Regular articleBiodegradation of benzo(a)pyrene by Microbacterium sp. strain under denitrification: Degradation pathway and effects of limiting electron acceptors or carbon source
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are one class of macromolecule organic compounds and are known to exert acutely toxic effects and/or possess mutagenic, teratogenic, or carcinogenic properties [1], [2]. Due to a variety of anthropogenic activities, PAHs have accumulated in the environment during recent decades. And studies have pointed out that PAHs have been detected in surface water, soil, sediment, even ground water [3], [4], [5]. Thus growing concerns over the rising concentrations and low removal rates of PAHs has spurred researchers to seek ways of treatment for their high bioaccumulation potential. As an cost-effective technology, bacterial biodegradation of PAHs have been studied for decades, and appreciable efforts have been put into biodegradation improvement either by isolating powerful degraders or by optimizing associated environmental conditions [6], [7]. The species white-rot fungi, Lasiodiplodia theobromae, algae, Rhodococcus, Alcaligenes denitricans, Sphingomonas sp. and Stenotrophomonas maltophilia have been extensively reported to be capable of degrading PAHs under aerobic conditions [8], [9], [10], [11], [12], [13]. Hamdi et al. [14] reported that complete degradation of three and four ring PAHs were observed after addition of sewage sludge compost into PAHs-contaminated soil for 15 months. Meanwhile, the addition of cometabolic substrate and surfactant amendments were proved to be effective in PAHs biodegradation enhancement [15]. In addition, investigations on degradation mechanisms of two- to five-ring PAHs by varying degraders were also conducted and showed that one or two oxygen atoms from O2 were introduced to yield dihydroxylated or hydroxylated compounds in the presence of oxygenases [16]. Although the degradation pathways of PAHs under aerobic conditions were widely proposed, limited studies were carried out to explore the anaerobic degradation pathway of PAHs, particularly the high molecular weight (HMW, 4-6 rings PAHs) PAHs [17]. However, it has been reported that half-lives of phenanthrene (three-ring) in soil and sediment may range from 16 to 126 days, while it may range from 229 to 1400 days for the five ring molecule benzo(a)pyrene [18]. Moreover, most HMW PAHs would be adsorbed onto the deep soil or sediments where oxygen is scarce [16]. Organisms have more difficulty in PAHs degradation without oxygen molecule since other oxidants are required for biochemical reactions to break the stable PAHs structures during degradation process. The discovery of PAHs-degrading organisms existed in anoxic and PAHs-contaminated habitats stimulated the interest of anaerobic PAHs biodegradation [19]. The previous investigations suggested that nitrate, sulfate, ferric iron and manganese (IV) could easily replace the molecular oxygen as electron acceptor in microbial respiration [20], [21], [22]. The anthracene was reported to be degraded successfully by species of genera Paracoccus, Herbaspirillum, Azotobacter and Rhodococcus under nitrate-reducing conditions [23]. The PAHs of two- to three-rings were degraded to CO2 as well by mixed bacterial population isolated from petroleum-contaminated harbor sediments under sulfate-reducing conditions [24]. And similar naphthalene degradation pathways by pure strain were proposed based on the detected metabolites under different redox conditions [25], [26]. Unfortunately, researches of HMW PAHs degradation by pure strain under anaerobic conditions were rarely reported [27], [28]. Also, the degradation pathways of HMW PAHs were still unclear due to the limited growth of microorganisms and the difficulty of determining trace levels of transient metabolites in the matrix [29].
Investigation of mechanisms of PAHs degradation by bacteria mainly rely on the detectable intermediates and related metabolic enzymes during degradation process [30], [31], [32]. And the pathways and level of degradation are always determined by bacterial species and electron acceptors available [33], [34]. Besides, the degradation pathways were supposed to be different for varying types of bacteria, and the level of degradation was controlled by the amount of electron acceptors [35]. Li et al. [36] studied the effect of NaHCO3 amendment on the anaerobic biodegradation of four mixed PAHs in mangrove sediment and found the NaHCO3 amendment did not significantly affect the biodegradation of PAHs and other parameters, except that an increased CO2 was observed in the experimental flasks. The phenomenon was attributed to the abundant presence of other electron acceptors which could be easier utilized by the anaerobic bacteria in the sediment. Thus a clear knowledge of applicable electron acceptors for specific bacteria would be important to a complete anaerobic degradation of PAHs. Furthermore, the reduction patterns of electron acceptors during degradation could be helpful to explore the degradation mechanism, whereas only few associated investigations without systematic elucidation were reported. Therefore, establishing a clear relationship between PAHs removal amount and electron acceptors consumption is essential to elucidate the complex anaerobic degradation mechanism and assist in the PAHs-contaminated soil/water bioremediation applications.
The present study aims to (1) investigate the degradation performance of benzo(a)pyrene (BaP, as a representative of HMW PAHs) by a strain of Microbacterium sp. through varying concentrations of electron acceptors under denitrifying conditions; (2) conclude the BaP degradation pathways through intermediate products monitored; (3) quantify the relationship between nitrate (nitrite) consumption and BaP removal amount under different ratios of C/N.
Section snippets
Bacterial and cultivation conditions
The BaP-degrading bacterium M.CSW3 was isolated from PAHs-contaminated soils under nitrate-reducing conditions and was identified as Microbacterium sp. (CGMCC: 1.15785; GenBank: KX496339). The strain was incubated at 35 °C in Luria-Bertani (LB) medium under argon condition for 18 h to obtain a microbial population size of around 106 cells/mL, and microbial cells were harvested by centrifugation at 12,000 r/min for 10 min. Then, the cells were washed three times to remove residual LB and resuspended
Degradation in control experiments
The microorganisms play a leading role in the degradation of toxic refractory organic pollutants, and the activities of microbial degraders determine the degradation efficiency [38]. The BaP loss by Microbacterium sp. M.CWS3 in the absence of nitrate or nitrite were 17.35% to 19.43% during the whole degradation process (data not shown). The BaP loss without inoculums or with inactivated inoculums in the presence of electron acceptors were negligible (only 0.2%) over a period of 10 days. And
Conclusions
The strain Microbacterium sp. M.CSW3 was isolated form PAHs-contaminated soils and could utilize BaP as carbon and energy sources under denitrification conditions. The regulation of nitrate and nitrite utilization as electron acceptors were investigated under different C/N ratios by quantitative analysis of BaP and electron acceptors. And several degradation pathways were proposed based on the intermediate products detected during BaP biodegradation.
The concentration ratios of BaP/nitrate and
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China [grant numbers 51579010]; and the Fundamental Research Funds for the Central Universities [grant numbers 2014NT32]
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