Enhanced biogas production from wheat straw with the application of synergistic microbial consortium pretreatment

Abstract

Lignocelluloses featuring complicated structure and poor degradability usually require pretreatments prior to their utilization; synergistic microbial systems are ubiquitous in nature and now exhibit appealing features that inspire mounting interest in developing functional microbial consortia for biotechnology development. In this study, we introduced a microbial consortium composed of bacteria and fungi that synergistically exhibited great abilities of lignocellulose degradation. A pretreatment of lignocelluloses by using this functional microbial consortium was designed and results showed that enhanced degradability in wheat straw and the broth containing amounts of organic materials (e.g. volatile fatty acids and carbohydrates) that could be used for biogas synthesis were detected in the pretreatment system. A subsequent anaerobic fermentation with the application of the pretreated system showed a 39.24% and 80.34% increase in total biogas and methane yield as well as a faster startup in a 20 day production process compared to the process based on the untreated system.

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Introduction

Lignocelluloses are a major component of biomass that make up about half of materials produced via photosynthesis and are primarily composed of cellulose, hemicelluloses and lignin, which interact strongly through covalent cross-linkages and non-covalent forces, resulting in low efficiency for biomass utilization.^1–3 So far, only a minority of biomass can be utilized in limited areas such as animal feed and organic fertilizer production, paper-making, and energy exploitation,^4,5 with the rest being directly burnt or buried, which is a substantial waste of natural resources and contamination of the environment.

With environmental problems becoming more exacerbated, interest in more efficient utilization of agro-industrial residues for the production of renewable energies has increased,^6,7 among which biogas (biomethane) has drawn attention and promises to be an ideal substitution for traditional fossil fuels.⁸ Generally, biogas production is divided into three phases: hydrolysis, acidogenesis and methanogenesis, among which the hydrolysis process is usually a rate-limiting step if the raw materials are rich in lignocelluloses,⁹ because lignocellulosic materials are usually resistant to being digested due to the tight associations in lignocelluloses.¹⁰ Hence, to highly-efficiently utilize these materials for biogas production, pretreatment prior to anaerobic digestion is required.

Up to now, extensive efforts have been made to develop efficient biomass pretreatment methods such as biological, physical, chemical or physicochemical methods,^11,12 among which the biological method has drawn increasing attention due to its lower energy requirement and milder operational conditions. Recently, biological pretreatment based on the use of enzymes (e.g. cellulase or laccase) has been reported and exhibited its advantages.⁴ However, this method needs a large amount of enzymes and may therefore increase the operation cost; additionally, the application of enzymes may be strictly limited by the operational conditions, and these drawbacks prevent it from being an ideal method for biomass pretreatment.

In comparison, pretreatment based on microorganisms is an economically promising alteration. The majority of current research focuses on pretreatment using fungi, especially those in single strain or pure cultures, which usually turn out to be low efficiency in view of the individual effect.^13–15 Recently, the application of a microbial consortium that imposes synergistic effects on biomass pretreatment has drawn mounting attention and achieved positive results.^16–18 However, many of these methods focus on the abilities of cellulose/hemicelluloses degradation while lignin removal is always obscure or ignored. In fact, research has confirmed that cellulase accessibility can be greatly improved by removing the lignin from lignocelluloses and pretreatment efficiency might be therefore enhanced if the microbial consortium could synergistically work on lignin degradation as well as cellulose/hemicellulose hydrolysis.^19,20

Hence, the objective of this study was to investigate the feasibility of wheat straw pretreatment by using a functional microbial consortium consisting of both bacteria and fungi; in addition, the microbial composition in this consortium as well as its effect on wheat straw biodegradability and biogas (methane) production were also studied to reveal the mechanism of synergistic microbial pretreatment. A laboratory-scale and continuous anaerobic biogas digester were applied to evaluate the biological methane potential (BMP) of pretreated wheat straw.

Experimental section

Materials

Agricultural wheat straw was collected in Siyang, China. After collection, the straw was air-dried to final moisture contents (MCs) of 7–8% and then cut into appropriate lengths of 5 cm. The samples were then packed in plastic bags and placed at 4 °C. Sodium chloride (NaCl), sodium monohydrogen phosphate heptahydrate (Na₂HPO₄·7H₂O), potassium dihydrogen phosphate (KH₂PO₄), ammonium chloride (NH₄Cl), ammonium bicarbonate (NH₄HCO₃) and sodium sulfide nonahydrate (Na₂S·9H₂O) were provided by Aladdin Reagent Company, China.

