronitrobenzene (with a final concentration = 100 mg L-1, and 5) 0.02% of each of the 2 reductants (VC, mannitol ) and 1% of beef extract. Control tests with no strains inoculation in the flask were conducted as well for all test sets. In all sets, the strains’ density (OD) and 4-Chloronitrobenzene concentrations were measured after 7 days; each experiment was replicated four times (n = 4). 2.3 Analytical methods 4-Chloronitrobenzene was measured using high-performance liquid chromatography (HPLC) (Agilent 1260, Wilmington, DE, USA) equipped with an Agilent Extend-C18 column (150 mm×4.6 mm). The analysis was performed with a flow of methanol/water (v/v, 7:3) at a rate of 1.0 mL min-1. Bacterial cell density was measured as OD600 and 4-chloronitrobenzene concentrations were determined by HPLC.
3. Results 3.1 Phylogenetic analysis of 4-Chloronitrobenzene-degrading strains The strains were isolated from different conditions and locations (Table1). Fig. 1A and Fig.1B show that the ten novel 4-chloronitrobenzene-degrading strains were Staphylococcus carnosus strain S2 (S.c S2), Bacillus amyloliquefaciens lihwang YX0 (B.a YX0), Bacillus subtilis lihwang strian YX3 (B.s YX3), Bacillus cereus lihwang Y10 (B.c Y10), Bacillus cereus lihwang YX2 (B.c YX2), Serratia marcescens LWJ1 (S.m LWJ1), Lysinibacillus fusiformis strain LWJ2 (L.f LWJ2), Stenotrophomonas maltophilia strain LWJ3 (S.m LWJ3), Xanthomonas retroflexus strain LWJ4 (X.r LWJ4) and Staphylococcus equorum strain LWJ5 (S.e LWJ5). In Fig. 1A, 5 4-chloronitrobenzene-degrading strains previously reported by others are also listed.
(Niu et al., 2009; Wu et al., 2006; Zhang et al.,
2013). Phylogenetic trees reveal the relationship of the fifteen strains, and all of them fall into the two phylums of firmicutes and proteobacteria. Fig. 1A shows a phylogenetic tree of proteobacteria grouped into 3 clusters. The first cluster of gammaproteobacterial class, pseudomonas putida ZWL73，S.m LWJ1，X.r LWJ4 and S.m LWJ3 falls into three orders (Pseudomonadales, Enterobacteriales，and Xanthomonadales), while the second cluster contains Acidovorax sp. LW1, comamonas testosteroni CNB-1 and burkholderia sp. CAN6, which fall into the class Betaproteobacteria, while the third cluster contains Sphingomonas sp. CNB3, falling into the gammaproteobacterial class. The seven strains in Fig. 1B exhibit low divergence in the 16S rRNA sequence. They are members of the Firmicutes, falling into the same Bacillales order. In this tree, the four strains B.c Y10, B.c YX2, B.a YX0 and B.s YX3 are of the Bacillus genus, while strains S.c S2 and
S.e LWJ5 are of the Staphylococcaceae genus, and strain L.f LWJ2 is of the Lysinibacillus genus. Thus, the seven target strains are closely related genetically, while the other three target strains (S.m LWJ1, X.r LWJ4 and S.m LWJ3) are not so closely related. 3.2 Effect of substrate on cometabolic biodegradation of 4-chloronitrobenzene by the strains As shown in Fig. 2A, the ten target strains’ 4-chloronitrobenzene-degrading ability was limited. 4-chloronitrobenzene removal was 7.1%, 7.6%, 18.9%, 8.4%,7.6%, 13.6%, 6.2%, 13.5%, 7.0% and 11.0% over the course of 7 days for the ten strains (B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3), respectively (Fig. 2A). The OD values of the strains exhibited a similar tendency as 4-chloronitrobenzene removal with the highest OD values of strain LWJ1 being 0.061 and the lowest OD values of strain LWJ2 being 0.031 (Fig. 2B), which indicates that 4-chloronitrobenzene could hardly support the target strains’ growth, possibly one major reason that the strains had a limited degradation of 4-chloronitrobenzene. One option to effectively enhance the strains’ 4-chloronitrobenzene-degrading capability is to increase the biomass of microorganisms. Thus, we firstly analyzed how the additional carbon source (i.e., glucose and sucrose) would impact the ten target strains’ 4-chloronitrobenzene degradation rate and the OD values of the strains. Fig. 2B shows that each of the additional carbon sources has little impact on strains’ growth as compared with the basic culture (MSM), which led to almost no variation in the 4-chloronitrobenzene removal of the ten target strains (Fig. 2A). With glucose addition, the OD values of the ten strains ranged from 0.031 to 0.094, and the highest degradation rate of strain LWJ3 was just
