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A novel approach for enhancing bacterial strains’ Nitrobenzene degradation rate
International Biodeterioration & Biodegradation 123 (2017) 63e69
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
A novel approach for enhancing bacterial strains’ Nitrobenzene degradation rate Tian Li a, Zhifeng Zhou b, Lin He a, * a b
College of Plant Protection, Southwest University, Chongqing 400715, PR China College of Resources and Environment, Southwest University, Chongqing 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2016 Received in revised form 26 May 2017 Accepted 2 June 2017
This study presents a novel approach that could efficiently enhance the rate of the strains' nitrobenzene biodegradation by utilizing the synergistic effect of organic reductants and a co-substrate. The authors found that the rates of strains' nitrobenzene biodegradation were increased along with the enhancement of the electron-donating power of organic additives. Combining the electron-donating power of organic reductants acting as catalyst with the ability of a co-substrate to increase the biomass of the strains improved the rate of nitrobenzene biodegradation. Employing the new approach, the rates of nitrobenzene biodegradation of the five targeted strains (Staphylococcus carnosus S12, Bacillus amyloliquefaciens YX0, Bacillus subtilis YX3, Bacillus cereus Y10, and Bacillus cereus YX2) were enhanced from 8.4%, 16.4%, 23.8%, 11.1% and 8.3% in a salt medium up to 85.1%, 88.6%, 95.8%, 60.5%, and 58.6%, respectively. The process described in this research may offer a protocol useful for enhancing the strains’ biodegradation rate of other nitroaromatic compounds. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Nitrobenzene Biodegradation Organic reductant Co-substrate Synergistic effect
1. Introduction Bioremediation is the use of microorganisms to remove pollutants from the soil or water. As an environmental remediation technology, it has great potential due to its economic and ecological advantages (Gavrilescu, 2005). A large number of organic compound-degrading microorganisms have been isolated to date. However, it is extremely difficult, time consuming, labor intensive, and costly to isolate highly efficient biodegrading microorganisms. Therefore, a method capable of efficiently enhancing the biodegradation rate of microorganisms with limited biodegradation ability will be valuable. Nitroaromatic compounds are ubiquitous aquatic contaminants because of their widespread use as pesticides, munitions, pharmaceuticals, and industrial chemical intermediates (Lin et al., 2013; Zeng et al., 2012). Nitrobenzene has been chosen in the research as a model nitroaromatic compound to establish a novel method for enhancing the biodegradation rate of nitroaromatic compound by microbiological means. Because of the strong electron affinity of the nitro group (Huang et al., 2012), nitrobenzene requires harsher
* Corresponding author. College of Plant Protection, Southwest University, Chongqing 400715, PR China. Tel.: 023 68251269; fax: 023 68251269. E-mail address: [email protected] (L. He). http://dx.doi.org/10.1016/j.ibiod.2017.06.004 0964-8305/© 2017 Elsevier Ltd. All rights reserved.
conditions than benzene to be degraded by microorganisms. Previous studies on the chemical reaction of nitroaromatic compounds demonstrated that electron donors of reducing agents (bisulfide, polysulfides, and Fe2þ) could contribute to the reduction of nitrobenzene and other nitroaromatic compounds (Klausen et al., 1995; Klupinski et al., 2004; Naka et al., 2006; Schmidt et al., 2010). These results are consistent with the recent study by Narayan Pradhan (Pradhan et al., 2002), showing that electron transfer resulted in nitroaromatic compound reduction. Microbial biodegradation is also a type of oxidation-reduction (or redox) reaction (Hambrick et al., 1980) that involves a transfer of electrons between two species. There have been reports on the enhancement of nitrobenzene bioremediation through the use of inorganic reductants as electron donors (Luan et al., 2009; Roy et al., 2013; Wang et al., 2011). However, few reports have been published on the use of organic reductants for enhancing the biodegradability of nitrobenzene by microorganisms despite the fact that both organic reductants and inorganic reductants have electron-donating groups. Furthermore, the fact that a large number of organic reductants in nature can be screened represents a significant advantage. The cometabolic removal of toxicants is also a well-established method to enhance their biodegradation by increasing the biomass of microorganisms (Joshi et al., 2010; Sahinkaya and Dilek, 2006; Tarighian et al., 2003). Biodegradable organic compounds
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can be categorized into primary substrates (growth substrates) and z and co-metabolized species (non-growth substrates) (Sae Rittmann, 1993; Wen et al., 2011). If the co-metabolism of cosubstrates and the catalysis of organic reductants have a synergistic effect on enhancing the rate of nitrobenzene biodegradation, this phenomenon could be utilized to effectively enhance nitrobenzene biodegradation rate. In the present study, five target strains (Staphylococcus carnosus S12, Bacillus amyloliquefaciens YX0, Bacillus subtilis YX3, Bacillus cereus Y10 and Bacillus cereus YX2), which degraded 8.4%, 16.4%, 23.8%, 11.1% and 8.3% of 200 mg L1 nitrobenzene, respectively, in mineral salts medium (MSM) in 5 days, were selected as candidates. It was first investigated how organic oxidizing agents (succinic acid and citric acid) and reducing agents (ascorbic acid, mannitol and Fe2þ) affect the rate of nitrobenzene removal by the five target strains. Then, four compounds (sucrose, glucose, peptone, and peptone plus yeast powder) were screened to identify the optimal co-substrate to enhance the rate of nitrobenzene biodegradation by the strains. To this end, experiments were set up with the cosubstrate as the growth substrate and the organic reductants as the catalyst enhancing the five target strains’ nitrobenzene biodegradation rate. 2. Materials and methods 2.1. Strains The soils used for the isolation of bacteria were collected from typical farmland of Chongqing and Wuhan, China, and from the activated sludge of Henan, China. One gram of the farm soil or activated sludge was added to 50 mL of lysogeny broth medium (LB) in a 250 mL sealed flask, which was supplemented with nitrobenzene to a final concentration of 200 mg L1, and the cultures were incubated on a rotary shaker (150 rpm) at 28 C for 7 days. Each culture was diluted and spread onto solid lysogeny broth medium (SLB). SLB plates were incubated at 28 C for 4e5 days. Colonies appearing under the same conditions were sub-cultured. Culture purity was confirmed by microscopic examination. Finally, the pure colonies were transferred to a mineral salt medium (MSM), which contained 200 mg L1 nitrobenzene. To investigate their ability to degrade nitrobenzene under aerobic conditions, five nitrobenzene-degrading strains that could degrade no more than 23.8% of 200 mg L1 nitrobenzene in 5 days were selected as candidates. A phylogenetic tree of the 16S rDNA of the strains compared with the sequences available in the GenBank database is shown in Table 1. 2.2. Experimental design Strains were pre-cultivated in the LB at 28 C and 200 r min1. One milliliter of LB-grown cells in the late exponential phase was
centrifuged at 8900 g for 10 min. The harvested cells were washed twice with 0.1 M phosphate buffer (pH 7.0), re-suspended in 1 mL of MSM and inoculated in 49 mL MSM in a 250 mL sealed flask. The flask was supplemented with 200 mg L1 nitrobenzene. The cultures were incubated on a rotary shaker (150 r min1) at 28 C. Uninoculated media with the same concentration of nitrobenzene served as controls. Samples were withdrawn after 5 days for measurements of nitrobenzene, nitrite, ammonia and cell mass. Next, the effect of organic reductants on the nitrobenzene biodegradation rate by the five strains was evaluated. Each 250 mL glass serum vial was supplemented with 200 mg L1 nitrobenzene, 49 mL of MSM, and 1 mL of LB-grown cell suspension at the late exponential growth phase along with 0.02% succinic acid, 0.02% citric acid, 0.02% ascorbic acid or 0.02% mannitol (%, w/v). Samples were withdrawn to analyze the residual nitrobenzene after 5 days. Uninoculated media with the same concentration of nitrobenzene served as controls. The effect of the co-substrates on the nitrobenzene biodegradation rate by the five strains was also investigated. Each 250 mL flask was supplemented with 200 mg L1 nitrobenzene, 49 mL of MSM and 1 mL of LB-grown cell suspension along with 1% sucrose, 1% glucose, 1% peptone or LB (1% peptone þ 0.5% yeast powder; %, w/v). Uninoculated media with the same concentration of nitrobenzene served as controls. Finally, we studied how the co-substrate (peptone) and different organic reductants work synergistically to affect nitrobenzene biodegradation by the five target strains. Each 250 mL flask was supplemented with 1% (%, w/w) peptone, 200 mg L1 nitrobenzene, 49 mL MSM and 1 mL LB-grown cell suspension along with 0.02% succinic acid, 0.02% citric acid, 0.02% ascorbic acid or 0.02% mannitol (%, w/w). Uninoculated media with the same concentration of nitrobenzene served as controls. Samples were withdrawn to analyze residual nitrobenzene after 5 days. Each experiment was replicated five times.