Screening and selection of the microbial consortium

In this study, microbial consortium screening and selection was conducted as follows (Fig. 1): firstly, raw materials collected from distributed areas such as straw composts, anaerobic digestion reactors, sludge in rivers, etc. around Nanjing Tech University were serially marked and stored in sealed sample bags at 4 °C. For screening, each sample was respectively mixed with sterile water at 10% (w/v) in a correspondingly numbered flask and cultivated at 37 °C for 2 h. After that, each sample solution with 1000-fold serial dilution was inoculated and cultivated in Luria–Bertani (LB) medium at 37 °C for 6 h, which was then respectively inoculated in the M9 screening medium (MSM) with 1% (v/v) seed volume. Here, MSM was prepared as the M9 minimal medium using wheat straw as the carbon source, which contained 12.8 g Na₂HPO₄·7H₂O, 3.0 g KH₂PO₄, 0.5 g NaCl, 1 g NH₄Cl, 10 g wheat straw and 1 L water (in preliminary screening, an extra piece of filter paper (1 × 5 cm) was added). Preliminary screening was performed in the capped flask at 37 °C for 3–5 days, and the groups presenting a high filter paper degradation rate were picked out. Here, the filter paper degradation rate was calculated according to the equation as follows:where the L_o (cm) stands for the original length of filter paper, L_p (cm) stands for the length of filter paper after pretreatment, T_p (d) stands for the time of pretreatment (time counting continued until the filter paper was completely degraded).

Fig. 1 Scheme for screening and selection of microbial consortium from different raw materials and its application on wheat straw pretreatment for biogas production.

For the microbial consortium selection, microbial flora chosen from the primary screening were respectively renumbered and inoculated in capped flasks containing fresh MSM. In addition, the chosen flora were mutually mixed with each other in the same ratio and inoculated in capped flasks containing fresh MSM. Selection was based on the overall analyses including lignocellulosic ingredient degradation of wheat straw, filter paper degradation rate and contents of water soluble carbohydrate and organic products in the fermentation broth. After selection, continuous domestication of the determined microbial consortium was carried out to maintain the stable community.

Wheat straw pretreatment with microbial consortium

30 g sterilized wheat straw mixed with 300 mL sterile M9 medium containing 3.84 g Na₂HPO₄·7H₂O, 0.9 g KH₂PO₄, 0.15 g NaCl and 0.03 g NH₄Cl was poured into a 1.5 L glass bottle, and the microbial consortium was then inoculated with a 5% seed volume. Each of the 3 bottles was immediately covered and sealed with a plastic film. The control bottle was not inoculated with the microbial consortium and it only contained 30 g sterilized wheat straw and 300 mL sterile M9 medium. All bottles were placed in a chamber under temperature 37 °C for 15 days.

Analysis method

During the pretreatment, wheat straw was periodically collected and washed with HCl–HNO₃ (v/v, 1/3) solution followed by water to remove non-cellulosic materials, and then dried at 50 °C. The composition of solid substrates in the wheat straw was analyzed by the APHA standard method.²¹ In addition, scanning electronic microscopy (SEM) of wheat straw was conducted to investigate the structural changes of lignocelluloses.²² Dissolved oxygen (DO) was detected using the dissolved oxygen meter (Milwaukee MW600, USA).

Determination of the volatile products in the pretreatment broth was conducted as follows: broth that was periodically collected was initially centrifuged at 5500 rpm for 10 min, with the supernatant being filtered with an aperture of 0.22 μm and analyzed with GC-MS (gas chromatography mass spectrometry, Trace GC Ultra DSQ, USA), which was performed online using a capillary column, CP-Chirasil-Dex CB (25 mm × 0.25 mm). The temperature program was set as: initial temperature 60 °C with 1 min holding and then increased to 100 °C with a rate of 7 °C min⁻¹, followed by a rise of 18 °C min⁻¹ to 195 °C with a final hold of 2 min.

Water-soluble carbohydrates in the broth were detected by using gas chromatography (GC) analysis of their corresponding alditol acetates, which were prepared using the method described by Blakeney.²³ The alditol acetate derivatives of sugars were quantified with the capillary column Alltech DB-225 (30 m × 0.25 mm × 0.25 μm film), and a temperature program with an initial temperature of 190 °C with a hold of 4 min, followed by a temperature rise of 2 °C min⁻¹ to 230 °C with a final hold of 25 min was designed.