16.2%, while the degradation rates of the other nine target strains appeared to be inhibited and ranged from 4.4% to 12.4%. With the additional sucrose, 4-chloronitrobenzene removal was slightly higher than those with the additional glucose, ranging from 14.1% to 27.6%, but the OD values showed a similar tendency, ranging from 0.075 to 0.146. This demonstrates that the additional carbon source could not effectively enhance the strains’ growth, resulting in the limited increase in 4-chloronitrobenzene-degradation rates of the ten strains. Then,
to assess the effect of
an
additional
nitrogen
source
on
the
strains’
4-chloronitrobenzene-degradation and their growth, 4-chloronitrobenzene removals were observed with the ten strains in the presence of 50 mg L-1 of 4-chloronitrobenzene and peptone. The OD values of the strains (B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3) were 1.24, 1.02, 1.46, 1.35, 1.17, 1.24, 1.78, 1.24, 1.39 and 1.12, respectively (Fig. 2B). At the same time, 4-chloronitrobenzene removal was found to increase to 47.7%, 45.0%, 62.9%, 44.0%, 45.9%, 64.8%, 53.4%, 55.8%, 57.8% and 52.7% for the strains of B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 or B.sYX3, respectively (Fig. 2A). This result indicates that the additional peptone source could support the ten strains’ growth, causing their 4-chloronitrobenzene-degradation rates to increase. Finally, we investigated how a primary substrate as an additional carbon and nitrogen source affects the strains’ 4-chloronitrobenzene degradation and their growth. With beef extract, the OD values of the ten strains were over 1.24, and all OD values were almost ten times those without beef extract and peptone addition (Fig. 2B). The 4-chloronitrobenzene removals of strains were increased, with six strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m
LWJ3, X.r LWJ4 and B.c YX2) having the 4-chloronitrobenzene removal over 60%. The lowest degrading rate of strain B.c Y10 was 43.1% and the rates of other three strains (Sc. S2, S.e LWJ5 and B.s YX3) were 52.0%, 52.6% and 54.2% (Fig. 2A). Fig. 2A clearly shows that the additional peptone or beef extract had a greater effect on the ten target strains’ biodegradation rates than additional glucose or sucrose. Taking into account all of these results, beef extract was screened for the optimal primary substrate in our following experiments. 3.3 Effects of reducing agent on biodegradation of 4-chloronitrobenzene by the strains According
to
previous
reports,
addition of
reducing
agents would
enhance
the biodegradation of nitroaromatic compounds (Li et al., 2017; Singh et al., 2014). Fig. 3A and Fig. 3B show that the 4-chloronitrobenzene removal of strains B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3 were increased to 19.7%, 25.0%, 24.7%, 22.8%, 13.7%, 23.1%, 34.2%, 21.5%, 24.4% and 23.6%, respectively, while their OD values did not vary significantly. In addition, Fig. 3B shows that, compared with the results from the MSM culture, the OD values had only limited enhancement with the addition of the other three reducing agents (Fe2+, Fe2+/Fe, and VC). Nevertheless, Fig. 3A shows that 4-chloronitrobenzene removal of some strains was increased with the addition of Fe2+, Fe2+/Fe, and VC. With the addition of Fe2+, the ten strains’ removal of 4-chloronitrobenzene increased to 9.2–31.6% as compared with 6.2–18.9% without Fe2+. When Fe2+/Fe (1:1) was added, all ten target strains’ 4-chloronitrobenzene degradation increased to 9.9–35.7% even though the corresponding OD values showed limited increase only, demonstrating that the addition of iron had limited
