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A new screened microbial consortium OEM2 for lignocellulosic biomass deconstruction and chlorophenols detoxification
Journal of Hazardous Materials 347 (2018) 341–348
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A new screened microbial consortium OEM2 for lignocellulosic biomass deconstruction and chlorophenols detoxification
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Jiajin Lianga, Xiuxiu Fanga,b, Yunqin Lina,b, , Dehan Wanga,b a b
College of Natural Resources and Environment, South China Agricultural University, Guangzhou, Guangdong 510642, PR China Integrate Microbiology Research Center, South China Agricultural University, Guangzhou, Guangdong 510642, PR China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Microbial consortium Lignocellulose Chlorophenols Pretreatment Detoxification
Recalcitrance limits biomass application in biorefinery. It is even more so when toxic chlorophenols are present. In this study, we screened a microbial consortium, OEM2, for lignocellulose deconstruction and chlorophenols detoxification through a short-term and efficient screening process. Microbial consortium OEM2 had a good buffer capability in the cultivation process and exhibited a high xylanase activity, with over 85% hemicellulose degradation within 12 days. Throughout the treatment process, 41.5% rice straw decomposition on day 12 and around 75% chlorophenols (MCP, 2,4-DCP, 2,4,6-TCP) removal on day 9, were recorded. Moreover, Fourier translation infrared spectroscopy (FTIR) analysis indicated that chemical bonds and groups (eg. hydrogen-bond, β-1,4 glycosidic bond, lignin-carbohydrate cross-linking) in the rice straw were broken. Cuticle and silica layer destruction and subsequent exposed cellulose fibers were observed by scanning electron microscopy (SEM). Microbial consortium OEM2 diversity analysis by 16S rRNA gene sequencing indicated that Proteobacteria (41.3%) was the most abundant phylum and the genera Paenibacillus and Pseudomonas played an important role in the lignocellulose decomposition and chlorophenols detoxification. This study developed a faster and more efficient strategy to screen a specific microbial consortium. And the new microbial consortium, OEM2, makes lignocellulose more accessible and complex pollutants unproblematic in the further biorefinery process.
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Corresponding author at: College of Natural Resources and Environment, South China Agricultural University, Guangzhou, Guangdong 510642, PR China. E-mail address: [email protected] (Y. Lin).
https://doi.org/10.1016/j.jhazmat.2018.01.023 Received 1 August 2017; Received in revised form 13 December 2017; Accepted 10 January 2018 Available online 11 January 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved.
Journal of Hazardous Materials 347 (2018) 341–348
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1. Introduction
lignocellulosic biomass deconstruction and chlorophenols detoxification; (ii) apply an advanced method (16S rRNA gene sequencing) to obtain more accurate and comprehensive information on its diversity. In this work, the potential performances on lignocellulosic biomass deconstruction and chlorophenols detoxification of the microbial consortium were also evaluated. More specifically, this study focused on microbial consortium construction, growth, metabolic characteristics, along with lignocellulose and chlorophenols bioconversion behaviors. This research would develop a short-term and efficient strategy for microbial consortium screening and obtain a new microorganism source to pretreat complex solid waste (lignocellulose co-existing with chlorophenols) that could enhance its downstream utilization and further clarified how this microbial consortium worked.
Lignocellulosic biomass is an abundant bioresource with a yield of over 200 billion dry metric tons available annually. Biomass has been gaining much attention as a candidate feedstock for the generation of bioenergy and biobased products [1,2]. Anaerobic digestion is a wellknown mature commercial process which converts non-sterile, diverse and complex organic substrates into energy-rich biogas. Anaerobic digestion has inherent and significant merits including the ability to use a varity of feedstocks and environmental friendliness. With thousands of full-scale plants currently in operation worldwide, anaerobic digestion is considered to be one of the promising alternatives to fossil-derived energy [2–4]. However, regarding lignocellulosic biomass, the interaction of the main components (cellulose, hemicellulose, and lignin) forms a resistant and recalcitrant structure. This structure limits the lignocellulose hydrolysis and consequently leads to a low degradation efficiency in traditional anaerobic digestion processes [5]. Previous research has focused on altering the recalcitrant structure of biomass through physical, chemical, biological and hybrid pretreatments to facilitate the biological conversion of biomass into bioenergy [6]. Compared to other pretreatments, biological pretreatment is a promising technology due to its obvious advantages like eco-friendliness, economic viability, moderate reaction conditions, lack of chemical consumption and less toxic compounds released [7]. Recently, microorganisms like white-, brown-, and soft- rot fungi are commonly used for lignin and hemicellulose pretreatments. And previous research has substantiated that these pretreatments enhanced the methane potential and/or the hydrolysis rate of lignocellulosic biomass during the downstream anaerobic digestion process [3,8,9]. Chlorophenols were widely used as wood preservatives. The logs were dipped into chlorophenol solutions in the manufacture process [10]. In addition, organo-chlorine compounds (measured as AOX) were produced involuntarily in the bleaching pulp generation process using chlorine dioxide as a bleaching agent. AOX were ubiquitous in pulp mill effluents and migrated to pulp and paper sludge during the waste water treatment process [11]. Chlorophenols, as persistent organic pollutants, co-existed with lignocellulosic biomass in logs, pulp & paper sludge, and result integrated into combined contaminants. These contaminants aggravate biomass utilization and chlorophenols safe disposal. The biological treatment performance of these contaminants is determined by a number of factors and Kim et al. [12] considered that the detoxification was the critical factor. Previous studies revealed that the toxic organic compounds significantly changed the microbial community structure, had a noticeably negative effect on the diversity of the microbial community, and decreased the activity of enzymes in the treatment systems [13,14]. The hydrolysis of lignocellulosic biomass and the detoxification of chlorophenols were crucial for enhancing the downstream utilization performance. Hence, a unique consortia is required for biological treatment of these combined contaminants. The latest research indicated that compared to the single specie bacterium/fungus or enzyme treatment, microbial consortium including various bacteria, actinomycetes and fungi exhibited many advantages during the biological treatment of lignocellulosic biomass via their synergistic action [7]. Microbial consortium has good adaptability in a complex environment, which could be flexible enough for a certain range of contaminants, easily controls the pH in the degradation process, and saves cost by operating under non-sterile conditions [7,15]. Our previous study had screened a microbial consortium, named OEM1, for lignocellulose & chlorophenols degradation and analyzed its diversity via PCR-DGGE combined with clone and sequence. However, the screening process of microbial consortium OEM1 spent about 8 months (48 generations successive transfer cultivation). And only 31 strains bacteria were observed due to the limitation of analytical method. The aims of this study were therefore to (i) develop a faster and more efficient strategy to screen a new microbial consortium for
2. Materials and methods 2.1. Experimental materials Rice straw used as the lignocellulose substrate in this study was obtained from agricultural plots of South China Agricultural University (Guangdong, China). Samples of rice straw were pretreated with 1% (W/V) sodium hydroxide solution for 24 h. Furthermore, rice straw was dried and cut into about 0.5 cm pieces prior to use in the experiment. This chemical pretreatment was conductive to the cuticle wax and silica layer of rice straw dissolution partially and led to a certain degree of damage of its surface [16]. It would offer more attachment points for microbial growth and make the rice straw fully exposed to them and their metabolic enzymes. This favorable feedstock would enhance the rice straw biological degradation efficiency. Two kinds of effective spent mushroom substrates (i.e. Pleurotus eryngii substrate and Volvariella volvacea substrate) obtained from Baiyun District (Guangzhou, China) were utilized as the microorganism source for the microbial consortium construction. The physico-chemical characteristics of spent mushroom substrates were provided in Supplement Table 1. Three representative kinds of chlorophenols including o-chlorophenol (MCP), 2,4-dichlorophenol (2,4-DCP), and 2,4,6-trichlorophenol (2,4,6-TCP) were selected for this experiment. A mixed chlorophenols solution (concentration of 50 g/L each chlorophenol) was prepared using ethanol as the solvent. The mixed chlorophenols solution was stored at 4 °C within a brown bottle for further use. The oat-spelt xylan was purchased from Sigma Aldrich Inc. (USA). The carboxymethylcellulose (CMC) was obtained from Sinopharm Chemical Reagent Co., Ltd. 2.2. Culture conditions The enrichment medium used for screening process and biodegradation experiment contained (g/L): peptone, 2.5; KH2PO4, 1.5; Na2HPO4, 1.5; CaCl2·2H2O, 0.8; MgCl2·6H2O, 0.8; and yeast extract, 0.8. Trace elements solution (1mL) was added into the medium. The constitution of trace elements solution was as described by Liang et al. [17]. All flasks were incubated at 28 °C under continuous shaking at 120 rpm/min. 2.3. The screening process for microbial consortium OEM2 In the screening process, rice straw (0.5g) and mixed chlorophenols stock solution (50 μL) were loaded into the medium (100 mL). Two kinds of SMS, Pleurotus eryngii substrate (3.0 g) and Pleurotus ostreatus substrate (3.0 g), were inoculated into the medium. Five replicates were carried out in this screening experiment. As the rice straw decomposed into filament, a 10% inoculum (vol/vol) from each of the well-blended culture fluids was transferred to a fresh medium. During the successive transfer cultivation process, the pH evolution of the medium was detected every day and the degradation ratios of rice straw and chlorophenols were determined after inoculation. The replicate would be eliminated during the successive transfer cultivation under one of these 342
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the DNS method [20] with a spectrophotometer at 540 nm. The xylanase activity was also measured by the DNS method using oat-spelt xylan as the substrate. The crude enzyme fluid (1 mL) was incubated for 10 min at 50 °C in 1 mL of 50 mM Na-citric acid buffer (pH 5.0) with 1.0% oat-spelt xylan. Calibration curves were established with glucose and xylose, respectively and the activity of CMCase/xylanase was expressed in U (μg of glucose/xylose released per min)/mL of crude enzyme fluidr (U/mL). The laccase activity was determined spectroscopically using 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as the substrate [18]. The activity of laccase was expressed in U (μmol of ABTS released per min)/L of crude enzyme fluid (U/L). For FTIR analysis, the dried samples were mixed with KBr with a 1/ 50 mass ratio and 100 mg was ground, then pressed into a lamellar form. FTIR spectra were recorded in the range of 4000–400 cm−1 with a resolution of 4 cm−1. The spectra were then used for characterizing the changes of chemical structure of rice straw matrix. Physical structure changes of rice straw were observed by SEM (Zeiss-Merlin) at a voltage of 5 kV. Dried samples were prepared by platinum plating to ensure electrical conductivity of the surface and then they were observed under different magnifications.