Biochemical methane potential (BMP) assay

The biochemical methane potential assay was based on the principles described as follows: after being pretreated by the microbial consortium for a certain number of days, the systems that contained both pretreated straw and pretreatment broth were mixed with 300 mL anaerobic slurry for a working volume of 600 mL, and the same conditions were used for the untreated system. Here, the anaerobic slurry that contained 18.9 g L⁻¹ TS and 7.8 g L⁻¹ VS was taken from an up-flow anaerobic sludge blanket (UASB) digester in the biogas pilot plant of Nanjing Tech University. The anaerobic slurry was collected from the same batch digester and domesticated for parallel biogas production assays. Ammonium bicarbonate (NH₄HCO₃) was added to adjust the carbon-to-nitrogen ratio (C/N) to 25, which was favorable to anaerobic microorganisms.²⁴ All bottles were gassed with nitrogen before adding sodium sulfide to ensure anaerobic conditions, and immediately sealed using a rubber plug and aluminum crimp caps with drilled holes where a glass tube was inserted to export gas for biogas and compositional analysis. Once sealed, the bottles were fixed in an incubator with a constant temperature of 37 °C. Two control bottles containing 300 mL of distilled water that was respectively mixed with 300 mL of anaerobic slurry and 300 mL of pretreated system were prepared to remove endogenous methane production from the assays. The daily biogas volume was monitored throughout the process by water displacement, with measured water volumes being converted to gas volume at standard conditions. The methane concentration in the biogas was periodically measured by gas chromatography (GC).

Microbial diversity analysis

Analysis of microbial diversity was conducted using the GS FLX Systems, Roche. Whole genome surveys with 454 sequencing systems have become the standard for ribosomal RNA identification (i.e. 16S, 18S) and enable a general view of microbial diversity in a particular environment.²⁵ Comparison of the genetic information was based on the BLAST (http://www.ncbi.nlm.nih.gov/) and the ribosomal database project classifier (http://rdp.cme.msu.edu/).

To investigate the microbial effect on biomass pretreatment, bacteria and fungi in the consortium were isolated as follows (Fig. 2a): to isolate bacteria, 1000-fold diluted broth was spread on the LB solid medium and then anaerobically cultured at 37 °C for 12 h; meanwhile, the diluted broth was evenly spread on potato dextrose agar (PDA) solid medium containing the antibiotic chloramphenicol (30 μg mL⁻¹) at 20 °C for 48 h to selectively cultivate fungi.²⁶ After that, single colonies were picked out according to their distinct morphologies. All the isolated bacteria were re-inoculated in LB medium at 37 °C for 12 h and then combined in the same ratio to construct bacteria consortia; the fungi consortia were constructed likewise. Reconstructed microbial consortia were prepared by mixing the bacteria and fungi consortia in the same ratio and cultivated at 37 °C for 48 h. To investigate the effects of microbial composition on pretreatment, three assembled consortia were each cultured and inoculated into fresh MSM to compare their overall pretreatment abilities. After a 3 day pretreatment, the three pretreated systems were each applied to the biogas production using the same conditions as described above.

Fig. 2 (a) Isolation of bacteria and fungi from microbial consortia and reconstruction of microbial consortia; (b) mechanism of synergetic effects of the microbial consortium on lignocellulose pretreatment and utilization.

Results and discussion

Microbial consortium screening and selection

In this study, microbial consortium screening and selection was performed as shown in Fig. 1. During the primary screening, the filter paper degradation rate was used to evaluate the lignocellulose utilization efficiency of a microbial consortium, and the groups presenting fast filter paper degradation rate might have potential capability for lignocellulose pretreatment.²⁷ Hereby, the three groups of microbial flora that originated from wheat straw composts (A), river sludge (F), and dairy manure (G) which presented the highest paper degradation rates (i.e. almost complete filter paper degradation in less than 4 days), suggesting the potential ability to degrade lignocelluloses, were chosen from the primary screening. Compared to the screened bacteria showing the highest filter paper degradation rate of 65.7% in 4 days reported by Gupta et al.,²⁸ these three screened microbial flora showed much better filter paper degradation ability.