2.3. Analytical methods For HPLC analysis, samples were filtered through a Syrasep polytetrafluoroethylene syringe filter (0.2 mm) and then analyzed. Nitrobenzene was measured using 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 min1. The absorbance wavelength for nitrobenzene was 280 nm, and the cell density was calculated based on the OD600 value with reference to a calibration curve constructed with scalar dilutions of a cell suspension. The ammonia was detected by Nessler's reagent colorimetric method and nitrite concentrations were analyzed by using diazotization method. (Administration, 2007).
Table 1 Identification and comparison of bacterial isolates from different locations. Isolate
GenBank Accession Number
Reference Strain
% identity
Isolation Source
Staphylococcus carnosus lihwang S12(S.c S12)
KM093862
99%
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)
KM403448
Staphylococcus carnosus subsp. carnosus TM300 Bacillus amyloliquefaciens LFB112
99%
the vegetable garden of Chongqing the farm soil of Wuhan
KM403447 KM093861 KM093863
Bacillus subtilis strain HDYM-28 Bacillus cereus F837/76 Bacillus cereus strain MT6
99% 99% 99%
the active sludge of Henan the cornfield of Chongqing the vegetable garden of Chongqing
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3. Results and discussion 3.1. Isolation and identification of nitrobenzene-degrading bacteria In the present study, we aimed to isolate a certain number of strains with limited nitrobenzene biodegradation ability. The bacterial colonies were examined for morphological characteristics, such as color, shape, size, and surface properties on an SLB plate, and 65 distinguishable strains were isolated. When tested in MSM supplemented with 200 mg L1 nitrobenzene, five strains that showed limited nitrobenzene removal ability met the research requirements. The morphological, physiological, and biochemical characteristics and partial 16S rDNA gene sequencing of these bacteria clearly showed that the five strains belong to the species Bacillus amyloliquefaciens, Staphylococcus carnosus, Bacillus cereus, and Bacillus subtilis (Table 1). In the study, Bacillus cereus lihwang Y10 and Bacillus cereus lihwang YX2 belong to the same genus and were isolated from different places. In previous research studies, Bacillus cereus, Bacillus subtilis, and Bacillus amyloliquefaciens were reported to degrade organic compounds, such as phenol, naphthalene, diesel oil, and dye (Banerjee and Ghoshal, 2010; Lon car et al., 2013), which have chemical structures similar to that of nitrobenzene. To our knowledge, no report exists concerning nitrobenzene biodegradation by Bacillus amyloliquefaciens, Staphylococcus carnosus, Bacillus cereus and Bacillus subtilis. 3.2. Pathways of nitrobenzene catabolism by the five strains under aerobic conditions Nitrobenzene could be used by the five strains as a sole carbon source, but it was insufficient for driving strain growth (Fig. 1). Furthermore, nitrobenzene biodegradation was inefficient. Identifying the catabolic pathways for nitrobenzene could contribute to understanding how reducing agents impact the rate of nitrobenzene biodegradation by strains. The pathways for nitrobenzene catabolism under aerobic conditions have been classified as oxidative or partial reductive pathways (Fig. 2) (Yin et al., 2010; Zheng et al., 2009). Partial reductive nitrobenzene pathways are
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characterized by the formation of the metabolite 2-aminophenol and ammonia release (Fig. 3). In contrast, the oxidative pathway is characterized by the formation of a catechol metabolite and nitrite release (He and Spain, 1999). A previous study demonstrated that the reduction of an aromatic NO2 group consists of a series of sequential electron and proton transfers before a first N-O bond is irreversibly broken (Lund, 2001), and nitroaromatic compounds only break the first N-O bond, yielding the nitroso compound (Hartenbach et al., 2006). The cleavage of the second N-O bond is the reduction of the hydroxylamino compound to the substituted aniline. Thus, both catabolic pathways of nitrobenzene include a reductive process. 3.3. Establishment of a novel method for enhancing a strain's nitrobenzene biodegradation rate 3.3.1. Effects of reducing agents or organic oxidation on nitrobenzene biodegradation by strains The five strains could use nitrobenzene as a sole carbon source, but the inefficient biodegradation of nitrobenzene is shown in Figs. 1 and 4. The rate of nitrobenzene removal by strains S12, YX0, YX3, Y10 and YX2 in 5 days was 8.4%, 16.4%, 23.8%, 11.1%, and 8.3%, respectively. Thus, all five strains have a weak ability to degrade nitrobenzene. To evaluate whether organic reductants or organic oxidation agents have an effect on nitrobenzene biodegradation, batch tests were conducted with the five strains to degrade nitrobenzene in the presence of succinic acid, citric acid, Fe2þ, ascorbic acid or mannitol (Fig. 4). When supplemented with succinic acid, the nitrobenzene removal rates for strains S12, YX0, YX3, Y10 and YX2 were 28.8%, 33.6%, 19.5%, 26.3% and 26.1%, respectively; when supplemented with citric acid, the five strains exhibited nitrobenzene biodegradation rates of 9.3%, 24.9%, 27.9%, 12.9%, and 15.3%, respectively. The results show that supplementation with succinic acid and citric acid cannot effectively enhance the rate of nitrobenzene reduction by the strains. In the presence of Fe2þ, all of the rates of nitrobenzene reduction by the strains were increased considerably, with nitrobenzene removal rates of 41.9%, 44.4%, 42.6%, 39.4% and 43.7% for strains S12, YX0, YX3, Y10 and YX2, respectively. When supplemented with ascorbic acid, 19.4%, 56.5%,
Fig. 1. Effect of time on the cell growth of strains S12, YX0, YX3, Y10, and YX2 and the percentage of nitrobenzene removal with 200 mg L1 nitrobenzene in MSM.
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Fig. 2. Release of ammonia or nitrite accompanied by the degradation of nitrobenzene.
Fig. 3. Partial reductive pathways for nitrobenzene catabolism under aerobic conditions.
Fig. 4. Effect of organic oxidation agents, organic reductants, and Fe2þ on the biodegradation of 200 mg L1 nitrobenzene by the five strains.
43.1%, 51.4%, and 69.2% of the nitrobenzene was removed by strains S12, YX0, YX3, Y10 and YX2, respectively. Fig. 4 shows that 69.2% of the nitrobenzene was removed by strain Y10 with ascorbic acid, whereas only 8.3% of the nitrobenzene was removed by strain Y10 in the absence of ascorbic acid. In contrast, the ability of strains YX3,
Y10 and YX2 to degrade nitrobenzene was inhibited in the presence of mannitol, with nitrobenzene removal rates of 8.1%, 8.2% and 6.8%, respectively. However, the nitrobenzene biodegradation ability of strains S12 and YX0 was improved considerably with mannitol, with rates of 47.3% and 39.2%, respectively.
