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Anaerobic digestion of lignocellulosic biomasses pretreated with Ceriporiopsis subvermispora
Journal of Environmental Management 193 (2017) 154e162
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
Anaerobic digestion of lignocellulosic biomasses pretreated with Ceriporiopsis subvermispora X. Liu a, S. Hiligsmann b, R. Gourdon a, R. Bayard a, * a b
Univ. Lyon, INSA-Lyon, DEEP Laboratory, EA4126, Bldg. S. Carnot, 20 Avenue A. Einstein, F-69621 Villeurbanne, France 3BIO-BioTech, Universit e Libre de Bruxelles, Av. F. Roosevelt 50, CP 165/61, Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2016 Received in revised form 26 January 2017 Accepted 28 January 2017
Fungal pretreatment by Ceriporiopsis subvermispora of two forest residues (hazel and acacia branches) and two agricultural lignocellulosic residues (barley straw and sugarcane bagasse) were studied as a pretreatment to improve their subsequent anaerobic digestion for methane production. Biomass samples were grinded to 2 ranges of particle sizes (<4 or 1 mm), autoclaved, inoculated with two strains of C. subvermispora (ATCC 90467 and ATCC 96608) and incubated at 28 C for 28 days. The effects of fungal pretreatment were assessed by analyzing the samples before and after incubations for dry solids mass, biochemical composition, bio-methane production (BMP) and availability of cellulose to hydrolysis. The production of ligninolytic enzymes MnP and/or laccase was observed with both strains during incubation on most of the samples tested. It almost doubled the hazel branches BMP per unit mass of dry solids but did not improve however the BMP of the agricultural residues and acacia branches. These observations were explained by the fact that although both strains were able to degrade 20e25% of lignin in <1 mm and <4 mm hazel branches samples, none of them was successful however to significantly degrade lignin in the other samples, except for sugarcane bagasse. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Ceriporiopsis subvermispora White-rot fungi Lignocellulosic biomass Biomethane potential Enzymatic hydrolysis Fungal pretreatment
1. Introduction Lignocellulosic biomass is the predominant organic resource on the surface of the planet and as such is considered as one of the most promising renewable energy resources (Bertrand et al., 2014; Monlau et al., 2015). It is not digestible by human beings and for a great part neither by animals. Therefore using it for energy recovery purposes does not raise competition with alimentary usages unlike first generation energy crops such as sugar beet, wheat or maize. In addition, available lignocellulosic biomass can be obtained in large amounts from forestry or agricultural residues, causing no pressure on arable land unlike energy crops (Bertrand et al., 2014; Monlau et al., 2015). Lignocellulosic biomass is mainly composed of cellulose, hemicelluloses and lignin. Although potentially biodegradable, cellulose and hemicelluloses are protected within a gangue of lignin, which is a 3D hydrophobic polymer of great chemical stability (Klein et al., 2016; Floudas et al., 2012). Bioconversion of lignocellulosic materials is therefore limited by the protective structure of recalcitrant lignin (Goshadrou et al., 2013; Teghammar et al., 2014). * Corresponding author. E-mail address: [email protected] (R. Bayard). http://dx.doi.org/10.1016/j.jenvman.2017.01.075 0301-4797/© 2017 Elsevier Ltd. All rights reserved.