For microbial consortium selection, the three determined groups as well as their mutually combined groups were respectively investigated. As seen in Table 1, microbial flora F always presented a negative effect on microbial reassembling, which was possibly due to the instability and adverseness of the microbial communities in flora F. In terms of the lignocellulose degradability, the microbial flora G as well as the mixed group A + G both showed outstanding advantages. Particularly, the mixed group A + G could completely degrade filter paper and achieve a wheat straw degradation rate of 31.7% in 2 days. Compared to the composite microbial system of MC1 reported by Cui et al. that exhibited a corn stalk degradation rate of 39.0% in 3 days,¹⁸ the mixed group A + G presented the same or even higher efficiency in lignocellulose degradation. Additionally, amounts of water-soluble carbohydrates and volatile products were detected in broth after being treated by the mixed group and these organic materials could be further used for downstream utilization.²⁹ As a result, the mixed microflora A + G was chosen to be the microbial consortium candidate applied for wheat straw pretreatment.

Table 1 Biochemical characterizations of microbial consortium (MC) selection^a

Group index	Resources	Filter paper degradation rate (cm d^−1)	Degradation of wheat straw (%)	Water soluble carbohydrate (g L^−1)	Volatile products (g L^−1)
a All the data in each index was detected until the filter paper was completely degraded.
MC 1	Microbial flora A	1.2 ± 0.1	13.3 ± 0.3	0.46 ± 0.03	0.26 ± 0.01
MC 2	Microbial flora F	1.8 ± 0.2	17.9 ± 0.7	0.54 ± 0.02	0.33 ± 0.03
MC 3	Microbial flora G	2.3 ± 0.1	22.5 ± 1.3	0.78 ± 0.06	0.71 ± 0.10
MC 4	Microbial flora A + F	1.5 ± 0.3	10.7 ± 0.3	0.43 ± 0.01	0.28 ± 0.06
MC 5	Microbial flora A + G	3.1 ± 0.1	31.7 ± 0.1	0.97 ± 0.03	1.12 ± 0.09
MC 6	Microbial flora F + G	1.9 ± 0.1	15.6 ± 0.2	0.58 ± 0.06	0.31 ± 0.05
MC 7	Microbial flora A + F + G	2.1 ± 0.2	19.3 ± 0.6	0.65 ± 0.11	0.45 ± 0.02

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Effect of biological pretreatment on wheat straw

Lignocelluloses, known as a complicated system, usually require some pretreatments that disrupt their outer/inner structures to promote their utilization efficiency.^30,31 In microbial pretreatment, the traditional method in the solid state usually takes a long time and induces great losses of carbohydrates or metabolism products, which is not beneficial to the subsequent utilization. To overcome these drawbacks, pretreatment by using the microbial consortium in the liquid state was introduced here.

As confirmed from the histogram for the degradation ratio (Fig. 3a), the wheat straw was degraded most expeditiously during the first 3 d, exhibiting a degradation ratio of about 47% on day 3, and then proceeded relatively gently, obtaining a degradation ratio of 69% on day 11. A similar result was reported by Cui et al. stating that the initial stage (∼3 days) was usually the period in which microorganisms propagated most vigorously and also the period in which biomass was degraded most intensively.¹⁸ According to periodic analyses, compositional variation in pretreated wheat straw was summarized as the initial lignin removal followed by cellulose and hemicellulose degradation: almost 20% of lignin was removed from wheat straw on day 1 while cellulose and hemicelluloses remained inactive; when 40% of lignin was removed on day 3, the hemicelluloses as well as cellulose exhibited an enhanced degradation rate. It’s known that the general structure of lignocelluloses is described as inner cellulose wrapped by hemicelluloses and lignin which are the major obstacles of cellulase, and the structural destruction by removing the lignin is favorable to cellulose utilization.^19,20 We therefore believed that the tight interactions in lignocelluloses were disrupted by degrading the majority of lignin and the accessibility of cellulose was greatly improved after being pretreated by the microbial consortium. This speculation could be verified by the SEM analyses of wheat straw (Fig. 4) showing that obvious structural destruction accompanied with larger specific surface area were achieved in pretreated straw and the disrupted structure improved the accessibility of remaining cellulose (hemicelluloses).

Fig. 3 (a) Degradation and composition analyses of wheat straw during pretreatment; (b) quantitative analyses of water soluble carbohydrates and volatile products in broth during pretreatment.