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Previous studies have proved that biodegradation is a fraction of a chemical reaction, which is, in essence, a process of gaining or losing electrons (Marcus, 1993; Pross, 1985; Semple et al., 2004). The rate of chlorinated organic compound biodegradation by a microorganism is often limited because of a lack of electron donors (Aulenta et al., 2006; Bradley, 2003). Besides, the nitro substituents on the aromatic ring are resistant to electrophilic attack by oxygenases (Boopathy and Melancon, 2004). Both halogenated functional groups and nitrobenzene functional groups have strong electron affinity, and therefore, the rate of nitroaromatic compound biodegradation by a microorganism is also limited. Recently, there has been increasing interest in the use of bioelectro-processes to improve nitroaromatic bioremediation efficiencies (Gao et al., 2016; Liang et al., 2013; Lohner and Tiehm, 2009). Furthermore, Liang et al. (2013) reported that a cathodic biofilm catalyzed nitroaromatic reduction with an enhancement of the cathodic current and a positive shift of the onset potential of the cathodic current. These observations suggest that the increase in the strain's nitrobenzene bioremediation rate in our experiment can be attributed to the growth of the electron-donating power of the succinic acid, citric acid, ascorbic acid and mannitol, and their electron-donating power was in the ascending order of citric acid > succinic acid > mannitol > ascorbic acid. There was no noticeable difference between citric acid and succinic acid as additives in the system because they have similar molecular structures and the same chemical functional groups. Succinic acid is a dicarboxylic acid with the chemical formula C4H6O4 and structural formula HOOCe(CH2)2eCOOH, and citric acid is a tricarboxylic acid with the chemical name 2hydroxypropane-1,2,3-tricarboxylic acid (Filgueiras et al., 2006). Because of the weak electron affinity of the carboxylic acid group, succinic acid and citric acid acted as organic oxidizing agents, which had only a slight positive impact on the strain's ability to biodegrade nitrobenzene, but as co-substrates, they increased the biomass of strains and thereby enhanced the rate of nitrobenzene removal. Mannitol is composed of carbon, hydrogen, and multiple hydroxyl groups (C6H8(OH)6) and has a tendency to lose a hydrogen ion in aqueous solutions. Thus, the five strains' rates of nitrobenzene biodegradation were expected to increase when supplemented with mannitol, but it is an interesting phenomenon that the effect of mannitol on the nitrobenzene biodegradation rates of strains YX3, Y10 and Y10 was negative. The reason for this negative trend may be that the effect is steric rather than electronic (Phillips et al., 2010). Ascorbic acid is classed as a reductone because it can transfer a single electron (Weis, 1975) due to the resonancestabilized nature of its own radical ion, semidehydroascorbate. Ascorbic acid is oxidized with the loss of one electron to form a radical cation and then with the loss of a second electron to form dehydroascorbic acid. Thus, compared with the other three compounds, ascorbic acid has a greater positive effect on the rate of nitrobenzene biodegradation due to its electron-donating power. In the presence of Fe2þ, the ability of the five strains to degrade nitrobenzene was improved, which demonstrated that the reducing agent could increase the redox reaction rate. The results in the literature agree with our result. For example, Sun et al. (2010) proposed an integrated Fe (0)esorbentemicroorganism remediation system as an in situ active capping technique to remediate nitrobenzene-contaminated sediment. Luan et al. (2009) suggested nitrobenzene reduction with biogenic-Fe(II). The experimental data demonstrate that the appropriate organic reducing agents could enhance nitrobenzene biodegradation more effectively than some inorganic reducing agents. Furthermore, a considerably greater number of organic reducing agents are available due to their wide variety and the many different spatial structures of the molecule.
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The effects of supplementation with the same organic reductant in MSM on different strains' ability to remove nitrobenzene may be different. For instance, when supplemented with mannitol, the rates of nitrobenzene biodegradation by strains S12 and YX0 were enhanced considerably, whereas there was nearly no impact on or inhibition of the other three strains' nitrobenzene biodegradation rate. Although strains Y10 and YX2 belong to the same species, the effects of supplementation with the same organic reductant on their nitrobenzene biodegradation ability were not entirely consistent. For instance, strain YX2's rate of nitrobenzene biodegradation was increased from 8.3% to 69.2% in the presence of ascorbic acid, whereas the rate of strain Y10's nitrobenzene biodegradation was only increased from 11.1% to 45.4%. Thus, when screening for the optimal organic reductant, we should consider not only the chemical characteristics and molecular structure of the organic reductant but also the different strains with which it will be used. 3.3.2. Effect of co-substrates on nitrobenzene biodegradation by the strains The results in Fig. 4 showed that organic reductants could be considered a promising tool for enhancing the strains’ nitrobenzene biodegradation rate, but there is still considerable room to increase their efficiency. In previous studies, co-substrates could enhance the biodegradation of refractory organic compounds simply by increasing the biomass of the degraders (Brandt et al., 2003; Khan et al., 2014). Indeed, there are reports on cosubstrates enhancing the efficiency of nitrobenzene biodegradation (Pradhan and Ingle, 2007; Wang et al., 2012). The nitrobenzene biodegradation ability of strain YX0 was improved considerably with sucrose supplementation, which increased nitrobenzene biodegradation from 16.4% in MSM to 43.6%, whereas the rate of nitrobenzene biodegradation by strains S12, YX3, YX10 and YX2 was increased to 8.2%, 25.8%, 34.5% and 37.4%, respectively, following sucrose supplementation (Fig. 5). Moreover, the addition of glucose as an auxiliary had only a slight influence on nitrobenzene biodegradation in the presence of the five different strains, with the nitrobenzene removal rate being increased to no more than 26.42%. When supplemented with peptone, 65.2%, 58.2%, 55.4%, 57.5% and 59.6% of the nitrobenzene was removed by strains S12, YX0, YX3, Y10 and YX2, respectively. Interestingly, it was discovered that the strains’ nitrobenzene biodegradation rate did not exhibit such a remarkable increase when supplemented with both peptone and yeast powder, with only the nitrobenzene removal rate by strain Y10 being increased to over 50%. This result showed that peptone had better effect on nitrobenzene biodegradation than peptone and yeast powder, which contradicts a previous study by our research group that peptone and yeast powder had a greater impact on nitrobenzene biodegradation than peptone alone as an additive (Li et al., 2014). In addition, different species or strains may exhibit a different effect on the nitrobenzene biodegradation rate with the same cosubstrate. 3.3.3. Synergistic effect of organic reductants and substratum on nitrobenzene biodegradation Because our experiment demonstrated that the appropriate organic reducing agents have a positive effect on the rate of nitrobenzene biodegradation due to their electronic donating power and that supplementation with peptone could enhance nitrobenzene biodegradation, we tested the hypothesis that organic reductants and a co-substrate (peptone) may have a synergistic impact on nitrobenzene biodegradation. Fig. 6 showed that the organic oxidizing agents and co-substrate have no synergistic impact on nitrobenzene biodegradation; in contrast, organic
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Fig. 5. Effect of a co-substrate (glucose, sucrose, peptone and yeast powder plus peptone) on the biodegradation of nitrobenzene by the five strains in an MSM.
Fig. 6. Synergistic effect of organic reductants (ascorbic acid, mannitol) or organic oxidizers (succinic acid, citric acid) and a co-substrate (peptone) on the biodegradation of nitrobenzene by the five strains. Symbols in the figures are as follows: mineral salt medium ( MSM), Peptone plus Ascorbic Acid ( PþA), Peptone plus Mannitol ( PþM), Peptone plus Succinic Acid ( PþS), Peptone plus Succinic Acid ( PþC).