three cases: (1) the degradation ratio of lignocellulose or chlorophenols decreased; (2) the degradation efficient of lignocellulose or chlorophenols maintained at a low level after several successive generations transfer cultivation; (3) the pH evolution was unstable. After successive transfer cultivation for more than 10 generations, when the degradation ratio of lignocellulose and total chlorophenols was constantly maintained at 35% and 75%, respectively, for three generations in 7 days. It was regarded that the microbial consortium had a stable and high efficient ability in lignocellulose and chlorophenols degradation. After 12 generations successive transfer cultivation, the desired microbial consortium was obtained and named as “OEM2”. Microbial consortium OEM2 was preserved in glycerol at −80 °C for further study. 2.4. Lignocellulose deconstruction and chlorophenols detoxification via microbial consortium OEM2 and its metabolism characteristics For the purpose of evaluating the potential performance of microbial consortium OEM2 on lignocellulose deconstruction and chlorophenols detoxification, and clarifying the metabolism characteristics of microbial consortium OEM2, the biodegradation experiment was carried out continuously for 12 days in a 250 mL conical flask. The preserved microbial consortium OEM2 was activated for two generations. The experiment was conducted on 0.5% rice straw load and 50 μL mixed chlorophenols solution in 100 mL enrichment medium. A seed volume of 10% was inoculated. During the bioconversion process, the pH of the fermentation broth was determined every day. The weight loss of rice straw was determined on days 1, 3, 6, 9, 12. The lignocellulosic component (cellulose, hemicellulose, and lignin) alteration were analyzed on day 12. The chlorophenols degradation were examined on days 3, 6, and 9. Samples were taken on days 0.5, 3, 6, 9, and 12 for carboxymethyl cellulase activity (CMCase), xylanase activity, and laccase activity analysis. Moreover, the alteration of structural characterization of rice straw in this pretreatment process was investigated using FTIR and SEM.
2.6. Microbial consortium OEM2 diversity analysis Microbial diversity analysis of microbial consortium OEM2 was carried out by 16S rRNA gene sequencing. The preserved sample in glycerol (2 mL) was used for total DNA extraction. For 16S rRNA genes sequencing, the V3-V4 hypervariable regions of the bacteria were amplified with primers 338F and 806R. PCR amplicons were sequenced by Illumina MiSeq platform to generate paired about 500 bp reads. The sequencing data was applied for operational taxonomic unit (OTU) picking at cutoff of 97% and uclust was applied to assign OTU at 70% confidence level against Silva database. The detail of the method about microbial diversity analysis was described in the Supplementary Information.
2.5. Analytical methods
3. Results and discussion
The methods for pH, weight loss of rice straw and chlorophenols determination corresponded to a previous study [17]. The quantification of MCP, 2,4-DCP and 2,4,6-TCP were determined using high performance liquid chromatograph (HPLC) system. The samples pretreatment methods, HPLC detection program, and the equation of chlorophenols degradation ratio were referred to our previous study. The retention time of 2,4,6-TCP, 2,4-DCP, and MCP was 2.79 min, 3.55 min, and 4.75 min, respectively. The limits of detection (LOD, S/ N = 3) and limits of quantification (LOQ, S/N = 10) in this experiment were 1.2–3.3 μg/L and 4.0–10.9 μg/L, respectively. What's more, the control treatment (Ctrl.) inoculated sterilized microbial consortium OEM2 was carried out to evaluate the influence of environmental factors (e.g. light) in the chlorophenols degradation process. Lignocellulosic contents (cellulose, hemicellulose and lignin) of the samples were determined according to the modified Van Soest method [18]. The degradation ratio of each lignocellulosic component was calculated with the following equation: X(k) = (M0(k) − MOEM2(k))/M0(k). The X(k) represents the degradation ratio of lignocellulosic component (k varies with cellulose, hemicellulose, and lignin in this experiment). M0(k) denotes the mass of component before degradation. MOEM2(k) is the mass of component after 12 days’ degradation by microbial consortium OEM2. For enzyme activity analysis, the fermentation broth was centrifuged at 5000 rpm/min (10 min) to prepare the crude enzyme fluid. The measurement of carboxymethyl cellulase activity (CMCase) was determined using the International Union of Pure and Applied Chemistry standard assay [19]. The crude enzyme fluid (1 mL) was incubated for 0.5 h at 50 °C in 4 mL of 20 mM Na-acetate buffer (pH 5.0) with 0.625% CMC. The quantify of sugars reduction was measured by
3.1. Microbial consortium OEM2 screening process As recorded in the screening process, microbial consortia had a well performance on lignocellulose and chlorophenols decomposition using spent mushroom substrates, as the initial source of microorganisms. The rice straw was decomposed into filament after 6 days’ cultivation (cf. Fig. S1). The weight loss of rice straw in the second generation reached 25.08% within 7 days. We believe this highly efficient lignocellulose decay was due to (i) spent mushroom substrates prepared by lignocellulosic material containing large amounts of microorganisms with lignocellulose decomposition capacity, and (ii) microorganisms forming a synergistic microbial consortium with lignocellulose degradation capability through long-term acclimation during the mushroom cultivation process. With continued the successive transfer cultivation, the capacity of lignocellulose decomposition of microbial consortia was improved after fierce competition in the new environment. The lignocellulose decomposition ability as measured by the dry matter loss of rice straw after 7 days leveled off above 35% after ten generations. Microbial consortiums exhibited a high efficiency on MCP and 2,4DCP degradation in the screening process. In the cultivation of generation 7 and generation 11, the degradation percentage of MCP was more than 80% and 2,4-DCP was 100% within 7 days. The 2,4,6-TCP degradation capability of microbial consortium was continuously enhanced from generation 7 to generation 11. The degradation percentage of total chlorophenols was up to 75% in the last three generations successive transfer precess. This may be due to the spent mushroom substrates containing many bacteria and fungus, secreting enzymes such as laccase which facilitates the chlorophenols degradation [21,22]. In the screening process, the trend of pH evolution had good 343
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Fig. 2. The mass loss of rice straw treated by microbial consortium OEM2 within 12 days.