Fig. 4 Scanning electronic microscopy (SEM) analysis of wheat straw before and after pretreatment: (a and b) untreated wheat straw; (c and d) wheat straw with 3 day pretreatment.

In microbial pretreatment of lignocellulosic biomass, a notable issue is how to efficiently utilize carbohydrates during pretreatment for downstream utilization.³² Analysis of the broth is shown in Fig. 3b and it showed that maximum of carbohydrates including glucose, arabinose, and xylose were accumulated in the first 3 d, achieving the maximum concentration of 5.94 ± 0.14 g L⁻¹. In the following process, carbohydrates were partially metabolized by the microbial consortium and converted into byproducts such as volatile fatty acids (VFA).³³ In this study, six volatile products were detected during pretreatment, among which acetic acid, propanoic acid, butanoic acid and glycerin were the four major compounds and their maximal total content of 9.12 ± 0.13 g L⁻¹ was detected on day 3. Compared to fungal pretreatment, usually in solid state, that takes a longer time inducing unnecessary carbohydrate consumption or loss, the microbial pretreatment in the liquid state not only facilitated the degradation of lignocellulose into carbohydrates but also specifically produced some byproducts (e.g. volatile fatty acids, glycerin) that can be used in acetogenesis for biogas production. Hence, all the merits made us believe that enhanced production yield and a shortened fermentation period would be achieved if this pretreatment system were applied to biogas production.

Enhancement of biogas production by microbial pretreatment

To verify if microbial pretreatment of wheat straw would promote biogas production efficiency, the pretreated systems with different pretreatment times were applied to the anaerobic fermentation. Results showed that when pretreated for 3, 5, and 10 days, the production systems respectively presented biogas yields of 363.39, 312.34, and 289.67 mL_N g⁻¹ VS, which were 41.23%, 21.38%, and 12.58% higher than the biogas yield from the untreated system, indicating that the microbial pretreatment could enhance the utilization efficiency of wheat straw. Here, we found that declining biogas (methane) yield was detected with increasing the pretreatment time and it might be due to the competition of carbohydrates between pretreatment internal fermentation and downstream utilization: during biological pretreatment, it’s known that part of the degraded product (carbohydrates) is not accumulated but consumed by the growing microorganisms, and this situation might be enhanced and result in decreasing contents of organic materials for acetogenesis or methanation if pretreatment time were extended. Similar results reported by Poszytek et al. also indicated that biogas production efficiency was affected by pretreatment time and the optimum time should be the period during which soluble chemical oxygen demand and concentration of VFA reached the maximum.³⁴ Besides, an alkaline environment would be generated due to the accumulation of byproducts (e.g. ammonia) with more pretreatment time, and this is adverse to anaerobic fermentation (biogas production).²⁸

As a result, wheat straw pretreated by the microbial consortium for three days was favorable to biogas production and the corresponding methane potential assays were performed, with the cumulative biogas/methane yield being shown in Fig. 5a. As seen, a more rapid increase in biogas/methane yields at the initial stage was achieved from the pretreatment. Zhong et al. previously reported a similar result and they thought the initial increase was due to the enhanced degradability of pretreated biomass.⁴ Here, according to the periodic analysis of the fermentation broth shown in Table 2, we found that much more volatile products and carbohydrates could be provided from the 3 day pretreated system compared to the 3 day fermentation of the untreated system and this difference led to a biogas yield of 143.20 ± 0.32 mL_N g⁻¹ VS from 5 day production based on the pretreated system, which was a 2-fold increase compared to the yield of 8 day production based on untreated system. We therefore believed that greater accumulation of organic compounds from pretreatment might be another reason for the faster startup in biogas production and it also verified the assumption that disrupted lignocelluloses accompanied by the rich-content pretreatment broth might greatly shorten the initial digestion period of biogas production.^11,31

Fig. 5 Characterization of biogas production: (a) cumulative biogas/methane yields for the untreated and pretreated systems (3 day pretreatment); (b) comparisons of the total biogas/methane yields of the untreated and pretreated system (3 day pretreatment) in 20 day production.