oxidizing agents have a negative effect on the rate of nitrobenzene biodegradation in combination with a co-substrate. The result is consistent with a previous study, which found that when iron (III) (hydr)oxides are added to systems containing nitroaromatic compounds, oxidizing agent reduction may compete with nitroaromatic compound reduction for the available electron donor (Luan et al., 2009). This provides indirect evidence that the oxidation properties of organic oxidizing agents have negative effects on strains’ nitrobenzene biodegradation ability. In the present study, mixtures of an organic reductant with a co-substrate (peptone and ascorbic acid & peptone and mannitol) had a significant positive influence on the biodegradation efficiency of the strain. In the presence of peptone and ascorbic acid, 66.6%, 73.9%, 95.8%, 60.5%, and 58.6% of the nitrobenzene was removed by strains S12, YX0,
YX3, Y10 and YX2, respectively. When supplemented with peptone and mannitol, 85.1%, 88.6%, 59.3, 52.7%, and 37.7% of the nitrobenzene was removed by strains S12, YX0, YX3, Y10 and YX2, respectively. The high rate of nitrobenzene removal in the experiment provides clear evidence that supplementation with organic reduction agents and a co-substrate might have a synergistic influence on nitrobenzene biodegradation. 4. Conclusions In this study, we present a novel approach that could efficiently enhance the rate of the strains’ nitrobenzene biodegradation by utilizing the synergistic effect of organic reductants (ascorbic acid or mannitol) and a co-substrate (peptone). Nitrobenzene as a
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targeted compound is a model nitroaromatic compound. Thus, the results may be demonstrated using the method to efficiently enhance the rate of nitroaromatic compound biodegradation. Acknowledgements We are grateful for the support of the Natural Science Foundation of China (Grant No. 41371477) and the Fundamental Research Funds for the Central Universities (SWU113007). References Administration, S.E.P., 2007. Standard Methods for the Examination of Water and Wastewater, fourth ed. Aulenta, F., Majone, M., Tandoi, V., 2006. Enhanced anaerobic bioremediation of chlorinated solvents: environmental factors influencing microbial activity and their relevance under field conditions. J. Chem. Technol. Biotechnol. 81, 1463e1474. Banerjee, A., Ghoshal, A.K., 2010. Phenol degradation by Bacillus cereus: pathway and kinetic modeling. Bioresour. Technol. 101, 5501e5507. Boopathy, R., Melancon, E., 2004. Metabolism of compounds with nitro-functions by Klebsiella pnuemoniae isolated from a regional wetland. Int. Biodeterior. Biodegrad. 54, 269e275. Bradley, P.M., 2003. History and ecology of chloroethene biodegradation: a review. Bioremediation J. 7, 81e109. Brandt, B.W., van Leeuwen, I.M.M., Kooijman, S.A.L.M., 2003. A general model for multiple substrate biodegradation. Application to co-metabolism of structurally non-analogous compounds. Water Res. 37, 4843e4854. Filgueiras, M., Mkhonto, D., De Leeuw, N., 2006. Computer simulations of the adsorption of citric acid at hydroxyapatite surfaces. J. Cryst. growth 294, 60e68. Gao, S.H., Peng, L., Liu, Y., Zhou, X., Ni, B.J., Bond, P.L., Liang, B., Wang, A.J., 2016. Bioelectrochemical reduction of an azo dye by a Shewanella oneidensis MR-1 formed biocathode. Int. Biodeterior. Biodegrad. 115, 250e256. Gavrilescu, M., 2005. Fate of pesticides in the environment and its bioremediation. Eng. Life Sci. 5, 497e526. Hambrick, G.A., DeLaune, R.D., Patrick, W., 1980. Effect of estuarine sediment pH and oxidation-reduction potential on microbial hydrocarbon degradation. Appl. Environ. Microbiol. 40, 365e369. Hartenbach, A., Hofstetter, T.B., Berg, M., Bolotin, J., Schwarzenbach, R.P., 2006. Using nitrogen isotope fractionation to assess abiotic reduction of nitroaromatic compounds. Environ. Sci. Technol. 40, 7710e7716. He, Z., Spain, J.C., 1999. Comparison of the downstream pathways for degradation of nitrobenzene by Pseudomonaspseudoalcaligenes JS45 (2-aminophenol pathway) and by Comamonas sp. JS765 (catechol pathway). Archives Microbiol. 171, 309e316. Huang, J., Wen, Y., Ding, N., Xu, Y., Zhou, Q., 2012. Fast start-up and stable performance coupled to sulfate reduction in the nitrobenzene bio-reduction system and its microbial community. Bioresour. Technol. 114, 201e206. Joshi, S.M., Inamdar, S.A., Telke, A.A., Tamboli, D.P., Govindwar, S.P., 2010. Exploring the potential of natural bacterial consortium to degrade mixture of dyes and textile effluent. Int. Biodeterior. Biodegrad. 64, 622e628. Khan, Z., Jain, K., Soni, A., Madamwar, D., 2014. Microaerophilic degradation of sulphonated azo dye e reactive Red 195 by bacterial consortium AR1 through co-metabolism. Int. Biodeterior. Biodegrad. 94, 167e175. Klausen, J., Troeber, S.P., Haderlein, S.B., Schwarzenbach, R.P., 1995. Reduction of substituted nitrobenzenes by Fe (II) in aqueous mineral suspensions. Environ. Sci. Technol. 29, 2396e2404. Klupinski, T.P., Chin, Y.-P., Traina, S.J., 2004. Abiotic degradation of pentachloronitrobenzene by Fe (II): reactions on goethite and iron oxide nanoparticles. Environ. Sci. Technol. 38, 4353e4360. Li, T., Deng, X., Wang, J., Chen, Y., He, L., Sun, Y., Song, C., Zhou, Z., 2014. Biodegradation of nitrobenzene in a lysogeny broth medium by a novel halophilic bacterium Bacillus licheniformis. Mar. Pollut. Bull. 89 (1e2), 384e389. Liang, B., Cheng, H.-Y., Kong, D.-Y., Gao, S.-H., Sun, F., Cui, D., Kong, F.-Y., Zhou, A.-J., Liu, W.-Z., Ren, N.-Q., 2013. Accelerated reduction of chlorinated nitroaromatic antibiotic chloramphenicol by biocathode. Environ. Sci. Technol. 47, 5353e5361.
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Lin, Y., Han, X., Lu, H., Zhou, J., 2013. Study of archaea community structure during the biodegradation process of nitrobenzene wastewater in an anaerobic baffled reactor. Int. Biodeterior. Biodegrad. 85, 499e505. Lohner, S.T., Tiehm, A., 2009. Application of electrolysis to stimulate microbial reductive PCE dechlorination and oxidative VC biodegradation. Environ. Sci. Technol. 43, 7098e7104. Lon car, N., Bo zi c, N., Lopez-Santin, J., Vuj ci c, Z., 2013. Bacillus amyloliquefaciens laccaseeFrom soil bacteria to recombinant enzyme for wastewater decolorization. Bioresour. Technol. 147, 177e183. Luan, F., Burgos, W.D., Xie, L., Zhou, Q., 2009. Bioreduction of nitrobenzene, natural organic matter, and hematite by Shewanella putrefaciens CN32. Environ. Sci. Technol. 44, 184e190. Lund, H., 2001. Cathodic reduction of nitro and related compounds. In: Lund, H., Hammerich, O. (Eds.), Organic Electrochemistry, fourth ed. Marcel Dekker, Inc., New York, pp. 379e409. Marcus, R.A., 1993. Electron transfer reactions in chemistry. Theory and experiment. Rev. Mod. Phys. 65, 599e610. Naka, D., Kim, D., Strathmann, T.J., 2006. Abiotic reduction of nitroaromatic compounds by aqueous iron (II)-catechol complexes. Environ. Sci. Technol. 40, 3006e3012. Phillips, K.L., Chiu, P.C., Sandler, S.I., 2010. Reduction rate constants for nitroaromatic compounds estimated from adiabatic electron affinities. Environ. Sci. Technol. 44, 7431e7436. Pradhan, N., Ingle, A.O., 2007. Mineralization of phenol by a Serratia plymuthica strain GC isolated from sludge sample. Int. Biodeterior. Biodegrad. 60, 103e108. Pradhan, N., Pal, A., Pal, T., 2002. Silver nanoparticle catalyzed reduction of aromatic nitro compounds. Colloids Surfaces A Physicochem. Eng. Aspects 196, 247e257. Pross, A., 1985. The single electron shift as a fundamental process in organic chemistry: the relationship between polar and electron-transfer pathways. Accounts Chem. Res. 18, 212e219. Roy, P., Periasamy, A.P., Liang, C.-T., Chang, H.-T., 2013. Synthesis of graphene-ZnOAu nanocomposites for efficient photocatalytic reduction of nitrobenzene. Environ. Sci. Technol. 47, 6688e6695. z, P.B., Rittmann, B.E., 1993. Biodegradation kinetics of a mixture containing a Sae primary substrate (phenol) and an inhibitory co-metabolite (4-chlorophenol). Biodegradation 4, 3e21. Sahinkaya, E., Dilek, F.B., 2006. Effect of biogenic substrate concentration on the performance of sequencing batch reactor treating 4-CP and 2, 4-DCP mixtures. J. Hazard. Mater. 128, 258e264. Schmidt, C., Behrens, S., Kappler, A., 2010. Ecosystem functioning from a geomicrobiological perspectiveea conceptual framework for biogeochemical iron cycling. Environ. Chem. 7, 399e405. Semple, K., Doick, K., Jones, K., Burauel, P., Craven, A., Harms, H., 2004. Defining bioavailability and bioaccessibility of contaminated soil and sediments. Environ. Sci. Technol. 