consortium OEM2 went into the balanced phase (4–12d) and the OD values were maintained at around 1.8. This characteristic would help to exert the lignocellulose and chlorophenols degradation function of microbial consortium OEM2 stably. Subsequently, further rice straw decomposition and metabolite accumulation especially the acidic products decreased the pH slightly from day 5 to day 9. Thereafter, in the late stage of the treatment process, the pH of the fermentation broth was maintained in the vicinity of 7.60. Throughout the biological treatment process, the fermentation broth remained at a near-neutralpH, and this ideal environment for microbial consortium OEM2 proliferation, microbial activity enhancement, lignocellulose decomposition and chlorophenols detoxification. 3.3. Rice straw decomposition characteristics The weight loss of rice straw biologically treated by microbial consortium OEM2 within 12 days is depicted in Fig. 2. The rice straw decomposition was recorded at points during the first 9 days and the accumulative dry matter loss reached 38.05%. The result was similar to the research by Guo et al. [27] that corn stalk decomposition was concentrated in the early stage of cultivation. The weight loss of the rice straw was 41.5% after 12 days’ biological treatment. Compared to the biomass conversion ability of a bacteria community NSC-7 screened from compost heaps degrading 73.6% of rice straw within 14 days, microbial consortium OEM2 had an anticipated potential to treat lignocellulosic biomass for downstream biorefinery [28]. In this study, the residue substance (nearly 60%) and the soluble products metabolized by microbial consortium OEM2 in an aqueous environment could offer enough favorable organic materials for the following fermentation process. Furthermore, after 12 days’ cultivation, cellulose, hemicellulose, and lignin were decomposed up to 75.95%, 85.38%, and 18.90%, respectively. It indicated that the utilization of hemicellulose by microbial consortium OEM2 was easier than cellulose and lignin. These differences may be due to at least two reasons. First, the decomposition of lignocellulosic materials especially lignin was hampered by its intrinsic recalcitrance owing to its complex structure [29,30]. A second reason was that lower CMCase and laccase concentrations were found in the degradation process compared to the xylanase concentration. The xylanase secreted by microbial consortium OEM2 was 120–155 U/mL from day 3 to 9. The enzyme activities of laccase and CMCase were inferior to xylanase, which were 0.6 U/L and 1.2 U/mL on average in the degradation process, respectively. The extent of hemicellulose biodegradation led to the lignin carbohydrate complex (LCC) linkages cleavage and subsequent the lignocellulose three-dimensional network structure collapse. Additionally, the degradation of LCC structures improved accessibility for other enzymes or microorganism, thus, leading to the further depolymerization of cellulose and lignin. These were
Fig. 1. The evolution of pH (a) and OD (b) values within 12 days’ biological treatment.
consistency. The pH of the fermentation broth rose from 5.90 to around 7.30 within 3 days and was maintained around 7.4 until the end of cultivation. The stability of pH evolution trend may be helpful to maintain the lignocellulose and chlorophenols degradation efficiency and the stability of microbial consortia composition. After 12-generation successive transfer, the most efficient microbial consortium on lignocellulosic materials deconstruction and chlorophenols detoxification was preserved for the further research and named as microbial consortium OEM2.
3.2. pH and OD evolution in the bioconversion process Previous studies have confirmed that pH had a profound influence on the growth characteristics and metabolism characteristics of microorganisms [23,24]. There are a number of characteristics (e.g., cell membrane potential, nutrient availability, enzymatic activity, organic C characteristics, and salinity) that are often directly or indirectly related to pH. Furthermore, the above factors may alter microbial consortium diversity, affect the metabolic pathways, and determine the degradation efficiency of substrates [25]. The pH evolution of the fermentation broth during the bioconversion process is depicted in Fig. 1(a). The pH rose from initial 5.97 to around 7.57 in the first 4 days. In this period, microbial consortium OEM2, being in the exponential phase, proliferated rapidly (Fig. 1(b)). The OD values of the culture rose from initial 0.562 to around 1.835 within 4 days. Microbial consortium OEM2 was likely to take a prior consumption of the favorable substrates in the medium (viz. peptone and yeast extract) at the early cultivation stage, leading to the production and accumulation of alkaline compounds (e.g. amines), and then pH increased [26]. After that, microbial 344
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Fig. 3. The degradation characteristics of chlorophenols treated by microbial consortium OEM2.
Fig. 4. FTIR spectra of rice straw biodegraded by microbial consortium OEM2.
confirmed by FTIR analysis and SEM observation.
days. Compared to the commercially obtained laccase, microbial consortium OEM2 had an obvious advantage on large-scale application because of its low cost operation, readily availability, complex environment adaptability, and excellent lignocellulose degradation capability. Thus, microbial consortium OEM2 could be utilized in persistent organic pollution or for AOX degradation and detoxification in paper mill sludge (containing adsorbable organic halides, AOX) pretreatment to enhance the downstream utilization performance.
3.4. Chlorophenols degradation characteristics Fig. 3 depicted the disappearance ratios of chlorophenols (viz. 2MCP, 2,4-DCP, and 2,4,6-TCP) in the course of the degradation process. Microbial consortium OEM2 had a high efficiency of 2,4-DCP degradation, which was completely degraded at 6 days’ culture. 2-MCP degradation was concentrated from day 6 to 9, in which about 60% of 2-MCP were removed. A gradual decrease of 2,4,6-TCP concentration was observed until day 9 when the residual level of approximately 55% was reached. The results illustrated that microbial consortium OEM2 had selectivity on chlorophenols degradation, and the removal ratio decreased in the following order 2,4-DCP > MCP > 2,4,6-TCP. It is generally believed that trichlorophenol and pentachlorophenol metabolized by aerobic microorganisms are more resistant than mono- and di- chlorophenols to degradation due to the increased toxicity and less effective aerobic attack with highly chlorinated compounds [31,32]. The toxicity study by Leontievsky et al. [33] indicated that 2,4,6-TCP would cause hyphal growth inhibition at concentrations between 15 and 50 mg/L, leading to a lower degradation efficiency. Moreover, the metabolismic characteristics were determined by many important factors like microorganism species, reaction medium, the presence of cosubstrates, and the effect of pH on enzymatic activities. Up to now, the mechanism of chlorophenols degradation by microbial consortium OEM2 is unclear. Hence, it needs to be further investigated to fundamentally understand the key factors for chlorophenols degradation efficiency improvement. In our previous work, microbial consortium OEM1 was screened through 48 generations successive transfer cultivation and could degrade three kinds of chlorophenols (MCP, 2,4-DCP, and 2,4,6-TCP) completely after 9 days` treatment when 0.5% rice straw was loaded into the medium [24]. Compared to the microbial consortium OEM1, microbial consortium OEM2 also exhibited a good chlorophenols detoxification capability although it was screened only through 12 generations successive transfer cultivation. After 9 days’ treatment by microbial consortium OEM2, the degradation ratios of MCP, 2,4-DCP, and 2,4,6-TCP were up to 78.5%, 100%, and 44.3%, respectively. It indicated that the short-term microbial consortium screening strategy for lignocellulose degradation and chlorophenols detoxification was effective. Although the adaptability of microbial consortium OEM2 on these three chlorophenols was variant, around 75% of the total amount of chlorophenols (MCP, 2,4-DCP, and 2,4,6-TCP) was removed after 9
3.5. Chemical structure variations of rice straw after microbial consortium OEM2 treatment The structural characteristics of rice straw in the bioconversion process were investigated by FTIR (Fig. 4). Some peaks for rice straw were summarized in Supplement Table 2. They were assigned according to previous literature [34–37]. The band at 3405 cm−1 (OeH stretching vibration) decreased after microbial consortium OEM2 loading into the medium, indicating variations in the inter- and intra-molecular hydrogen-bond structure of rice straw. The peak at 2918 cm−1 was assigned to CH2– groups comprising the majority of the aliphatic fractions of waxes. After 12 days treatment, the band from 1642 cm−1 (absorbed OeH and conjugated CeO) was evolved into two bands, viz. 1654 cm−1 (HeOeH bending of adsorption) and 1605 cm−1 (aromatic skeleton vibrations), reflecting the decomposition of carbohydrate especially hemicellulose. The relative intensity in CeH (897 cm−1) deformation of cellulose was slightly decreased. This could be explained by partial cleavage of the β-1,4 glycosidic bond linkages and decreasing the degree of cellulose polymerization. However, the peak at 1062 cm−1 for CeO stretching mainly from C(3)eO(3)H in cellulose I increased during the decay process, which may be due to increased cellulose exposure because of broken hydrogen-bond and destruction the surface of rice straw [36,37]. Compared with the raw rice straw, the intensity of several typical peaks at 1512 cm−1 (C]C), 1429 cm−1 (CeH), and 1108 cm−1 (CeO) assigned to aromatic skeleton vibrations in lignin was significantly increased after microbial consortium OEM2 treatment. Moreover, the bands at 1605 cm−1, 1319 cm−1, and 1262 cm−1 associated with syringyl units in lignin molecule were observed. This sequence implied that microbial consortium OEM2 had selectivity on lignin degradation and the guaiacyl structure of the lignin molecule was more easily to be degraded. The changes in the adsorption bands after microbial consortium OEM2 treatment suggested that some chemical bonds and groups (eg. hydrogen-bond, β-1,4 glycosidic bond, and LCC linkages) 345
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Fig. 5. SEM images of rice straw before (A–C) and after (E–G) microbial consortium OEM2 treatment for 12 days. Magnification of images given in Figure A and E are 500 X; Figure B and F are 1000 ×; Figure C and G are 3000 ×.
were broken. This led to the disintegration of the resistant and recalcitrant lignocellulose, the separation of carbohydrate and lignin, and the exposure of more cellulose. Thus, the biological treatment of rice straw by microbial consortium OEM2 was beneficial to the downstream utilization.
3.6. SEM histological changes of rice straw after microbial consortium OEM2 treatment In the present study, we investigated the histological changes to the rice straw epidermal surface via SEM analysis for further understanding of substrates decomposition. Representative SEM images at various magnification are depicted in Fig. 5. SEM images show that the untreated controls had a very smooth and compact epidermal surface (Fig. 6 (A–C)). Moreover, cells and vascular bundles covered with the
Fig. 6. The main phyla of bacterial communities of microbial consortium OEM2.