effect on strains’ growth. When VC was added, the OD values of the ten target strains ranged from 0.034 to 0.063, showing little variation. But the ten strains could degrade 4-chloronitrobenzene more effectively with the biodegradation efficiency of strains B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.sYX3 being 14.6%, 9.6%, 17.6%, 11.2%, 10.1%, 14.3%, 8.2%, 29.9%, 19.7% and 18.4%, respectively. 3.4
Synergistic
effect
of
the
organic
reductant
and
the
substrate
on
4-chloronitrobenzene biodegradation Our previous study found that the rates of strains’ nitrobenzene biodegradation were increased by utilizing the synergistic effect of organic reductants and a substrate. (Li et al., 2017). In the present study, the addition of organic reductant and substrate was tested to see if it has synergistic effect on 4-chloronitrobenzene degradation. Fig. 4 shows that with Fe2+ and beef extract, 4-chloronitrobenzene was completely removed by strain S2 and were 84.7%, 83.2%, 89.8%, 76.8%, 84.6%, 64.7%, 80%, 62.2%, and 82.5% for strains of B.a YX0 , S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c Y10, B.c YX2 and B.sYX3, respectively. With the addition of Fe2+/Fe (1:1) and beef extract, the degradation efficiencies were more effectively enhanced than those with the addition of both Fe2+ and beef extract (except that for B.c Y10), with 86.3%, 100%, 95.9%, 90.7, 78.4%, 96.8%, 73.9%, 92.7% and 87.5% for strains B.a YX0, S.c S2, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2 and B.sYX3, respectively. When VC and beef extract were added, 4-chloronitrobenzene could be completely removed by five strains (S.c S2, X.r LWJ4, S.e LWJ5, B.c YX2, B.s YX3) in 7 days, while
strains B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3 and B.c Y10 had a removal of
90.2%,
88.7%, 82.6%, 70% and 82%, respectively. Even with a higher concentration of 4-chloronitrobenzene (100 mgL-1), 100% was removed by strain S.c S2, and the other strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3, X.r LWJ4, S.e LWJ5, B.c YX2, B.c Y10 and B.s YX3) had a removal of 77.8%, 81.7%, 72.6%, 51.3%, 86.3%,98.4%, 99.0%, 66.3% and 85.0%, respectively (Fig. 5). With mannitol and beef extract, all the strains’ degrading abilities were efficiently enhanced, with the degradation of strains S.c S2, S.m LWJ1, L.f LWJ2, X.r LWJ4, S.e LWJ5, B.c YX2 and B.c Y10 reaching up to 100% after 7 days, and none of the other removals were less than 90%. When with 4-chloronitrobenzene of 100 mgL-1, the removal of strains S.c S2, X.r LWJ4 and B.c YX2 was 100% after 7 days, while the removal of the other seven strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3, S.e LWJ5, B.c Y10 and B.s YX3 ) was 92.5%, 97.3%, 94.6%, 91.4%, 95.2%, 79.8% and 88.1%, respectively. With the addition of four different mixtures (Fe2+/Fe + beef extract, Fe2+ + beef extract, VC + beef extract, or mannitol + beef extract ), the strains’ 4-chloronitrobenzene removal showed a similar tendency, i.e., a reductant and a substrate working better together than alone. However, Fig. 4 and Fig. 5 demonstrate that mixtures of organic reductant and substrate have a better synergistic effect on 4-chloronitrobenzene degradation than inorganic reductant and substrate. Since the synergistic effect of mannitol and beef extract was the most efficient on enhancing the strains’ 4-chloronitrobenzene biodegradation, we recommend the optimal additive of mannitol and beef extract.