15, 229Ae231A. Sun, H., Xu, X., Gao, G., Zhang, Z., Yin, P., 2010. A novel integrated active capping technique for the remediation of nitrobenzene-contaminated sediment. J. Hazard. Mater. 182, 184e190. Tarighian, A., Hill, G., Headley, J., Pedras, S., 2003. Enhancement of 4-chlorophenol biodegradation using glucose. Clean Technol. Environ. policy 5, 61e65. Wang, A.-J., Cheng, H.-Y., Liang, B., Ren, N.-Q., Cui, D., Lin, N., Kim, B.H., Rabaey, K., 2011. Efficient reduction of nitrobenzene to aniline with a biocatalyzed cathode. Environ. Sci. Technol. 45, 10186e10193. Wang, D., Zheng, G., Zhou, L., 2012. Isolation and characterization of a nitrobenzenedegrading bacterium Klebsiella ornithinolytica NB1 from aerobic granular sludge. Bioresour. Technol. 110, 91e96. Weis, W., 1975. Ascorbic acid and electron transport. Ann. N. Y. Acad. Sci. 258, 190e200. Wen, J., Gao, D., Zhang, B., Liang, H., 2011. Co-metabolic degradation of pyrene by indigenous white-rot fungus Pseudotrametes gibbosa from the northeast China. Int. Biodeterior. Biodegrad. 65, 600e604. Yin, Y., Xiao, Y., Liu, H.-Z., Hao, F., Rayner, S., Tang, H., Zhou, N.-Y., 2010. Characterization of catabolic meta-nitrophenol nitroreductase from Cupriavidus necator JMP134. Appl. Microbiol. Biotechnol. 87, 2077e2085. Zeng, T., Chin, Y.-P., Arnold, W.A., 2012. Potential for abiotic reduction of pesticides in prairie pothole porewaters. Environ. Sci. Technol. 46, 3177e3187. Zheng, C., Qu, B., Wang, J., Zhou, J., Lu, H., 2009. Isolation and characterization of a novel nitrobenzene-degrading bacterium with high salinity tolerance: Micrococcus luteus. J. Hazard. Mater. 165, 1152e1158.
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
A novel approach for enhancing bacterial strains’ Nitrobenzene degradation rate Tian Li a, Zhifeng Zhou b, Lin He a, * a b
College of Plant Protection, Southwest University, Chongqing 400715, PR China College of Resources and Environment, Southwest University, Chongqing 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2016 Received in revised form 26 May 2017 Accepted 2 June 2017
This study presents a novel approach that could efficiently enhance the rate of the strains' nitrobenzene biodegradation by utilizing the synergistic effect of organic reductants and a co-substrate. The authors found that the rates of strains' nitrobenzene biodegradation were increased along with the enhancement of the electron-donating power of organic additives. Combining the electron-donating power of organic reductants acting as catalyst with the ability of a co-substrate to increase the biomass of the strains improved the rate of nitrobenzene biodegradation. Employing the new approach, the rates of nitrobenzene biodegradation of the five targeted strains (Staphylococcus carnosus S12, Bacillus amyloliquefaciens YX0, Bacillus subtilis YX3, Bacillus cereus Y10, and Bacillus cereus YX2) were enhanced from 8.4%, 16.4%, 23.8%, 11.1% and 8.3% in a salt medium up to 85.1%, 88.6%, 95.8%, 60.5%, and 58.6%, respectively. The process described in this research may offer a protocol useful for enhancing the strains’ biodegradation rate of other nitroaromatic compounds. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Nitrobenzene Biodegradation Organic reductant Co-substrate Synergistic effect
1. Introduction Bioremediation is the use of microorganisms to remove pollutants from the soil or water. As an environmental remediation technology, it has great potential due to its economic and ecological advantages (Gavrilescu, 2005). A large number of organic compound-degrading microorganisms have been isolated to date. However, it is extremely difficult, time consuming, labor intensive, and costly to isolate highly efficient biodegrading microorganisms. Therefore, a method capable of efficiently enhancing the biodegradation rate of microorganisms with limited biodegradation ability will be valuable. Nitroaromatic compounds are ubiquitous aquatic contaminants because of their widespread use as pesticides, munitions, pharmaceuticals, and industrial chemical intermediates (Lin et al., 2013; Zeng et al., 2012). Nitrobenzene has been chosen in the research as a model nitroaromatic compound to establish a novel method for enhancing the biodegradation rate of nitroaromatic compound by microbiological means. Because of the strong electron affinity of the nitro group (Huang et al., 2012), nitrobenzene requires harsher
* Corresponding author. College of Plant Protection, Southwest University, Chongqing 400715, PR China. Tel.: 023 68251269; fax: 023 68251269. E-mail address: [email protected] (L. He). http://dx.doi.org/10.1016/j.ibiod.2017.06.004 0964-8305/© 2017 Elsevier Ltd. All rights reserved.
conditions than benzene to be degraded by microorganisms. Previous studies on the chemical reaction of nitroaromatic compounds demonstrated that electron donors of reducing agents (bisulfide, polysulfides, and Fe2þ) could contribute to the reduction of nitrobenzene and other nitroaromatic compounds (Klausen et al., 1995; Klupinski et al., 2004; Naka et al., 2006; Schmidt et al., 2010). These results are consistent with the recent study by Narayan Pradhan (Pradhan et al., 2002), showing that electron transfer resulted in nitroaromatic compound reduction. Microbial biodegradation is also a type of oxidation-reduction (or redox) reaction (Hambrick et al., 1980) that involves a transfer of electrons between two species. There have been reports on the enhancement of nitrobenzene bioremediation through the use of inorganic reductants as electron donors (Luan et al., 2009; Roy et al., 2013; Wang et al., 2011). However, few reports have been published on the use of organic reductants for enhancing the biodegradability of nitrobenzene by microorganisms despite the fact that both organic reductants and inorganic reductants have electron-donating groups. Furthermore, the fact that a large number of organic reductants in nature can be screened represents a significant advantage. The cometabolic removal of toxicants is also a well-established method to enhance their biodegradation by increasing the biomass of microorganisms (Joshi et al., 2010; Sahinkaya and Dilek, 2006; Tarighian et al., 2003). Biodegradable organic compounds
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can be categorized into primary substrates (growth substrates) and z and co-metabolized species (non-growth substrates) (Sae Rittmann, 1993; Wen et al., 2011). If the co-metabolism of cosubstrates and the catalysis of organic reductants have a synergistic effect on enhancing the rate of nitrobenzene biodegradation, this phenomenon could be utilized to effectively enhance nitrobenzene biodegradation rate. In the present study, five target strains (Staphylococcus carnosus S12, Bacillus amyloliquefaciens YX0, Bacillus subtilis YX3, Bacillus cereus Y10 and Bacillus cereus YX2), which degraded 8.4%, 16.4%, 23.8%, 11.1% and 8.3% of 200 mg L1 nitrobenzene, respectively, in mineral salts medium (MSM) in 5 days, were selected as candidates. It was first investigated how organic oxidizing agents (succinic acid and citric acid) and reducing agents (ascorbic acid, mannitol and Fe2þ) affect the rate of nitrobenzene removal by the five target strains. Then, four compounds (sucrose, glucose, peptone, and peptone plus yeast powder) were screened to identify the optimal co-substrate to enhance the rate of nitrobenzene biodegradation by the strains. To this end, experiments were set up with the cosubstrate as the growth substrate and the organic reductants as the catalyst enhancing the five target strains’ nitrobenzene biodegradation rate. 2. Materials and methods 2.1. Strains The soils used for the isolation of bacteria were collected from typical farmland of Chongqing and Wuhan, China, and from the activated sludge of Henan, China. One gram of the farm soil or activated sludge was added to 50 mL of lysogeny broth medium (LB) in a 250 mL sealed flask, which was supplemented with nitrobenzene to a final concentration of 200 mg L1, and the cultures were incubated on a rotary shaker (150 rpm) at 28 C for 7 days. Each culture was diluted and spread onto solid lysogeny broth medium (SLB). SLB plates were incubated at 28 C for 4e5 days. Colonies appearing under the same conditions were sub-cultured. Culture purity was confirmed by microscopic examination. Finally, the pure colonies were transferred to a mineral salt medium (MSM), which contained 200 mg L1 nitrobenzene. To investigate their ability to degrade nitrobenzene under aerobic conditions, five nitrobenzene-degrading strains that could degrade no more than 23.8% of 200 mg L1 nitrobenzene in 5 days were selected as candidates. A phylogenetic tree of the 16S rDNA of the strains compared with the sequences available in the GenBank database is shown in Table 1. 2.2. Experimental design Strains were pre-cultivated in the LB at 28 C and 200 r min1. One milliliter of LB-grown cells in the late exponential phase was