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This biological treatment increased the lignocellulose bio-accessibility and decreased the complex pollutants toxicity, which has the potential to enhance downstream biorefinery performance. What’s more, microbial consortium OEM2 diversity analysis indicated that Proteobacteria (41.3%) was the most abundant phylum and the genera Paenibacillus and Pseudomonas played an important role in the lignocellulose decomposition and chlorophenols detoxification.
cuticle layers exhibited an intact morphology. During the biological treatment process, microbial consortium OEM2 stuck to the surface of the rice straw and aggressed their various issues and cells. This resulted in a clear view of the lump pits on the epidermal surface and the disruption of parenchyma cells. This treatment resulted in the exposed cellulose fibers on the surface and depositied the silica body in the inner lumen spaces and outside the cell walls, suggesting that the cuticle and silica layers were destructed and removed completely. The cellulose fibers were also dissociated and broken. Thereby, the cuticle and silica layers fragmentation, and the cellulose fibers exposure, along with the recalcitrant structure destruction, increased the accessibility to microorganisms and enzymes.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 51108195); Science and Technology Project of Guangdong Province under Contract No. 2015A010106012 and 2016A020210085.
3.7. Microbial consortium OEM2 diversity analysis
Appendix A. Supplementary data
The community composition of microbial consortium OEM2 was investigated via 16S rRNA gene sequencing. The bacterial OTU numbers and Chao1 estimators were 203 at 97% identity level. As Fig. 6 depicted, the composition of microbial consortium OEM2 mainly included six bacterial phyla: Chloroflexi, Firmicutes, Saccharibacteria, Bacteroidetes, Cyanobacteria, and Proteobacteria. Proteobacteria (41.3%) was the most abundant phylum across all samples. The amplicon library of microbial consortium OEM2 included sequences of 137 genera, and 18 of them reached 1% of relative abundance. SM2D12_norank, Saccharibacteria_norank, Geobacillus, Lactococcus and Cyanobacteria_norank were the dominant bacteria detected in this study, and the sum of their abundance accounted for more than 50%. Notably, the relative abundance of unclassified genera was particularly high. These unclassified microbial taxa suggested that they worked together with others in the degradation process. The composition of microbial consortium OEM2 at genus level was shown in Supplement Fig. 2. Leadbetterella belonging to the phylum of Bacteroidetes was isolated from mushroom substrate previously [38] and participated in the process of phenolic compounds degradation [39]. Geobacillus and Bacillus were the member of the phylum of Firmicutes, which accounted for about 6.0% and 1.1% of microbial consortium OEM2, respectively. Previous study indicated that they could accelerate cellulose degradation with a high cellulase activity [40]. In this study, carboxymethyl cellulase (CMCase), the representative enzyme for cellulose degradation, was detected and the concentration achieved a maxima on day 9. The glucose derived from cellulose degradation by cellulase was probably an ideal substrate for the fermentation of Lactococcus. Paenibacillus and Pseudomonas were regarded as the bacteria which had a good potential on lignocellulosic biomass deconstruction and chlorophenols detoxification [41–44]. Moreover, multiple literatures widely reported that Pseudomonas and Desulfovibrio were the main 2-chlorophenol-degrading bacteria [45,46]. The diversity of bacterial communities not only played an important role in the lignocellulose decomposition and chlorophenols detoxification, but also was helpful for maintaining the stability during the biodegradation process. Our further studies will be focused on the evolution of the microbial consortium OEM2 community structure during the biological treatment process under various culture conditions (e.g. lignocellulose loads and chlorophenols concentrations). Combined with the metabolic products analysis, it would further clarify the mechanism of how microbial consortium OEM2 worked in the degradation process.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2018.01.023. References [1] R. Kumar, S. Singh, O.V. Singh, Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives, J. Ind. Microbiol. Biotechnol. 35 (2008) 377–391. [2] C. Sawatdeenarunat, K.C. Surendra, D. Takara, H. Oechsner, S.K. Khanal, Anaerobic digestion of lignocellulosic biomass: challenges and opportunities, Bioresour. Technol. 178 (2015) 178–186. [3] H. Carrere, G. Antonopoulou, R. Affes, F. Passos, A. Battimelli, G. Lyberatos, I. Ferrer, Review of feedstock pretreatment strategies for improved anaerobic digestion: from lab-scale research to full-scale application, Bioresour. Technol. 199 (2016) 386–397. [4] P. Kaparaju, M. Serrano, A. Thomsen, P. Kongjan, I. Angelidaki, Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept, Bioresour. Technol. 100 (2009) 2562–2568. [5] S.K. Khanal, Anaerobic Biotechnology for Bioenergy Production: Principles and Applications, Wiley-Blackwell, 2008. [6] F. Passos, E. Uggetti, H. Carrère, I. Ferrer, Pretreatment of microalgae to improve biogas production: a review, Bioresour. Technol. 172 (2014) 403–412. [7] R. Sindhu, P. Binod, A. Pandey, Biological pretreatment of lignocellulosic biomass an overview, Bioresour. Technol. 199 (2016) 76. [8] X. Ge, T. Matsumoto, L. Keith, Y. Li, Fungal pretreatment of Albizia chips for enhanced biogas production by solid-state anaerobic digestion, Energy Fuels 29 (2015) 200–204. [9] Z. Jia, Z. Yi, Y. Li, Fungal pretreatment of yard trimmings for enhancement of methane yield from solid-state anaerobic digestion, Bioresour. Technol. 156 (2014) 176–181. [10] C. Aeppli, M. Tysklind, H. Holmstrand, Ö. Gustafsson, Use of Cl and C isotopic fractionation to identify degradation and sources of polychlorinated phenols: mechanistic study and field application, Environ. Sci. Technol. 47 (2013) 790–797. [11] A. Sharma, V.V. Thakur, A. Shrivastava, R.K. Jain, R.M. Mathur, R. Gupta, R.C. Kuhad, Xylanase and laccase based enzymatic kraft pulp bleaching reduces adsorbable organic halogen (AOX) in bleach effluents: a pilot scale study, Bioresour. Technol. 169 (2014) 96–102. [12] S. Kim, P. Eichhorn, J.N. Jensen, A.S. Weber, D.S. Aga, Removal of antibiotics in wastewater: effect of hydraulic and solid retention times on the fate of tetracycline in the activated sludge process, Environ. Sci. Technol. 39 (2005) 5816–5823. [13] H. Li, T.T. Shen, X.L. Wang, K.F. Lin, Y.D. Liu, S.G. Lu, J.D. Gu, P. Wang, Q. Lu, X.M. Du, Biodegradation of perchloroethylene and chlorophenol co-contamination and toxic effect on activated sludge performance, Bioresour. Technol. 137 (2013) 286–293. [14] Y.L. Yao, J. Guan, P. Tang, H.P. Jiao, C. Lin, J.J. Wang, Z.M. Lu, H. Min, H.C. Gao, Assessment of toxicity of tetrahydrofuran on the microbial community in activated sludge, Bioresour. Technol. 101 (2010) 5213–5221. [15] D. Kalyani, K.M. Lee, T.S. Kim, J. Li, S.S. Dhiman, Y.C. Kang, J.K. Lee, Microbial consortia for saccharification of woody biomass and ethanol fermentation, Fuel 107 (2013) 815–822. [16] J.K. Wang, J.X. Liu, J.Y. Li, Y.M. Wu, J.A. Yea, Histological and rumen degradation changes of rice straw stem epidermis as influenced by chemical pretreatment, Anim. Feed Sci. Tech. 136 (2007) 51–62. [17] J. Liang, X. Peng, D. Yin, B. Li, D. Wang, Y. Lin, Screening of a microbial consortium for highly simultaneous degradation of lignocellulose and chlorophenols, Bioresour. Technol. 190 (2015) 381–387. [18] S. Liu, Screening of Straw Degradation Strains Under Medium - Low Temperature and Their Degradation Effects on Crop Straw (D), Chinese Academy of Agricultural Sciences Dissertation, 2011. [19] IUPAC, Measurement of cellulase activities, Pure Appl. Chem. 59 (2009) 257–268. [20] G.L. Miller, R. Blum, W.E. Glennon, A.L. Burton, Measurement of carboxymethylcellulase activity, Anal. Biochem. 1 (1960) 127–132.