Table 2 Periodic characterizations of biogas (methane) production process

Stage	Fermentation period^a	Component analysis^b		Biogas production (mLN g^−1 VS)	Methane production (mLN g^−1 VS)
		Volatile fatty acids (g L^−1)	Water-soluble carbohydrate (g L^−1)
a “Pretreated-based Fermentation” represents the fermentation (biogas production) based on the 3 day pretreated system; “Untreated-based Fermentation” represents the fermentation based on the untreated system; times in brackets are the detection times during biogas production.b Periodic component analysis of the fermentation broth during biogas production.
Stage 1	Pretreated-based fermentation (0 day)	5.08 ± 0.06	3.12 ± 0.02	—	—
	Untreated-based fermentation (3^rd day)	2.35 ± 0.02	1.24 ± 0.01	24.54 ± 0.21	7.62 ± 0.11
Stage 2	Pretreated-based fermentation (5^th day)	5.65 ± 0.14	4.03 ± 0.04	143.20 ± 0.32	74.33 ± 0.78
	Untreated-based fermentation (8^th day)	1.76 ± 0.07	1.65 ± 0.04	59.71 ± 0.98	25.63 ± 0.67
Stage 3	Pretreated-based fermentation (10^th day)	3.23 ± 0.05	1.32 ± 0.06	287.50 ± 0.88	146.33 ± 0.34
	Untreated-based fermentation (13^th day)	0.85 ± 0.02	0.79 ± 0.03	162.42 ± 1.11	78.57 ± 0.78
Stage 4	Pretreated-based fermentation (20^th day)	1.67 ± 0.11	0.76 ± 0.03	363.39 ± 0.56	246.20 ± 0.89
	Untreated-based fermentation (23^rd day)	0.34 ± 0.01	0.32 ± 0.01	260.31 ± 1.56	136.54 ± 0.88

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In addition, a total biogas yield of 363.39 ± 0.56 mL_N g⁻¹ VS after 20 day fermentation was obtained from the pretreated system, which was 39.24% higher than the untreated system for 23 day fermentation; the methane yield shown in Fig. 5b indicates that the pretreated system could lead to a methane yield of 246.20 ± 0.89 mL_N g⁻¹ VS in 20 day production, which was 80.34% higher than that from the production based on untreated system, suggesting that pretreatment is capable of enhancing not only the biomass utilization efficiency but also energy conversion.

Effect of microbial composition on pretreatment

As discussed above, pretreatment by the microbial consortium could improve biomass utilization and energy conversion efficiency. However, unlike pretreatment merely by using fungi or bacteria, processing with the application of a consortium involving groups of microorganisms is unknown. Here, we tried to reveal the underlying effects of the microorganism communities and their integral properties on biomass pretreatment.

According to the microbial analyses shown in Table 3, the consortium was mainly composed of bacteria and fungi. The bacteria could be classified into the classes of Proteobacteria (2 types), Firmicutes (3 types), Bacteroidetes (1 type). The fungi Coriolus versicolor and Gloeophyllum trabeum were identified and they both have been reported for biomass pretreatment or lignin degradation.^35,36 In this consortium, the majority of bacteria were facultative anaerobic types and according to dissolved oxygen detection during pretreatment, we found that oxygen was initially consumed by the microorganisms and the environment could remain microaerophilic (DO = 0.9 ± 0.1 mg L⁻¹) throughout the process to maintain fungi metabolism and lignin degradation.

Table 3 Microorganism identification in the microbial consortium

Microbial index	Phylum or class of nearest neighbor	Nearest neighbor sequence^a	Identity
a Nearest neighbor sequences is identified by running BLAST against the GenBank database.
Bacteria
1^#	Γ-Proteobacteria	Escherichia sp. clone out-X1-5	99%
2^#	Bacteroidetes	Ruminofilibacter xylanolyticum strain S1	98%
3^#	B-Proteobacteria	Alcaligenes faecalis strain G	98%
4^#	Firmicutes	Bacillus sp. MHS037	97%
5^#	Firmicutes	Bacillus amyloliquefaciens BDH 27	99%
6^#	Firmicutes	Bacillus subtilis DCY-1	99%
Fungi
7^#	White-rot fungi	Coriolus versicolor	91%
8^#	Brown-rot fungi	Gloeophyllum trabeum	93%