centrifuged at 8900 g for 10 min. The harvested cells were washed twice with 0.1 M phosphate buffer (pH 7.0), re-suspended in 1 mL of MSM and inoculated in 49 mL MSM in a 250 mL sealed flask. The flask was supplemented with 200 mg L1 nitrobenzene. The cultures were incubated on a rotary shaker (150 r min1) at 28 C. Uninoculated media with the same concentration of nitrobenzene served as controls. Samples were withdrawn after 5 days for measurements of nitrobenzene, nitrite, ammonia and cell mass. Next, the effect of organic reductants on the nitrobenzene biodegradation rate by the five strains was evaluated. Each 250 mL glass serum vial was supplemented with 200 mg L1 nitrobenzene, 49 mL of MSM, and 1 mL of LB-grown cell suspension at the late exponential growth phase along with 0.02% succinic acid, 0.02% citric acid, 0.02% ascorbic acid or 0.02% mannitol (%, w/v). Samples were withdrawn to analyze the residual nitrobenzene after 5 days. Uninoculated media with the same concentration of nitrobenzene served as controls. The effect of the co-substrates on the nitrobenzene biodegradation rate by the five strains was also investigated. Each 250 mL flask was supplemented with 200 mg L1 nitrobenzene, 49 mL of MSM and 1 mL of LB-grown cell suspension along with 1% sucrose, 1% glucose, 1% peptone or LB (1% peptone þ 0.5% yeast powder; %, w/v). Uninoculated media with the same concentration of nitrobenzene served as controls. Finally, we studied how the co-substrate (peptone) and different organic reductants work synergistically to affect nitrobenzene biodegradation by the five target strains. Each 250 mL flask was supplemented with 1% (%, w/w) peptone, 200 mg L1 nitrobenzene, 49 mL MSM and 1 mL LB-grown cell suspension along with 0.02% succinic acid, 0.02% citric acid, 0.02% ascorbic acid or 0.02% mannitol (%, w/w). Uninoculated media with the same concentration of nitrobenzene served as controls. Samples were withdrawn to analyze residual nitrobenzene after 5 days. Each experiment was replicated five times.
2.3. Analytical methods For HPLC analysis, samples were filtered through a Syrasep polytetrafluoroethylene syringe filter (0.2 mm) and then analyzed. Nitrobenzene was measured using 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 min1. The absorbance wavelength for nitrobenzene was 280 nm, and the cell density was calculated based on the OD600 value with reference to a calibration curve constructed with scalar dilutions of a cell suspension. The ammonia was detected by Nessler's reagent colorimetric method and nitrite concentrations were analyzed by using diazotization method. (Administration, 2007).
Table 1 Identification and comparison of bacterial isolates from different locations. Isolate
GenBank Accession Number
Reference Strain
% identity
Isolation Source
Staphylococcus carnosus lihwang S12(S.c S12)
KM093862
99%
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)
KM403448
Staphylococcus carnosus subsp. carnosus TM300 Bacillus amyloliquefaciens LFB112
99%
the vegetable garden of Chongqing the farm soil of Wuhan
KM403447 KM093861 KM093863
Bacillus subtilis strain HDYM-28 Bacillus cereus F837/76 Bacillus cereus strain MT6
99% 99% 99%
the active sludge of Henan the cornfield of Chongqing the vegetable garden of Chongqing
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3. Results and discussion 3.1. Isolation and identification of nitrobenzene-degrading bacteria In the present study, we aimed to isolate a certain number of strains with limited nitrobenzene biodegradation ability. The bacterial colonies were examined for morphological characteristics, such as color, shape, size, and surface properties on an SLB plate, and 65 distinguishable strains were isolated. When tested in MSM supplemented with 200 mg L1 nitrobenzene, five strains that showed limited nitrobenzene removal ability met the research requirements. The morphological, physiological, and biochemical characteristics and partial 16S rDNA gene sequencing of these bacteria clearly showed that the five strains belong to the species Bacillus amyloliquefaciens, Staphylococcus carnosus, Bacillus cereus, and Bacillus subtilis (Table 1). In the study, Bacillus cereus lihwang Y10 and Bacillus cereus lihwang YX2 belong to the same genus and were isolated from different places. In previous research studies, Bacillus cereus, Bacillus subtilis, and Bacillus amyloliquefaciens were reported to degrade organic compounds, such as phenol, naphthalene, diesel oil, and dye (Banerjee and Ghoshal, 2010; Lon car et al., 2013), which have chemical structures similar to that of nitrobenzene. To our knowledge, no report exists concerning nitrobenzene biodegradation by Bacillus amyloliquefaciens, Staphylococcus carnosus, Bacillus cereus and Bacillus subtilis. 3.2. Pathways of nitrobenzene catabolism by the five strains under aerobic conditions Nitrobenzene could be used by the five strains as a sole carbon source, but it was insufficient for driving strain growth (Fig. 1). Furthermore, nitrobenzene biodegradation was inefficient. Identifying the catabolic pathways for nitrobenzene could contribute to understanding how reducing agents impact the rate of nitrobenzene biodegradation by strains. The pathways for nitrobenzene catabolism under aerobic conditions have been classified as oxidative or partial reductive pathways (Fig. 2) (Yin et al., 2010; Zheng et al., 2009). Partial reductive nitrobenzene pathways are
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characterized by the formation of the metabolite 2-aminophenol and ammonia release (Fig. 3). In contrast, the oxidative pathway is characterized by the formation of a catechol metabolite and nitrite release (He and Spain, 1999). A previous study demonstrated that the reduction of an aromatic NO2 group consists of a series of sequential electron and proton transfers before a first N-O bond is irreversibly broken (Lund, 2001), and nitroaromatic compounds only break the first N-O bond, yielding the nitroso compound (Hartenbach et al., 2006). The cleavage of the second N-O bond is the reduction of the hydroxylamino compound to the substituted aniline. Thus, both catabolic pathways of nitrobenzene include a reductive process. 3.3. Establishment of a novel method for enhancing a strain's nitrobenzene biodegradation rate 3.3.1. Effects of reducing agents or organic oxidation on nitrobenzene biodegradation by strains The five strains could use nitrobenzene as a sole carbon source, but the inefficient biodegradation of nitrobenzene is shown in Figs. 1 and 4. The rate of nitrobenzene removal by strains S12, YX0, YX3, Y10 and YX2 in 5 days was 8.4%, 16.4%, 23.8%, 11.1%, and 8.3%, respectively. Thus, all five strains have a weak ability to degrade nitrobenzene. To evaluate whether organic reductants or organic oxidation agents have an effect on nitrobenzene biodegradation, batch tests were conducted with the five strains to degrade nitrobenzene in the presence of succinic acid, citric acid, Fe2þ, ascorbic acid or mannitol (Fig. 4). When supplemented with succinic acid, the nitrobenzene removal rates for strains S12, YX0, YX3, Y10 and YX2 were 28.8%, 33.6%, 19.5%, 26.3% and 26.1%, respectively; when supplemented with citric acid, the five strains exhibited nitrobenzene biodegradation rates of 9.3%, 24.9%, 27.9%, 12.9%, and 15.3%, respectively. The results show that supplementation with succinic acid and citric acid cannot effectively enhance the rate of nitrobenzene reduction by the strains. In the presence of Fe2þ, all of the rates of nitrobenzene reduction by the strains were increased considerably, with nitrobenzene removal rates of 41.9%, 44.4%, 42.6%, 39.4% and 43.7% for strains S12, YX0, YX3, Y10 and YX2, respectively. When supplemented with ascorbic acid, 19.4%, 56.5%,
Fig. 1. Effect of time on the cell growth of strains S12, YX0, YX3, Y10, and YX2 and the percentage of nitrobenzene removal with 200 mg L1 nitrobenzene in MSM.