4. Conclusions Microbial consortium OEM2 with a stable pH buffer capability and high microbial activity was screened for lignocellulosic biomass deconstruction and chlorophenols detoxification. In this biological treatment process, OEM2 removed around 75% chlorophenols after 9 days and decomposed 41.5% rice straw after 12 days. FTIR analysis verified that OEM2 was able to dramatically break the chemical bonds of rice straw, resulting in recalcitrant lignocellulose deconstruction. SEM analysis supported our interpretation of the lignocellulose modification. 347
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[35] X. Xiao, B. Chen, L. Zhu, Transformation, morphology, and dissolution of silicon and carbon in rice straw-derived biochars under different pyrolytic temperatures, Environ. Sci. Technol. 48 (2014) 3411–3419. [36] F. Li, H. Hu, R. Yao, H. Wang, M. Li, Structure and saccharification of rice straw pretreated with microwave-Assisted Dilute Lye, Ind. Eng. Chem. Res. 51 (2012) 6270–6274. [37] P. Carmenmihaela, P. Mariacristina, V. Cornelia, Structural changes in biodegraded lime wood, Carbohydr. Polym. 79 (2010) 362–372. [38] H.Y. Weon, B.Y. Kim, S.W. Kwon, I.C. Park, I.B. Cha, B.J. Tindall, E. Stackebrandt, H.G. Trüper, S.J. Go, Leadbetterella byssophila gen. nov., sp. nov., isolated from cotton-waste composts for the cultivation of oyster mushroom, Int. J. Syst. Evol. Micr. 55 (2005) 2297–2302. [39] S. Gómez-Acata, I. Esquivel-Ríos, M.V. Pérez-Sandoval, Y. Navarro-Noya, A. RojasValdez, F. Thalasso, M. Luna-Guido, L. Dendooven, Bacterial community structure within an activated sludge reactor added with phenolic compounds, Appl. Microbiol. Biot. 101 (2017) 3405–3414. [40] B. Unal, A. Baykal, M. Senel, H. Sözeri, Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains, Bioresour. Technol. 101 (2010) 8798–8806. [41] S. Singh, B.B. Singh, R. Chandra, Biodegradation of phenol in batch culture by pure and mixed strains of Paenibacillus sp. and Bacillus cereus, Pol. J. Microbiol. 58 (2009) 319–325. [42] S.L. Mathews, A.M. Grunden, J. Pawlak, Degradation of lignocellulose and lignin by Paenibacillus glucanolyticus, Int. Biodeter. Biodegr. 110 (2016) 79–86. [43] C.L. Cheng, J.S. Chang, Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production, Bioresour. Technol. 102 (2011) 8628–8634. [44] H. Kiyohara, T. Hatta, Y. Ogawa, T. Kakuda, H. Yokoyama, N. Takizawa, Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols, Appl. Environ. Microbiol. 58 (1992) 1276–1283. [45] A. Farrell, B. Quilty, The enhancement of 2-chlorophenol degradation by a mixed microbial community when augmented with Pseudomonas putida CP1, Water Res. 36 (2002) 2443–2450. [46] B. Sun, J.R. Cole, R.A. Sanford, J.M. Tiedje, Isolation and characterization of desulfovibrio dechloracetivorans sp. nov., a marine dechlorinating bacterium growing by coupling the oxidation of acetate to the reductive dechlorination of 2-chlorophenol, Appl. Environ. Microbiol. 66 (2000) 2408–2413.
[21] K.T. Semple, B.J. Reid, T.R. Fermor, Impact of composting strategies on the treatment of soils contaminated with organic pollutants, Environ. Pollut. 112 (2001) 269–283. [22] W.M. Law, W.N. Lau, K.L. Lo, L.M. Wai, S.W. Chiu, Removal of biocide pentachlorophenol in water system by the spent mushroom compost of Pleurotus pulmonarius, Chemosphere 52 (2009) 1531–1537. [23] C. Lin, L. Gan, Z.L. Chen, Biodegradation of naphthalene by strain Bacillus fusiformis (BFN), J. Hazard. Mater. 182 (2010) 771–777. [24] J.J. Liang, Y.Q. Lin, T. Li, F.Y. Mo, Microbial consortium OEM1 cultivation for higher lignocellulose degradation and chlorophenol removal, RSC Adv. 7 (2017) 39011–39017. [25] C.L. Lauber, M. Hamady, R. Knight, N. Fierer, Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale, Appl. Environ. Microbiol. 75 (2009) 5111. [26] C. Allison, G.T. Macfarlane, Influence of pH, nutrient availability, and growth rate on amine production by Bacteroides fragilis and Clostridium perfringens, Appl. Environ. Microb. 55 (1989) 2894–2898. [27] P. Guo, K. Mochidzuki, W. Cheng, M. Zhou, H. Gao, D. Zheng, X. Wang, Z. Cui, Effects of different pretreatment strategies on corn stalk acidogenic fermentation using a microbial consortium, Bioresour. Technol. 102 (2011) 7526–7531. [28] C.L. Liu, The WANG, Cultural characteristics and stability of the dual-functional bacteria community NSC-7, Microbiol. 35 (2008) 725–730. [29] H. Li, Y. Qu, Y. Yang, S. Chang, J. Xu, Microwave irradiation–A green and efficient way to pretreat biomass, Bioresour. Technol. 199 (2016) 34–41. [30] H.W. Blanch, B.A. Simmons, D. Kleinmarcuschamer, Biomass deconstruction to sugars, Biotechnol. J. 6 (2011) 1086–1102. [31] L. Gonzalez, V. Sarria, O. Sanchez, Degradation of chlorophenols by sequential biological-advanced oxidative process using Trametes pubescens and TiO2/UV, Bioresour. Technol. 101 (2010) 3493–3499. [32] G.R. Chaudhry, S. Chapalamadugu, Biodegradation of halogenated organic compounds, Microbiol. Rev. 55 (1991) 59–79. [33] A.A. Leontievsky, N.M. Myasoedova, B.P. Baskunov, C.S. Evans, L.A. Golovleva, Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor, Biodegradation 11 (2000) 331–340. [34] M. Jahan, D. Chowdhury, M. Islam, S. Moeiz, Characterization of lignin isolated from some nonwood available in Bangladesh, Bioresour. Technol. 98 (2007) 465–469.
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A new screened microbial consortium OEM2 for lignocellulosic biomass deconstruction and chlorophenols detoxification
T
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Jiajin Lianga, Xiuxiu Fanga,b, Yunqin Lina,b, , Dehan Wanga,b a b
College of Natural Resources and Environment, South China Agricultural University, Guangzhou, Guangdong 510642, PR China Integrate Microbiology Research Center, South China Agricultural University, Guangzhou, Guangdong 510642, PR China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Microbial consortium Lignocellulose Chlorophenols Pretreatment Detoxification
Recalcitrance limits biomass application in biorefinery. It is even more so when toxic chlorophenols are present. In this study, we screened a microbial consortium, OEM2, for lignocellulose deconstruction and chlorophenols detoxification through a short-term and efficient screening process. Microbial consortium OEM2 had a good buffer capability in the cultivation process and exhibited a high xylanase activity, with over 85% hemicellulose degradation within 12 days. Throughout the treatment process, 41.5% rice straw decomposition on day 12 and around 75% chlorophenols (MCP, 2,4-DCP, 2,4,6-TCP) removal on day 9, were recorded. Moreover, Fourier translation infrared spectroscopy (FTIR) analysis indicated that chemical bonds and groups (eg. hydrogen-bond, β-1,4 glycosidic bond, lignin-carbohydrate cross-linking) in the rice straw were broken. Cuticle and silica layer destruction and subsequent exposed cellulose fibers were observed by scanning electron microscopy (SEM). Microbial consortium OEM2 diversity analysis by 16S rRNA gene sequencing indicated that Proteobacteria (41.3%) was the most abundant phylum and the genera Paenibacillus and Pseudomonas played an important role in the lignocellulose decomposition and chlorophenols detoxification. This study developed a faster and more efficient strategy to screen a specific microbial consortium. And the new microbial consortium, OEM2, makes lignocellulose more accessible and complex pollutants unproblematic in the further biorefinery process.
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Corresponding author at: College of Natural Resources and Environment, South China Agricultural University, Guangzhou, Guangdong 510642, PR China. E-mail address: [email protected] (Y. Lin).
https://doi.org/10.1016/j.jhazmat.2018.01.023 Received 1 August 2017; Received in revised form 13 December 2017; Accepted 10 January 2018 Available online 11 January 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved.
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1. Introduction
lignocellulosic biomass deconstruction and chlorophenols detoxification; (ii) apply an advanced method (16S rRNA gene sequencing) to obtain more accurate and comprehensive information on its diversity. In this work, the potential performances on lignocellulosic biomass deconstruction and chlorophenols detoxification of the microbial consortium were also evaluated. More specifically, this study focused on microbial consortium construction, growth, metabolic characteristics, along with lignocellulose and chlorophenols bioconversion behaviors. This research would develop a short-term and efficient strategy for microbial consortium screening and obtain a new microorganism source to pretreat complex solid waste (lignocellulose co-existing with chlorophenols) that could enhance its downstream utilization and further clarified how this microbial consortium worked.