4. Discussion
In our study, ten novel 4-chloronitrobenzene degrading bacteria were screened, with six of them falling into Bacillales order, which were previously reported to degrade nitrobenzene (Hu et al., 2008; Li et al., 2014; Wang et al., 2012; Zhao et al., 2011; Zheng et al., 2008). The microbial cometabolism, i.e. transformation of a non-growth substrate, namely co-substrate, in the obligate presence of a growth substrate by growing cells, or by resting cells in the absence of a growth substrate, is a significant part of the total biodegradative activity toward xenobiotic compounds (Nava et al., 2007). Hence, 4-chloronitrobenzene was biodegraded via cometabolic biodegradation pathway. As shown in Fig. 2A and Fig. 2B, the addition of a nitrogen source enhanced the strains’ removal of 4-chloronitrobenzene while increasing the strains’ biomass. In contrast, the addition of a carbon source had little effect on the strains’ removal of 4-chloronitrobenzene without increasing the strains’ biomass. These results indicate that nitrogen from 4-chloronitrobenzene was utilized by the strains to generate energy to drive metabolic reactions, not for the production of new biomass, while carbon from 4-chloronitrobenzene was utilized by the strains for the production of new biomass. Similar results were reported in previous studies. Bacillus licheniformis strainYX2 could not utilize nitrobenzene as the sole carbon and nitrogen source to grow. The addition of a nitrogen source (peptone) stimulated the bioreduction of nitrobenzene, whereas the addition of a carbon source had little effect (Li et al., 2014). On the other hand, M. odoratimimus strainYX6 and K. ornithinolytica strain NB1 could utilize nitrobenzene as the sole carbon and nitrogen source to grow, and the addition of carbon (glucose ) stimulated the bioreduction of nitrobenzene (Li et al., 2013; Wang et al., 2012). These reports demonstrate that whether the
bacteria could utilize nitroaromatic compound as a sole carbon and nitrogen source may decide the effectiveness of adding carbon source or nitrogen source to enhance strain’s biodegradation of nitroaromatic compounds. With the nitro and chloride groups, 4-chloronitrobenzene is both a chlorinated organic compound and a nitroaromatic compound. The removal of chlorinated organic compound by a microorganism is often limited because of a lack of electron donors (Aulenta et al., 2006; Bradley, 2003). Besides, oxygenases, which play a crucial role in the aerobic degradation of all aromatic compounds, cannot act on electron-deficient compounds, such as chlorinated organic compounds and nitroaromatic compounds (Arora et al., 2009). Under aerobic conditions, the reductive pathway of 4-chloronitrobenzene microbial degradation is initiated (Arora et al., 2012). In previous studies, 4-chloronitrobenzene was converted to an unstable intermediate 1-hydroxylamino-4-chlorobenzene by strain LW1 and strain CNB-1 (Katsivela et al., 1999; Wu et al., 2006); and 4-chloronitrobenzene could be transformed by Pseudomonas
sp.
strain
CBS3
to
4-chloroaniline,
N-acetyl-4-chloroaniline,
and
4-chloronitrosobenzene at low rates without any further degradation (Schackmann and Müller, 1991). Since oxidation and reduction are the processes of losing and gaining of electrons, the ion-exchange effect has been recognized as the main mechanism for remediation technology (Zeng et al., 2017). In addition, the addition of electron donors (e.g., glucose) could accelerate reduction of nitroaromatic antibiotic chloramphenicol, which has the same groups (chloro and nitro) as 4-Chloronitrobenzene (Liang et al., 2013). Fig. 3A shows that 4-chloronitrobenzene removal of the eight strains with an inorganic reducing agent were higher than those with an organic reducing agent, while the other two
strains had opposite results. The individual additional inorganic reducing agent can have a better effect on strains’ biodegradation of nitroaromatic comounds than the individual additional organic reducing agent, which may be a major reason why there is such limited research about the utilization of an organic reducing agent to reduce nitroaromatic compounds. In our experiments, the additional reducing agents could enhance the ten strains’ 4-chloronitrobenzene removal rates, but they had little impact on the strains’ growth at same time.