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Generally, the application of bacteria or fungi in biomass pretreatment was focused on cellulose/hemicellulose degradation or lignin removal.^35–37 Here, to investigate the mechanism of pretreatment using a microbial consortium composed of bacteria and fungi, we investigated their individual and combined effects on pretreatment. As seen in Table 4, after pretreatment by the bacteria consortium for 3 days, wheat straw showed cellulose/hemicellulose degradation of 31% while lignin almost remained inactive. Compared with the microbial consortium, the bacteria consortium showed weakened cellulose/hemicellulose degradation ability in the absence of fungi, indicating that lignin degradation caused by the fungi might promote the cellulose or hemicellulose utilization efficiency.^19,20 This result was consistent with a previous study by Huang et al. stating that enzymatic saccharification of cellulose is facilitated by degrading the lignin.³⁸ Similarly, pretreatment merely by the fungi consortium also exhibited low wheat straw and cellulose/hemicellulose degradation rate even if a lignin degradation of 28% was achieved. Comparatively, a reconstructed consortium could basically retain the degradation ability of the original consortium and the decreased effect might be attributed to the absence of some minor microorganisms that couldn’t be isolated from the original consortium for reconstruction. In addition, biogas production from wheat straw pretreated by different consortia was compared. As shown in Table 4, wheat straw pretreated by the bacteria and fungi consortia respectively gave biogas yields of 278.67 ± 0.34 and 237.78 ± 1.05 mL_N g⁻¹ VS, which were 23.31% and 34.57% decreased compared to the yield of wheat straw pretreated by original consortium; when pretreated by the reconstructed consortium, wheat straw presented a biogas yield of 312.45 ± 0.89 mL_N g⁻¹ VS, which was a slight decrease compared to that of the original consortium.

Table 4 Characterization of pretreatment and BMP assay using different consortia

Characterization analyses	Microbial consortium	Bacteria consortium	Fungi consortium	Reconstructed consortium
a Wheat straw pretreatment time was set to be 3 days.b All BMP assays were conducted with the application of the 3 day pretreated systems.
Wheat straw pretreatment^a
Wheat straw degradation (%)	48.4 ± 1.6	28.4 ± 0.3	7.2 ± 0.5	37.5 ± 0.7
Cellulose/hemicellulose degradation (%)	45.5 ± 0.4	31.1 ± 0.5	3.4 ± 0.1	40.3 ± 1.1
Lignin degradation (%)	30.6 ± 1.1	1.2 ± 0.2	28.6 ± 0.6	29.4 ± 1.4
Biochemical methane potential (BMP) assay^b
Biogas production (mLN g^−1 VS)	363.39 ± 0.56	278.67 ± 0.34	237.78 ± 1.05	312.45 ± 0.89
Methane production (mLN g^−1 VS)	246.20 ± 0.89	152.45 ± 0.21	134.67 ± 0.45	178.56 ± 0.56

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As a result, the synergetic effects of bacteria and fungi on biomass pretreatment that focused on lignin degradation as well as carbohydrate transformation is indeed an efficient way to fully promote biomass utilization efficiency. Hereby, a supposed mechanism of lignocellulosic biomass pretreatment by using the microbial consortium was made as follows (Fig. 2b): during pretreatment, fungi in the consortium initially interact and break down the lignin; when lignin is partially removed, the digestible fractions of cellulose fibers are more accessible to microorganisms and bacteria in the consortium can convert cellulose/hemicelluloses into some nutrients for self-growth and downstream utilization.^35,37

Conclusion

In this paper, to overcome the disadvantages generated from traditional pretreatment of biomass, a microbial consortium with great lignocellulose degradation ability was screened and applied to biomass (wheat straw) pretreatment. The results showed that the microbial consortium composed of bacteria and fungi could synergistically degrade the lignocelluloses, leading to enhanced biodegradability in wheat straw and great accumulations of organic materials (e.g. VFA and carbohydrates) that can be used for biogas production in the broth after pretreatment. When applying this pretreatment system to biogas production, greatly enhanced efficiency (39.24% higher in total biogas yield and 80.34% higher in methane yield) accompanied by a faster startup were achieved compared to the production based on the untreated system.

In conclusion, pretreatment of lignocellulosic biomass with the application of a microbial consortium is found to be an efficient way to promote biomass utilization efficiency.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

The research was supported financially by the State Key Basic Research and Development Plan of China (2013CB733500), National Key Technology R&D Program (2014BAC33B00), National Hi-Tech R&D Program (2012AA021405), Open Project of State Key Laboratory of Motor Vehicle Oriented Biofuels Technology (2013024), Key Technology R&D Project of Guangxi Zhuang Autonomous Region (GKZ1348004-4), PCSIRT (IRT1066) and PAPD.

Notes and references

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