Lignocellulosic biomass is an abundant bioresource with a yield of over 200 billion dry metric tons available annually. Biomass has been gaining much attention as a candidate feedstock for the generation of bioenergy and biobased products [1,2]. Anaerobic digestion is a wellknown mature commercial process which converts non-sterile, diverse and complex organic substrates into energy-rich biogas. Anaerobic digestion has inherent and significant merits including the ability to use a varity of feedstocks and environmental friendliness. With thousands of full-scale plants currently in operation worldwide, anaerobic digestion is considered to be one of the promising alternatives to fossil-derived energy [2–4]. However, regarding lignocellulosic biomass, the interaction of the main components (cellulose, hemicellulose, and lignin) forms a resistant and recalcitrant structure. This structure limits the lignocellulose hydrolysis and consequently leads to a low degradation efficiency in traditional anaerobic digestion processes [5]. Previous research has focused on altering the recalcitrant structure of biomass through physical, chemical, biological and hybrid pretreatments to facilitate the biological conversion of biomass into bioenergy [6]. Compared to other pretreatments, biological pretreatment is a promising technology due to its obvious advantages like eco-friendliness, economic viability, moderate reaction conditions, lack of chemical consumption and less toxic compounds released [7]. Recently, microorganisms like white-, brown-, and soft- rot fungi are commonly used for lignin and hemicellulose pretreatments. And previous research has substantiated that these pretreatments enhanced the methane potential and/or the hydrolysis rate of lignocellulosic biomass during the downstream anaerobic digestion process [3,8,9]. Chlorophenols were widely used as wood preservatives. The logs were dipped into chlorophenol solutions in the manufacture process [10]. In addition, organo-chlorine compounds (measured as AOX) were produced involuntarily in the bleaching pulp generation process using chlorine dioxide as a bleaching agent. AOX were ubiquitous in pulp mill effluents and migrated to pulp and paper sludge during the waste water treatment process [11]. Chlorophenols, as persistent organic pollutants, co-existed with lignocellulosic biomass in logs, pulp & paper sludge, and result integrated into combined contaminants. These contaminants aggravate biomass utilization and chlorophenols safe disposal. The biological treatment performance of these contaminants is determined by a number of factors and Kim et al. [12] considered that the detoxification was the critical factor. Previous studies revealed that the toxic organic compounds significantly changed the microbial community structure, had a noticeably negative effect on the diversity of the microbial community, and decreased the activity of enzymes in the treatment systems [13,14]. The hydrolysis of lignocellulosic biomass and the detoxification of chlorophenols were crucial for enhancing the downstream utilization performance. Hence, a unique consortia is required for biological treatment of these combined contaminants. The latest research indicated that compared to the single specie bacterium/fungus or enzyme treatment, microbial consortium including various bacteria, actinomycetes and fungi exhibited many advantages during the biological treatment of lignocellulosic biomass via their synergistic action [7]. Microbial consortium has good adaptability in a complex environment, which could be flexible enough for a certain range of contaminants, easily controls the pH in the degradation process, and saves cost by operating under non-sterile conditions [7,15]. Our previous study had screened a microbial consortium, named OEM1, for lignocellulose & chlorophenols degradation and analyzed its diversity via PCR-DGGE combined with clone and sequence. However, the screening process of microbial consortium OEM1 spent about 8 months (48 generations successive transfer cultivation). And only 31 strains bacteria were observed due to the limitation of analytical method. The aims of this study were therefore to (i) develop a faster and more efficient strategy to screen a new microbial consortium for
2. Materials and methods 2.1. Experimental materials Rice straw used as the lignocellulose substrate in this study was obtained from agricultural plots of South China Agricultural University (Guangdong, China). Samples of rice straw were pretreated with 1% (W/V) sodium hydroxide solution for 24 h. Furthermore, rice straw was dried and cut into about 0.5 cm pieces prior to use in the experiment. This chemical pretreatment was conductive to the cuticle wax and silica layer of rice straw dissolution partially and led to a certain degree of damage of its surface [16]. It would offer more attachment points for microbial growth and make the rice straw fully exposed to them and their metabolic enzymes. This favorable feedstock would enhance the rice straw biological degradation efficiency. Two kinds of effective spent mushroom substrates (i.e. Pleurotus eryngii substrate and Volvariella volvacea substrate) obtained from Baiyun District (Guangzhou, China) were utilized as the microorganism source for the microbial consortium construction. The physico-chemical characteristics of spent mushroom substrates were provided in Supplement Table 1. Three representative kinds of chlorophenols including o-chlorophenol (MCP), 2,4-dichlorophenol (2,4-DCP), and 2,4,6-trichlorophenol (2,4,6-TCP) were selected for this experiment. A mixed chlorophenols solution (concentration of 50 g/L each chlorophenol) was prepared using ethanol as the solvent. The mixed chlorophenols solution was stored at 4 °C within a brown bottle for further use. The oat-spelt xylan was purchased from Sigma Aldrich Inc. (USA). The carboxymethylcellulose (CMC) was obtained from Sinopharm Chemical Reagent Co., Ltd. 2.2. Culture conditions The enrichment medium used for screening process and biodegradation experiment contained (g/L): peptone, 2.5; KH2PO4, 1.5; Na2HPO4, 1.5; CaCl2·2H2O, 0.8; MgCl2·6H2O, 0.8; and yeast extract, 0.8. Trace elements solution (1mL) was added into the medium. The constitution of trace elements solution was as described by Liang et al. [17]. All flasks were incubated at 28 °C under continuous shaking at 120 rpm/min. 2.3. The screening process for microbial consortium OEM2 In the screening process, rice straw (0.5g) and mixed chlorophenols stock solution (50 μL) were loaded into the medium (100 mL). Two kinds of SMS, Pleurotus eryngii substrate (3.0 g) and Pleurotus ostreatus substrate (3.0 g), were inoculated into the medium. Five replicates were carried out in this screening experiment. As the rice straw decomposed into filament, a 10% inoculum (vol/vol) from each of the well-blended culture fluids was transferred to a fresh medium. During the successive transfer cultivation process, the pH evolution of the medium was detected every day and the degradation ratios of rice straw and chlorophenols were determined after inoculation. The replicate would be eliminated during the successive transfer cultivation under one of these 342
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the DNS method [20] with a spectrophotometer at 540 nm. The xylanase activity was also measured by the DNS method using oat-spelt xylan as the substrate. The crude enzyme fluid (1 mL) was incubated for 10 min at 50 °C in 1 mL of 50 mM Na-citric acid buffer (pH 5.0) with 1.0% oat-spelt xylan. Calibration curves were established with glucose and xylose, respectively and the activity of CMCase/xylanase was expressed in U (μg of glucose/xylose released per min)/mL of crude enzyme fluidr (U/mL). The laccase activity was determined spectroscopically using 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as the substrate [18]. The activity of laccase was expressed in U (μmol of ABTS released per min)/L of crude enzyme fluid (U/L). For FTIR analysis, the dried samples were mixed with KBr with a 1/ 50 mass ratio and 100 mg was ground, then pressed into a lamellar form. FTIR spectra were recorded in the range of 4000–400 cm−1 with a resolution of 4 cm−1. The spectra were then used for characterizing the changes of chemical structure of rice straw matrix. Physical structure changes of rice straw were observed by SEM (Zeiss-Merlin) at a voltage of 5 kV. Dried samples were prepared by platinum plating to ensure electrical conductivity of the surface and then they were observed under different magnifications.
three cases: (1) the degradation ratio of lignocellulose or chlorophenols decreased; (2) the degradation efficient of lignocellulose or chlorophenols maintained at a low level after several successive generations transfer cultivation; (3) the pH evolution was unstable. After successive transfer cultivation for more than 10 generations, when the degradation ratio of lignocellulose and total chlorophenols was constantly maintained at 35% and 75%, respectively, for three generations in 7 days. It was regarded that the microbial consortium had a stable and high efficient ability in lignocellulose and chlorophenols degradation. After 12 generations successive transfer cultivation, the desired microbial consortium was obtained and named as “OEM2”. Microbial consortium OEM2 was preserved in glycerol at −80 °C for further study. 2.4. Lignocellulose deconstruction and chlorophenols detoxification via microbial consortium OEM2 and its metabolism characteristics For the purpose of evaluating the potential performance of microbial consortium OEM2 on lignocellulose deconstruction and chlorophenols detoxification, and clarifying the metabolism characteristics of microbial consortium OEM2, the biodegradation experiment was carried out continuously for 12 days in a 250 mL conical flask. The preserved microbial consortium OEM2 was activated for two generations. The experiment was conducted on 0.5% rice straw load and 50 μL mixed chlorophenols solution in 100 mL enrichment medium. A seed volume of 10% was inoculated. During the bioconversion process, the pH of the fermentation broth was determined every day. The weight loss of rice straw was determined on days 1, 3, 6, 9, 12. The lignocellulosic component (cellulose, hemicellulose, and lignin) alteration were analyzed on day 12. The chlorophenols degradation were examined on days 3, 6, and 9. Samples were taken on days 0.5, 3, 6, 9, and 12 for carboxymethyl cellulase activity (CMCase), xylanase activity, and laccase activity analysis. Moreover, the alteration of structural characterization of rice straw in this pretreatment process was investigated using FTIR and SEM.