The
results
demonstrate
that
reducing
agents
can
enhance
the
strains’
4-chloronitrobenzene degrading ability. The iron (Fe2+ or Fe2+/Fe) as electron donors could enhance 4-chloronitrobenzene bioremediation, and the growth of the electron-donating power could contribute to increasing the strains’ 4-chloronitrobenzene bioremediation, which is consistent with the reports about catalytic reductants contributing to the nitroaromatic compound degradation (An et al., 2012; Luan et al., 2010; Salter-Blanc et al., 2015; Zeng et al., 2013). The ten target strains belong to eight different families, but the addition of reducing agents had similar effects on their growth and 4-chloronitrobenzene degradation, demonstrating that adding a reducing agent may generally promote the strains’ 4-Chloronitrobenzene-degrading ability. The additional reductant (mannitol) as electron donors catalyzed 4-chloronitrobenzene biodegradation without supporting the strains’ growth, and it didn’t utilize cometabolic method to enhance the 4-chloronitrobenzene biodegradation rate. The addition of beef extraction as the obligatory substrate to enhance the biomass of the strains induced enhancement of the 4-chloronitrobenzene cometabolic biodegradation rate. Therefore, the addition of mannitol and beef extract had synergistic effect on 4-chloronitrobenzene
degradation. Employing the new method, the rates of 50 mgL-1 4-Chloronitrobenzene biodegradation of seven target strains (S.c S2, S.m LWJ1, L.f LWJ2, X.r LWJ4, S.e LWJ5, B.c YX2 and B.c Y10) were enhanced from 7.6%, 18.9%, 8.4%, 13.6%, 6.2%, 13.5% and 7.0% in MSM up to 100%, respectively. In addition, three strains (S.c S2, X.r LWJ4 and B.c YX2) could completely remove 100 mgL-1 4-chloronitrobenzene, while the removal of five strains (B.a YX0, S.m LWJ1, L.f LWJ2, S.m LWJ3, S.e LWJ5) ranged from 90% to 100%. Therefore, as long as a strain can degrade 4-chloronitrobenzene without using 4-chloronitrobenzene as its sole carbon and nitrogen source to support growth, this method of utilizing the synergistic effect of mannitol and beef extract to increase its degradation rate is effective. Because the ten strains were isolated from various environments, this method has the potential to deal with different 4-chloronitrobenzene-contaminated environments and could deal with wastewater with high 4-chloronitrobenzene concentrations. What’s more, the addition of mannitol and beef extract is non-toxic to the environment. Thus, the method has a good prospect to enhance remediation in nitroaromatic compounds contaminated environments. In addition, results of this study provide a clue for development of novel approaches to enrichment and culturing of microbes with enhanced biodegradation ability of refractory organic pollutants.
Acknowledgements We are grateful for the support of the National Key Research and Development Program of China (No：2017YFD 0200301).
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Figure Captions Fig. 1. Neighbour-joining dendrogram of 16S rDNA sequences of 4-Chloronitrobenzene -degrading strains. Bootstrap values (in percentage of 1000 replicates) and DNA database accession numbers are indicated. Fig. 1
Proteobacteria
Pseudomonas putida ZWL73
89
Serratia marcescens LWJ1
73
Xanthomonas retroflexus LWJ4
100
Stenotrophomonas maltophili LWJ3 Burkholderia sp. CAN6 Acidovorax sp. LW1
99
Comamonas testosteroni CNB-1
98
Sphingomonas sp. CNB3
A
0.1
Firmicutes 10
Bacillus amyloliquefaciens YX0
0
6
Bacillus subtilis YX3 Staphylococcus carnosus S2
1 10
Staphylococcus equorum LWJ5
0 Bacillus cereus Y10 10 0
0.01
Bacillus cereus YX2 Lysinibacillus fusiformis LWJ2
B
Fig. 2. Effect of substrate (
MSM,
glucose,
sucrose,
peptone and
beef extration ) on the
% 4-Chloronitrobenzene removal
degradation of 4-Chloronitrobenzene and OD600 by the ten target strains. (Set 1 and Set 2) Fig. 2
A
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2 S.m LWJ3
X.r LWJ4
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Strain
OD600
B
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2 S.m LWJ3
Strain
X.r LWJ4
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Fig. 3. Effect of reductant (
MSM,
Fe2+,
Fe2+/Fe,
Vc,
mannitol) on the biodegradation of 50 mg
L-1 nitrobenzene and OD60 by the ten target strains. (Set 1 and Set 3)
% 4-Chloronitrobenzene removal
Fig. 3
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2 S.m LWJ3
X.r LWJ4 S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
B.c Y10
B.s YX3
OD600
Strain
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2
S.m LWJ3 X.r LWJ4
Strain
S.e LWJ5
B.c YX2
Fig. 4. Synergistic effect of reductant and substrate ( extract,
Vc and beef extract,
MSM,
Fe2+ and beef extract,
Fe2+/Fe and beef
Mannitol and beef extract) on the degradation of 50 mgL-1
4-Chloronitrobenzene by the ten strains. (Set 4)
% 4-Chloronitrobenzene removal
Fig. 4
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2
S.m LWJ3 X.r LWJ4
Strain
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Fig. 5. Synergistic effect of reductant and substrate (
Vc and beef extract,
Mannitol and beef extract) on
-1
the degradation of 100 mgL 4-Chloronitrobenzene by the ten strains. (Set 5)
%4-Chloronitrobenzene removal
Fig. 5
B.a YX0
S.c S2
S.m LWJ1
L.f LWJ2
S.m LWJ3 X.r LWJ4
Strain
S.e LWJ5
B.c YX2
B.c Y10
B.s YX3
Table. 1. Identification and comparison of bacterial isolates from different locations Table 1.