A variety of physical, chemical and biological pretreatment methods have been evaluated in the literature with the objective to break down the lignin gangue and increase the bioavailability of cellulose and hemicelluloses which bear the biomethane (or bioethanol) potential of the material (Zheng et al., 2014). Biological pretreatments seem particularly attractive because they require mild operating conditions, involve relatively low operational costs and are often environmentally friendly. However their main drawbacks are the relatively slow action and the lack of specificity (Rico et al., 2014). Fungal pretreatment with selective white-rot fungi is one of the most promising processes of pretreatment (Alexandropoulou et al., 2016; Cardona et al., 2010). White-rot fungi are known to produce ligninolytic enzymes (predominantly lignin peroxidase - LiP, laccase or manganese-peroxidase eMnP) that break down the network of lignin with relatively little degradation of cellulose and hemicelluloses (Amirta et al., 2006; Tanaka et al., 2009). Among the various selective white-rot fungi studied in the literature (Amirta et al., 2006; Tian et al., 2012; Tuyen et al., 2012), Ceriporiopsis subvermispora was reported to exhibit a high lignin-degrading selectivity (Fernandez-Fueyo et al., 2012; Itoh et al., 2003). Wan and Li (2010), reported close to 40% lignin biodegradation over 42 days of cultivation on corn stover while cellulose degradation was
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less than 5%. Other authors reported similar trends (Sasaki et al., 2011; Tuyen et al., 2012). Some studies however reported variable efficiencies depending on the considered strain of C. subvermispora. For example, Amirta et al. (2006), observed a better efficiency of C. subvermispora ATCC 90467 as compared to ATCC 96608 in the capacity to increase methane potential of Japanese cedar wood. Based on these considerations, the objective of the present study was to investigate the efficiency of the 2 strains on lignocellulosic biomasses which were not yet investigated. Two ranges of particle sizes were studied (<1 and < 4 -mm) as this parameter is known to affect the performance of microbial pretreatment (Wan and Li, 2012). The pretreatment with each strain was evaluated by monitoring ligninolytic and cellulolytic enzyme activities, substrates’ mass reductions, the cellulose availability to hydrolysis in the substrates, and the efficiency of subsequent anaerobic digestion in terms of biomethane potential (BMP) and methane production kinetics. 2. Materials and methods 2.1. Samples collection and preparation Two forest residues and two agricultural residues with currently low added value were selected. Hazel and acacia branches were gny, Rho ^ne-Alpes, France. collected from a private forest in Re Barley straw and sugar cane bagasse were selected for their abundant production and their distinctive biochemical characteristics. They were collected respectively from a private farm located in Picardie, France and from an artisanal distillery in Plaine de Cul-deSac, Port-au-Prince, Haiti. About 10 kg of each biomass material were sampled in autumn, coarsely crushed on site and carefully homogenized. They were then immediately taken to the laboratory where they were dried in an oven at 60 C for 3 days, shredded three times with a low-speed shredder (Blik® monorotor M420, ^t, France) and sieved at 10 mm to obtain homogenous Milly-la-Fore stock powders. The stock powders were then further grinded using a cutting mill (Retsch® SM 200, Haan, Germany) into 2 subfractions of particle sizes below 4-mm and below 1-mm, respectively. All samples thus prepared were stored at 2 C before analysis or experiments. Autoclaved samples were also prepared in the same manner but not incubated in order to investigate the effect of the thermal treatment. 2.2. Fungal strains and inoculum preparation Cultures of Ceriporiopsis subvermispora ATCC 90467 (hereafter named F1) and Ceriporiopsis subvermispora ATCC 96608 (hereafter named F2) were grown on 33.6 g L1 malt extract agar at 28 C for 7 days. Ten discs of 10 mm diameter of the agar medium of each culture were then transferred into 500 mL sterile Erlenmeyer flasks containing 50 mL of 33.6 g L1 malt-extract medium, and the flasks were incubated (unshaken) at 28 C. After 7 days, each culture was aseptically blended in three cycles of 15 s and the mycelium suspensions were used as an inoculum (Wan and Li, 2010).