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Fig. 2. Release of ammonia or nitrite accompanied by the degradation of nitrobenzene.
Fig. 3. Partial reductive pathways for nitrobenzene catabolism under aerobic conditions.
Fig. 4. Effect of organic oxidation agents, organic reductants, and Fe2þ on the biodegradation of 200 mg L1 nitrobenzene by the five strains.
43.1%, 51.4%, and 69.2% of the nitrobenzene was removed by strains S12, YX0, YX3, Y10 and YX2, respectively. Fig. 4 shows that 69.2% of the nitrobenzene was removed by strain Y10 with ascorbic acid, whereas only 8.3% of the nitrobenzene was removed by strain Y10 in the absence of ascorbic acid. In contrast, the ability of strains YX3,
Y10 and YX2 to degrade nitrobenzene was inhibited in the presence of mannitol, with nitrobenzene removal rates of 8.1%, 8.2% and 6.8%, respectively. However, the nitrobenzene biodegradation ability of strains S12 and YX0 was improved considerably with mannitol, with rates of 47.3% and 39.2%, respectively.
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Previous studies have proved that biodegradation is a fraction of a chemical reaction, which is, in essence, a process of gaining or losing electrons (Marcus, 1993; Pross, 1985; Semple et al., 2004). The rate of chlorinated organic compound biodegradation by a microorganism is often limited because of a lack of electron donors (Aulenta et al., 2006; Bradley, 2003). Besides, the nitro substituents on the aromatic ring are resistant to electrophilic attack by oxygenases (Boopathy and Melancon, 2004). Both halogenated functional groups and nitrobenzene functional groups have strong electron affinity, and therefore, the rate of nitroaromatic compound biodegradation by a microorganism is also limited. Recently, there has been increasing interest in the use of bioelectro-processes to improve nitroaromatic bioremediation efficiencies (Gao et al., 2016; Liang et al., 2013; Lohner and Tiehm, 2009). Furthermore, Liang et al. (2013) reported that a cathodic biofilm catalyzed nitroaromatic reduction with an enhancement of the cathodic current and a positive shift of the onset potential of the cathodic current. These observations suggest that the increase in the strain's nitrobenzene bioremediation rate in our experiment can be attributed to the growth of the electron-donating power of the succinic acid, citric acid, ascorbic acid and mannitol, and their electron-donating power was in the ascending order of citric acid > succinic acid > mannitol > ascorbic acid. There was no noticeable difference between citric acid and succinic acid as additives in the system because they have similar molecular structures and the same chemical functional groups. Succinic acid is a dicarboxylic acid with the chemical formula C4H6O4 and structural formula HOOCe(CH2)2eCOOH, and citric acid is a tricarboxylic acid with the chemical name 2hydroxypropane-1,2,3-tricarboxylic acid (Filgueiras et al., 2006). Because of the weak electron affinity of the carboxylic acid group, succinic acid and citric acid acted as organic oxidizing agents, which had only a slight positive impact on the strain's ability to biodegrade nitrobenzene, but as co-substrates, they increased the biomass of strains and thereby enhanced the rate of nitrobenzene removal. Mannitol is composed of carbon, hydrogen, and multiple hydroxyl groups (C6H8(OH)6) and has a tendency to lose a hydrogen ion in aqueous solutions. Thus, the five strains' rates of nitrobenzene biodegradation were expected to increase when supplemented with mannitol, but it is an interesting phenomenon that the effect of mannitol on the nitrobenzene biodegradation rates of strains YX3, Y10 and Y10 was negative. The reason for this negative trend may be that the effect is steric rather than electronic (Phillips et al., 2010). Ascorbic acid is classed as a reductone because it can transfer a single electron (Weis, 1975) due to the resonancestabilized nature of its own radical ion, semidehydroascorbate. Ascorbic acid is oxidized with the loss of one electron to form a radical cation and then with the loss of a second electron to form dehydroascorbic acid. Thus, compared with the other three compounds, ascorbic acid has a greater positive effect on the rate of nitrobenzene biodegradation due to its electron-donating power. In the presence of Fe2þ, the ability of the five strains to degrade nitrobenzene was improved, which demonstrated that the reducing agent could increase the redox reaction rate. The results in the literature agree with our result. For example, Sun et al. (2010) proposed an integrated Fe (0)esorbentemicroorganism remediation system as an in situ active capping technique to remediate nitrobenzene-contaminated sediment. Luan et al. (2009) suggested nitrobenzene reduction with biogenic-Fe(II). The experimental data demonstrate that the appropriate organic reducing agents could enhance nitrobenzene biodegradation more effectively than some inorganic reducing agents. Furthermore, a considerably greater number of organic reducing agents are available due to their wide variety and the many different spatial structures of the molecule.
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The effects of supplementation with the same organic reductant in MSM on different strains' ability to remove nitrobenzene may be different. For instance, when supplemented with mannitol, the rates of nitrobenzene biodegradation by strains S12 and YX0 were enhanced considerably, whereas there was nearly no impact on or inhibition of the other three strains' nitrobenzene biodegradation rate. Although strains Y10 and YX2 belong to the same species, the effects of supplementation with the same organic reductant on their nitrobenzene biodegradation ability were not entirely consistent. For instance, strain YX2's rate of nitrobenzene biodegradation was increased from 8.3% to 69.2% in the presence of ascorbic acid, whereas the rate of strain Y10's nitrobenzene biodegradation was only increased from 11.1% to 45.4%. Thus, when screening for the optimal organic reductant, we should consider not only the chemical characteristics and molecular structure of the organic reductant but also the different strains with which it will be used. 3.3.2. Effect of co-substrates on nitrobenzene biodegradation by the strains The results in Fig. 4 showed that organic reductants could be considered a promising tool for enhancing the strains’ nitrobenzene biodegradation rate, but there is still considerable room to increase their efficiency. In previous studies, co-substrates could enhance the biodegradation of refractory organic compounds simply by increasing the biomass of the degraders (Brandt et al., 2003; Khan et al., 2014). Indeed, there are reports on cosubstrates enhancing the efficiency of nitrobenzene biodegradation (Pradhan and Ingle, 2007; Wang et al., 2012). The nitrobenzene biodegradation ability of strain YX0 was improved considerably with sucrose supplementation, which increased nitrobenzene biodegradation from 16.4% in MSM to 43.6%, whereas the rate of nitrobenzene biodegradation by strains S12, YX3, YX10 and YX2 was increased to 8.2%, 25.8%, 34.5% and 37.4%, respectively, following sucrose supplementation (Fig. 5). Moreover, the addition of glucose as an auxiliary had only a slight influence on nitrobenzene biodegradation in the presence of the five different strains, with the nitrobenzene removal rate being increased to no more than 26.42%. When supplemented with peptone, 65.2%, 58.2%, 55.4%, 57.5% and 59.6% of the nitrobenzene was removed by strains S12, YX0, YX3, Y10 and YX2, respectively. Interestingly, it was discovered that the strains’ nitrobenzene biodegradation rate did not exhibit such a remarkable increase when supplemented with both peptone and yeast powder, with only the nitrobenzene removal rate by strain Y10 being increased to over 50%. This result showed that peptone had better effect on nitrobenzene biodegradation than peptone and yeast powder, which contradicts a previous study by our research group that peptone and yeast powder had a greater impact on nitrobenzene biodegradation than peptone alone as an additive (Li et al., 2014). In addition, different species or strains may exhibit a different effect on the nitrobenzene biodegradation rate with the same cosubstrate. 3.3.3. Synergistic effect of organic reductants and substratum on nitrobenzene biodegradation Because our experiment demonstrated that the appropriate organic reducing agents have a positive effect on the rate of nitrobenzene biodegradation due to their electronic donating power and that supplementation with peptone could enhance nitrobenzene biodegradation, we tested the hypothesis that organic reductants and a co-substrate (peptone) may have a synergistic impact on nitrobenzene biodegradation. Fig. 6 showed that the organic oxidizing agents and co-substrate have no synergistic impact on nitrobenzene biodegradation; in contrast, organic
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Fig. 5. Effect of a co-substrate (glucose, sucrose, peptone and yeast powder plus peptone) on the biodegradation of nitrobenzene by the five strains in an MSM.