2.6. Microbial consortium OEM2 diversity analysis Microbial diversity analysis of microbial consortium OEM2 was carried out by 16S rRNA gene sequencing. The preserved sample in glycerol (2 mL) was used for total DNA extraction. For 16S rRNA genes sequencing, the V3-V4 hypervariable regions of the bacteria were amplified with primers 338F and 806R. PCR amplicons were sequenced by Illumina MiSeq platform to generate paired about 500 bp reads. The sequencing data was applied for operational taxonomic unit (OTU) picking at cutoff of 97% and uclust was applied to assign OTU at 70% confidence level against Silva database. The detail of the method about microbial diversity analysis was described in the Supplementary Information.
2.5. Analytical methods
3. Results and discussion
The methods for pH, weight loss of rice straw and chlorophenols determination corresponded to a previous study [17]. The quantification of MCP, 2,4-DCP and 2,4,6-TCP were determined using high performance liquid chromatograph (HPLC) system. The samples pretreatment methods, HPLC detection program, and the equation of chlorophenols degradation ratio were referred to our previous study. The retention time of 2,4,6-TCP, 2,4-DCP, and MCP was 2.79 min, 3.55 min, and 4.75 min, respectively. The limits of detection (LOD, S/ N = 3) and limits of quantification (LOQ, S/N = 10) in this experiment were 1.2–3.3 μg/L and 4.0–10.9 μg/L, respectively. What's more, the control treatment (Ctrl.) inoculated sterilized microbial consortium OEM2 was carried out to evaluate the influence of environmental factors (e.g. light) in the chlorophenols degradation process. Lignocellulosic contents (cellulose, hemicellulose and lignin) of the samples were determined according to the modified Van Soest method [18]. The degradation ratio of each lignocellulosic component was calculated with the following equation: X(k) = (M0(k) − MOEM2(k))/M0(k). The X(k) represents the degradation ratio of lignocellulosic component (k varies with cellulose, hemicellulose, and lignin in this experiment). M0(k) denotes the mass of component before degradation. MOEM2(k) is the mass of component after 12 days’ degradation by microbial consortium OEM2. For enzyme activity analysis, the fermentation broth was centrifuged at 5000 rpm/min (10 min) to prepare the crude enzyme fluid. The measurement of carboxymethyl cellulase activity (CMCase) was determined using the International Union of Pure and Applied Chemistry standard assay [19]. The crude enzyme fluid (1 mL) was incubated for 0.5 h at 50 °C in 4 mL of 20 mM Na-acetate buffer (pH 5.0) with 0.625% CMC. The quantify of sugars reduction was measured by
3.1. Microbial consortium OEM2 screening process As recorded in the screening process, microbial consortia had a well performance on lignocellulose and chlorophenols decomposition using spent mushroom substrates, as the initial source of microorganisms. The rice straw was decomposed into filament after 6 days’ cultivation (cf. Fig. S1). The weight loss of rice straw in the second generation reached 25.08% within 7 days. We believe this highly efficient lignocellulose decay was due to (i) spent mushroom substrates prepared by lignocellulosic material containing large amounts of microorganisms with lignocellulose decomposition capacity, and (ii) microorganisms forming a synergistic microbial consortium with lignocellulose degradation capability through long-term acclimation during the mushroom cultivation process. With continued the successive transfer cultivation, the capacity of lignocellulose decomposition of microbial consortia was improved after fierce competition in the new environment. The lignocellulose decomposition ability as measured by the dry matter loss of rice straw after 7 days leveled off above 35% after ten generations. Microbial consortiums exhibited a high efficiency on MCP and 2,4DCP degradation in the screening process. In the cultivation of generation 7 and generation 11, the degradation percentage of MCP was more than 80% and 2,4-DCP was 100% within 7 days. The 2,4,6-TCP degradation capability of microbial consortium was continuously enhanced from generation 7 to generation 11. The degradation percentage of total chlorophenols was up to 75% in the last three generations successive transfer precess. This may be due to the spent mushroom substrates containing many bacteria and fungus, secreting enzymes such as laccase which facilitates the chlorophenols degradation [21,22]. In the screening process, the trend of pH evolution had good 343
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Fig. 2. The mass loss of rice straw treated by microbial consortium OEM2 within 12 days.
consortium OEM2 went into the balanced phase (4–12d) and the OD values were maintained at around 1.8. This characteristic would help to exert the lignocellulose and chlorophenols degradation function of microbial consortium OEM2 stably. Subsequently, further rice straw decomposition and metabolite accumulation especially the acidic products decreased the pH slightly from day 5 to day 9. Thereafter, in the late stage of the treatment process, the pH of the fermentation broth was maintained in the vicinity of 7.60. Throughout the biological treatment process, the fermentation broth remained at a near-neutralpH, and this ideal environment for microbial consortium OEM2 proliferation, microbial activity enhancement, lignocellulose decomposition and chlorophenols detoxification. 3.3. Rice straw decomposition characteristics The weight loss of rice straw biologically treated by microbial consortium OEM2 within 12 days is depicted in Fig. 2. The rice straw decomposition was recorded at points during the first 9 days and the accumulative dry matter loss reached 38.05%. The result was similar to the research by Guo et al. [27] that corn stalk decomposition was concentrated in the early stage of cultivation. The weight loss of the rice straw was 41.5% after 12 days’ biological treatment. Compared to the biomass conversion ability of a bacteria community NSC-7 screened from compost heaps degrading 73.6% of rice straw within 14 days, microbial consortium OEM2 had an anticipated potential to treat lignocellulosic biomass for downstream biorefinery [28]. In this study, the residue substance (nearly 60%) and the soluble products metabolized by microbial consortium OEM2 in an aqueous environment could offer enough favorable organic materials for the following fermentation process. Furthermore, after 12 days’ cultivation, cellulose, hemicellulose, and lignin were decomposed up to 75.95%, 85.38%, and 18.90%, respectively. It indicated that the utilization of hemicellulose by microbial consortium OEM2 was easier than cellulose and lignin. These differences may be due to at least two reasons. First, the decomposition of lignocellulosic materials especially lignin was hampered by its intrinsic recalcitrance owing to its complex structure [29,30]. A second reason was that lower CMCase and laccase concentrations were found in the degradation process compared to the xylanase concentration. The xylanase secreted by microbial consortium OEM2 was 120–155 U/mL from day 3 to 9. The enzyme activities of laccase and CMCase were inferior to xylanase, which were 0.6 U/L and 1.2 U/mL on average in the degradation process, respectively. The extent of hemicellulose biodegradation led to the lignin carbohydrate complex (LCC) linkages cleavage and subsequent the lignocellulose three-dimensional network structure collapse. Additionally, the degradation of LCC structures improved accessibility for other enzymes or microorganism, thus, leading to the further depolymerization of cellulose and lignin. These were
Fig. 1. The evolution of pH (a) and OD (b) values within 12 days’ biological treatment.
consistency. The pH of the fermentation broth rose from 5.90 to around 7.30 within 3 days and was maintained around 7.4 until the end of cultivation. The stability of pH evolution trend may be helpful to maintain the lignocellulose and chlorophenols degradation efficiency and the stability of microbial consortia composition. After 12-generation successive transfer, the most efficient microbial consortium on lignocellulosic materials deconstruction and chlorophenols detoxification was preserved for the further research and named as microbial consortium OEM2.
3.2. pH and OD evolution in the bioconversion process Previous studies have confirmed that pH had a profound influence on the growth characteristics and metabolism characteristics of microorganisms [23,24]. There are a number of characteristics (e.g., cell membrane potential, nutrient availability, enzymatic activity, organic C characteristics, and salinity) that are often directly or indirectly related to pH. Furthermore, the above factors may alter microbial consortium diversity, affect the metabolic pathways, and determine the degradation efficiency of substrates [25]. The pH evolution of the fermentation broth during the bioconversion process is depicted in Fig. 1(a). The pH rose from initial 5.97 to around 7.57 in the first 4 days. In this period, microbial consortium OEM2, being in the exponential phase, proliferated rapidly (Fig. 1(b)). The OD values of the culture rose from initial 0.562 to around 1.835 within 4 days. Microbial consortium OEM2 was likely to take a prior consumption of the favorable substrates in the medium (viz. peptone and yeast extract) at the early cultivation stage, leading to the production and accumulation of alkaline compounds (e.g. amines), and then pH increased [26]. After that, microbial 344
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Fig. 3. The degradation characteristics of chlorophenols treated by microbial consortium OEM2.
Fig. 4. FTIR spectra of rice straw biodegraded by microbial consortium OEM2.
confirmed by FTIR analysis and SEM observation.
days. Compared to the commercially obtained laccase, microbial consortium OEM2 had an obvious advantage on large-scale application because of its low cost operation, readily availability, complex environment adaptability, and excellent lignocellulose degradation capability. Thus, microbial consortium OEM2 could be utilized in persistent organic pollution or for AOX degradation and detoxification in paper mill sludge (containing adsorbable organic halides, AOX) pretreatment to enhance the downstream utilization performance.