Identification and comparison of bacterial isolates from different locations Isolate
Staphylococcus carnosus strain S2 （S.c S2） Bacillus amyloliquefaciens lihwang YX0（B.a YX0） Bacillus subtilis lihwang strian YX3 ( B.s YX3) Bacillus cereus lihwang Y10 ( B.c Y10) Bacillus cereus lihwang YX2 (B.c YX2) Serratia marcescens LWJ1 (S.m LWJ1 ) Lysinibacillus fusiformis strain LWJ2 (L.f LWJ2) Stenotrophomonas maltophilia strain LWJ3 (S.m LWJ3) Xanthomonas retroflexus strain LWJ4 (X.r LWJ4) Staphylococcus equorum strain LWJ5 (S.e LWJ5)
GenBank number KT932959
Reference strain
Bacterial taxonomy
% identity 99
Isolation source
Staphylococcus carnosus subsp. carnosus TM300 Bacillus amyloliquefaciens LFB112
Firmicutes; Bacilli; Bacillales; Staphylococcaceae ; Staphylococcus; Staphylococcus carnosus Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus amyloliquefaciens Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus subtilis Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus;Bacillus cereus
99
KM093861
Bacillus subtilis strain HDYM-28 Bacillus cereus F837/76
99
The active sludge of Henan The cornfield of Chongqing
KM093863
Bacillus cereus strain MT6
Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus; Bacillus cereus
99
The vegetable garden of Chongqing
KT821502
Serratia marcescens WW4
Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Serratia; Serratia marcescens
99
Jialingjiang river shore soil
KT861858
Lysinibacillus fusiformis strain S-1
99
The farm soil of Wuhan
KT932956
Stenotrophomonas maltophilia
Firmicutes; Bacilli; Bacillales; Bacillaceae; Lysinibacillus Lysinibacillus fusiformis Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Stenotrophomonas; Stenotrophomonas maltophilia
99
The Soil in orange grove
KT932957
Xanthomonas retroflexus
Proteobacteria; Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae; Xanthomonas; Xanthomonas retroflexus
99
The pool of Chongqing
KT932958
Staphylococcus equorum strain CTSP8
Firmicutes; Bacilli; Bacillales; Staphylococcaceae; Staphylococcus; Staphylococcus equorum
99
The meadow soil of Chongqing
KM403448 KM403447
99
The vegetable garden of Chongqing The farm soil of Chongqing
Table. 2. Experimental sets and conditions Table 2. Experimental sets and conditions Substrate
Reductant
4-Chloronitrobenzene
Experimental purpose
Set 1
-
-
+
for testing the biodegradation of the strain for 4-chloronitrobenzene and the results were used as a benchmark
Set 2
+
-
+
for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of additional substrates
Set 3
-
+
+
for testing the biodegradation of the strains for 4-chloronitrobenzene under the influence of each of the selected reductants
Set 4
+
+
+
for testing the biodegradation of the strain for 4-chloronitrobenzene under the influence of adding one of the 4 reductants and one substrate (beef extraction)
Set 5
+
+
+
for testing the biodegradation of the strain for 4-chloronitrobenzene of a higher concentration and under the influence of additional substrate and reductant
Note: All of the 50 mL reaction solutions contained 1) the strain re-suspended in 1 mL of MSM; and 2) 49 mL mineral salt medium (MSM). + represents the additional substance; － represents without the additional substance. In Set 2, substrate represents 1% sucrose, or 1% glucose, or 1% peptone or 1% beef extraction (W/W); in Set 3, reductant represents 0.02% Fe2+, or 0.02% Fe2+/Fe (1:1 ratio), or 0.02% VC (= L-lactose) or 0.02% mannitol (W/W); In set 4, substrate represents 1% beef extraction (W/W), and reductant represents 0.02% of each of the 4 reductants [i.e., Fe2+, Fe2+/Fe (1:1 ratio), VC, mannitol]; in Set 5, substrate represents 1% beef extraction (W/W), and reductant represents 0.02% of VC or mannitol (W/W). The concentration of 4-Chloronitrobenzene was 50 mg L-1 for Sets 1-4, and 100 mg L-1 for Set 5.