Fig. 6. Synergistic effect of organic reductants (ascorbic acid, mannitol) or organic oxidizers (succinic acid, citric acid) and a co-substrate (peptone) on the biodegradation of nitrobenzene by the five strains. Symbols in the figures are as follows: mineral salt medium ( MSM), Peptone plus Ascorbic Acid ( PþA), Peptone plus Mannitol ( PþM), Peptone plus Succinic Acid ( PþS), Peptone plus Succinic Acid ( PþC).
oxidizing agents have a negative effect on the rate of nitrobenzene biodegradation in combination with a co-substrate. The result is consistent with a previous study, which found that when iron (III) (hydr)oxides are added to systems containing nitroaromatic compounds, oxidizing agent reduction may compete with nitroaromatic compound reduction for the available electron donor (Luan et al., 2009). This provides indirect evidence that the oxidation properties of organic oxidizing agents have negative effects on strains’ nitrobenzene biodegradation ability. In the present study, mixtures of an organic reductant with a co-substrate (peptone and ascorbic acid & peptone and mannitol) had a significant positive influence on the biodegradation efficiency of the strain. In the presence of peptone and ascorbic acid, 66.6%, 73.9%, 95.8%, 60.5%, and 58.6% of the nitrobenzene was removed by strains S12, YX0,
YX3, Y10 and YX2, respectively. When supplemented with peptone and mannitol, 85.1%, 88.6%, 59.3, 52.7%, and 37.7% of the nitrobenzene was removed by strains S12, YX0, YX3, Y10 and YX2, respectively. The high rate of nitrobenzene removal in the experiment provides clear evidence that supplementation with organic reduction agents and a co-substrate might have a synergistic influence on nitrobenzene biodegradation. 4. Conclusions In this study, we present a novel approach that could efficiently enhance the rate of the strains’ nitrobenzene biodegradation by utilizing the synergistic effect of organic reductants (ascorbic acid or mannitol) and a co-substrate (peptone). Nitrobenzene as a
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targeted compound is a model nitroaromatic compound. Thus, the results may be demonstrated using the method to efficiently enhance the rate of nitroaromatic compound biodegradation. Acknowledgements We are grateful for the support of the Natural Science Foundation of China (Grant No. 41371477) and the Fundamental Research Funds for the Central Universities (SWU113007). References Administration, S.E.P., 2007. Standard Methods for the Examination of Water and Wastewater, fourth ed. Aulenta, F., Majone, M., Tandoi, V., 2006. Enhanced anaerobic bioremediation of chlorinated solvents: environmental factors influencing microbial activity and their relevance under field conditions. J. Chem. Technol. Biotechnol. 81, 1463e1474. Banerjee, A., Ghoshal, A.K., 2010. Phenol degradation by Bacillus cereus: pathway and kinetic modeling. Bioresour. Technol. 101, 5501e5507. Boopathy, R., Melancon, E., 2004. Metabolism of compounds with nitro-functions by Klebsiella pnuemoniae isolated from a regional wetland. Int. Biodeterior. Biodegrad. 54, 269e275. Bradley, P.M., 2003. History and ecology of chloroethene biodegradation: a review. Bioremediation J. 7, 81e109. Brandt, B.W., van Leeuwen, I.M.M., Kooijman, S.A.L.M., 2003. A general model for multiple substrate biodegradation. Application to co-metabolism of structurally non-analogous compounds. Water Res. 37, 4843e4854. Filgueiras, M., Mkhonto, D., De Leeuw, N., 2006. Computer simulations of the adsorption of citric acid at hydroxyapatite surfaces. J. Cryst. growth 294, 60e68. Gao, S.H., Peng, L., Liu, Y., Zhou, X., Ni, B.J., Bond, P.L., Liang, B., Wang, A.J., 2016. Bioelectrochemical reduction of an azo dye by a Shewanella oneidensis MR-1 formed biocathode. Int. Biodeterior. Biodegrad. 115, 250e256. Gavrilescu, M., 2005. Fate of pesticides in the environment and its bioremediation. Eng. Life Sci. 5, 497e526. Hambrick, G.A., DeLaune, R.D., Patrick, W., 1980. Effect of estuarine sediment pH and oxidation-reduction potential on microbial hydrocarbon degradation. Appl. Environ. Microbiol. 40, 365e369. Hartenbach, A., Hofstetter, T.B., Berg, M., Bolotin, J., Schwarzenbach, R.P., 2006. Using nitrogen isotope fractionation to assess abiotic reduction of nitroaromatic compounds. Environ. Sci. Technol. 40, 7710e7716. He, Z., Spain, J.C., 1999. Comparison of the downstream pathways for degradation of nitrobenzene by Pseudomonaspseudoalcaligenes JS45 (2-aminophenol pathway) and by Comamonas sp. JS765 (catechol pathway). Archives Microbiol. 171, 309e316. Huang, J., Wen, Y., Ding, N., Xu, Y., Zhou, Q., 2012. Fast start-up and stable performance coupled to sulfate reduction in the nitrobenzene bio-reduction system and its microbial community. Bioresour. Technol. 114, 201e206. Joshi, S.M., Inamdar, S.A., Telke, A.A., Tamboli, D.P., Govindwar, S.P., 2010. Exploring the potential of natural bacterial consortium to degrade mixture of dyes and textile effluent. Int. Biodeterior. Biodegrad. 64, 622e628. Khan, Z., Jain, K., Soni, A., Madamwar, D., 2014. Microaerophilic degradation of sulphonated azo dye e reactive Red 195 by bacterial consortium AR1 through co-metabolism. Int. Biodeterior. Biodegrad. 94, 167e175. Klausen, J., Troeber, S.P., Haderlein, S.B., Schwarzenbach, R.P., 1995. Reduction of substituted nitrobenzenes by Fe (II) in aqueous mineral suspensions. Environ. Sci. Technol. 29, 2396e2404. Klupinski, T.P., Chin, Y.-P., Traina, S.J., 2004. Abiotic degradation of pentachloronitrobenzene by Fe (II): reactions on goethite and iron oxide nanoparticles. Environ. Sci. Technol. 38, 4353e4360. Li, T., Deng, X., Wang, J., Chen, Y., He, L., Sun, Y., Song, C., Zhou, Z., 2014. Biodegradation of nitrobenzene in a lysogeny broth medium by a novel halophilic bacterium Bacillus licheniformis. Mar. Pollut. Bull. 89 (1e2), 384e389. Liang, B., Cheng, H.-Y., Kong, D.-Y., Gao, S.-H., Sun, F., Cui, D., Kong, F.-Y., Zhou, A.-J., Liu, W.-Z., Ren, N.-Q., 2013. Accelerated reduction of chlorinated nitroaromatic antibiotic chloramphenicol by biocathode. Environ. Sci. Technol. 47, 5353e5361.
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