3.4. Chlorophenols degradation characteristics Fig. 3 depicted the disappearance ratios of chlorophenols (viz. 2MCP, 2,4-DCP, and 2,4,6-TCP) in the course of the degradation process. Microbial consortium OEM2 had a high efficiency of 2,4-DCP degradation, which was completely degraded at 6 days’ culture. 2-MCP degradation was concentrated from day 6 to 9, in which about 60% of 2-MCP were removed. A gradual decrease of 2,4,6-TCP concentration was observed until day 9 when the residual level of approximately 55% was reached. The results illustrated that microbial consortium OEM2 had selectivity on chlorophenols degradation, and the removal ratio decreased in the following order 2,4-DCP > MCP > 2,4,6-TCP. It is generally believed that trichlorophenol and pentachlorophenol metabolized by aerobic microorganisms are more resistant than mono- and di- chlorophenols to degradation due to the increased toxicity and less effective aerobic attack with highly chlorinated compounds [31,32]. The toxicity study by Leontievsky et al. [33] indicated that 2,4,6-TCP would cause hyphal growth inhibition at concentrations between 15 and 50 mg/L, leading to a lower degradation efficiency. Moreover, the metabolismic characteristics were determined by many important factors like microorganism species, reaction medium, the presence of cosubstrates, and the effect of pH on enzymatic activities. Up to now, the mechanism of chlorophenols degradation by microbial consortium OEM2 is unclear. Hence, it needs to be further investigated to fundamentally understand the key factors for chlorophenols degradation efficiency improvement. In our previous work, microbial consortium OEM1 was screened through 48 generations successive transfer cultivation and could degrade three kinds of chlorophenols (MCP, 2,4-DCP, and 2,4,6-TCP) completely after 9 days` treatment when 0.5% rice straw was loaded into the medium [24]. Compared to the microbial consortium OEM1, microbial consortium OEM2 also exhibited a good chlorophenols detoxification capability although it was screened only through 12 generations successive transfer cultivation. After 9 days’ treatment by microbial consortium OEM2, the degradation ratios of MCP, 2,4-DCP, and 2,4,6-TCP were up to 78.5%, 100%, and 44.3%, respectively. It indicated that the short-term microbial consortium screening strategy for lignocellulose degradation and chlorophenols detoxification was effective. Although the adaptability of microbial consortium OEM2 on these three chlorophenols was variant, around 75% of the total amount of chlorophenols (MCP, 2,4-DCP, and 2,4,6-TCP) was removed after 9
3.5. Chemical structure variations of rice straw after microbial consortium OEM2 treatment The structural characteristics of rice straw in the bioconversion process were investigated by FTIR (Fig. 4). Some peaks for rice straw were summarized in Supplement Table 2. They were assigned according to previous literature [34–37]. The band at 3405 cm−1 (OeH stretching vibration) decreased after microbial consortium OEM2 loading into the medium, indicating variations in the inter- and intra-molecular hydrogen-bond structure of rice straw. The peak at 2918 cm−1 was assigned to CH2– groups comprising the majority of the aliphatic fractions of waxes. After 12 days treatment, the band from 1642 cm−1 (absorbed OeH and conjugated CeO) was evolved into two bands, viz. 1654 cm−1 (HeOeH bending of adsorption) and 1605 cm−1 (aromatic skeleton vibrations), reflecting the decomposition of carbohydrate especially hemicellulose. The relative intensity in CeH (897 cm−1) deformation of cellulose was slightly decreased. This could be explained by partial cleavage of the β-1,4 glycosidic bond linkages and decreasing the degree of cellulose polymerization. However, the peak at 1062 cm−1 for CeO stretching mainly from C(3)eO(3)H in cellulose I increased during the decay process, which may be due to increased cellulose exposure because of broken hydrogen-bond and destruction the surface of rice straw [36,37]. Compared with the raw rice straw, the intensity of several typical peaks at 1512 cm−1 (C]C), 1429 cm−1 (CeH), and 1108 cm−1 (CeO) assigned to aromatic skeleton vibrations in lignin was significantly increased after microbial consortium OEM2 treatment. Moreover, the bands at 1605 cm−1, 1319 cm−1, and 1262 cm−1 associated with syringyl units in lignin molecule were observed. This sequence implied that microbial consortium OEM2 had selectivity on lignin degradation and the guaiacyl structure of the lignin molecule was more easily to be degraded. The changes in the adsorption bands after microbial consortium OEM2 treatment suggested that some chemical bonds and groups (eg. hydrogen-bond, β-1,4 glycosidic bond, and LCC linkages) 345
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Fig. 5. SEM images of rice straw before (A–C) and after (E–G) microbial consortium OEM2 treatment for 12 days. Magnification of images given in Figure A and E are 500 X; Figure B and F are 1000 ×; Figure C and G are 3000 ×.
were broken. This led to the disintegration of the resistant and recalcitrant lignocellulose, the separation of carbohydrate and lignin, and the exposure of more cellulose. Thus, the biological treatment of rice straw by microbial consortium OEM2 was beneficial to the downstream utilization.
3.6. SEM histological changes of rice straw after microbial consortium OEM2 treatment In the present study, we investigated the histological changes to the rice straw epidermal surface via SEM analysis for further understanding of substrates decomposition. Representative SEM images at various magnification are depicted in Fig. 5. SEM images show that the untreated controls had a very smooth and compact epidermal surface (Fig. 6 (A–C)). Moreover, cells and vascular bundles covered with the
Fig. 6. The main phyla of bacterial communities of microbial consortium OEM2.
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This biological treatment increased the lignocellulose bio-accessibility and decreased the complex pollutants toxicity, which has the potential to enhance downstream biorefinery performance. What’s more, microbial consortium OEM2 diversity analysis indicated that Proteobacteria (41.3%) was the most abundant phylum and the genera Paenibacillus and Pseudomonas played an important role in the lignocellulose decomposition and chlorophenols detoxification.
cuticle layers exhibited an intact morphology. During the biological treatment process, microbial consortium OEM2 stuck to the surface of the rice straw and aggressed their various issues and cells. This resulted in a clear view of the lump pits on the epidermal surface and the disruption of parenchyma cells. This treatment resulted in the exposed cellulose fibers on the surface and depositied the silica body in the inner lumen spaces and outside the cell walls, suggesting that the cuticle and silica layers were destructed and removed completely. The cellulose fibers were also dissociated and broken. Thereby, the cuticle and silica layers fragmentation, and the cellulose fibers exposure, along with the recalcitrant structure destruction, increased the accessibility to microorganisms and enzymes.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 51108195); Science and Technology Project of Guangdong Province under Contract No. 2015A010106012 and 2016A020210085.
3.7. Microbial consortium OEM2 diversity analysis
Appendix A. Supplementary data
The community composition of microbial consortium OEM2 was investigated via 16S rRNA gene sequencing. The bacterial OTU numbers and Chao1 estimators were 203 at 97% identity level. As Fig. 6 depicted, the composition of microbial consortium OEM2 mainly included six bacterial phyla: Chloroflexi, Firmicutes, Saccharibacteria, Bacteroidetes, Cyanobacteria, and Proteobacteria. Proteobacteria (41.3%) was the most abundant phylum across all samples. The amplicon library of microbial consortium OEM2 included sequences of 137 genera, and 18 of them reached 1% of relative abundance. SM2D12_norank, Saccharibacteria_norank, Geobacillus, Lactococcus and Cyanobacteria_norank were the dominant bacteria detected in this study, and the sum of their abundance accounted for more than 50%. Notably, the relative abundance of unclassified genera was particularly high. These unclassified microbial taxa suggested that they worked together with others in the degradation process. The composition of microbial consortium OEM2 at genus level was shown in Supplement Fig. 2. Leadbetterella belonging to the phylum of Bacteroidetes was isolated from mushroom substrate previously [38] and participated in the process of phenolic compounds degradation [39]. Geobacillus and Bacillus were the member of the phylum of Firmicutes, which accounted for about 6.0% and 1.1% of microbial consortium OEM2, respectively. Previous study indicated that they could accelerate cellulose degradation with a high cellulase activity [40]. In this study, carboxymethyl cellulase (CMCase), the representative enzyme for cellulose degradation, was detected and the concentration achieved a maxima on day 9. The glucose derived from cellulose degradation by cellulase was probably an ideal substrate for the fermentation of Lactococcus. Paenibacillus and Pseudomonas were regarded as the bacteria which had a good potential on lignocellulosic biomass deconstruction and chlorophenols detoxification [41–44]. Moreover, multiple literatures widely reported that Pseudomonas and Desulfovibrio were the main 2-chlorophenol-degrading bacteria [45,46]. The diversity of bacterial communities not only played an important role in the lignocellulose decomposition and chlorophenols detoxification, but also was helpful for maintaining the stability during the biodegradation process. Our further studies will be focused on the evolution of the microbial consortium OEM2 community structure during the biological treatment process under various culture conditions (e.g. lignocellulose loads and chlorophenols concentrations). Combined with the metabolic products analysis, it would further clarify the mechanism of how microbial consortium OEM2 worked in the degradation process.
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