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substrate and strain. After incubation, three of them were used for the determination of ligninolytic and cellulolytic enzymatic activities. Three others were used for chemical and biochemical analyses of the solid samples, cellulose enzymatic hydrolysis and BMP determinations. Four control flasks were followed. Two of them served as controls for enzymatic activities, and the other two were used for the other tests and analyses. After 28 days of incubation, the residual solids were collected and dried at 60 C for 72 h prior to analyses. The dry samples were weighed and the mass compared to initial mass to determine the extent of total solids biodegradation. In the assays however, the masses of the samples after incubation included the associated fungal biomass which could not be separated from the samples. This caused an overestimation of the residual masses of substrates. 2.4. Measurements of fungal enzymatic activities The residual solids collected after the incubations (assays and controls) were suspended in 120 mL of sodium acetate buffer (4.1 g L1, pH 4.5) and stirred in an orbital shaker at 150 r min1 at 28 C (Wan and Li, 2010). After for 4 h, the suspensions were filtered through 0.45 mm pore size cellulose acetate membranes and the solutions analyzed for enzymatic activities. Manganese-dependent peroxidase activity (MnP) was determined by phenol red oxidation using ε ¼ 22,000 M1 cm1 as the molar absorbance coefficient (Vicentim and Ferraz, 2007). Laccase activity was determined by oxidation of 2,2'-azino-bis-3-ethyl benzothiazoline-6-sulphonate (ABTS), using ε ¼ 36,000 M1 cm1 (Bourbonnais and Paice, 1990; Wan and Li, 2010). Cellulase activity was measured according to IUPAC guidelines (Ghose, 1987) using filter paper as a substrate. For these analyses, absorbance was measured with a SHIMADZU UV-2450 spectrophotometer. All analyses were triplicated. The digestibility of cellulose was determined as follows:
% cellulose degradation ðECD %Cell Þ ¼
cellulose digested ðgÞ 100 cellulose added ðgÞ (1)
The effect of fungal incubation on cellulose accessibility within the substrates was evaluated by comparing the efficiency of cellulose enzymatic hydrolysis before and after incubation following the NREL procedure (Selig et al., 2008). The suspensions were filtered through 0.22 mm pore size cellulose acetate membranes and the solutions analyzed for glucose by HPLC using a Waters® chromatograph equipped with a Bio-Rad HPX-87H column and a refractive index detector (RID) maintained at 45 C and 30 C, respectively. An aqueous H2SO4 solution at 0.49 g$L1was used as the mobile phase at a constant flow rate of 0.6 mL min1. 2.5. Chemical and biochemical analyses
2.3. Fungal pretreatment
2.5.1. Elemental analyses Total carbon (C), hydrogen (H) and nitrogen (N) contents were determined following standard procedures at CNRS analytical center SCA of Villeurbanne, France.
10 g of powdered samples of the required particle size were transferred into 250 mL Erlenmeyer flasks containing 30 mL deionized water. The flasks were then autoclaved at 121 C for 30 min, cooled to room temperature, and inoculated with 2 mL F1 or F2 mycelium suspension. Control flasks were prepared in the same manner but water was added instead of inoculum. All flasks were incubated at 28 C. Six replicates were incubated for each
2.5.2. Cellulose, hemicelluloses and lignin contents Analyses were done in triplicates following NREL Laboratory Analytical Procedure (Sluiter et al., 2005). The samples were firstly extracted successively with HPLC-grade water and with ethanol using the Dionex ASE 350 system to remove soluble components, then hydrolyzed with a concentrated sulphuric acid aqueous solution (72% w/w) at 30 C for 1 h. The suspensions were diluted to
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4% with de-ionized water, autoclaved at 121 C for 1 h, cooled to room temperature and filtered. The solutions were analyzed for acid-soluble lignin by measuring UV absorbance at 205 nm and for monomeric sugars (glucose, xylose, galactose, arabinose and mannose) using a Waters® HPLC equipped with a Bio-Rad Aminex HPX-87P column and a refractive index detector (RID) operated at 85 C and 49 C respectively, with HPLC-grade water as the mobile phase at a flow rate of 0.6 mL min1. Cellulose and hemicelluloses contents of the samples were calculated from the concentrations of the corresponding monomers analyzed in the solutions. The acid-insoluble solid residues were dried at 105 C overnight, weighed, calcinated at 575 C for 4 h, and the ashes were weighed after cooling. The mass loss was considered as acid-insoluble lignin. 2.6. Mass balance on total solids and biochemical fractions All substrates were weighed and analyzed for their biochemical compositions (i) in their powdered initial form as prepared for the experiments, (ii) after autoclaving, (iii) after autoclaving and abiotic incubation (controls), and (iv) after autoclaving and fungal incubation (assays). In order to evaluate the extent of degradation of the substrates specifically due to the action of the fungus, the mass reductions observed in the assays on the dry solids and on their biochemical fractions were corrected with the respective variations observed on the same parameters in the autoclaving and/or the abiotic controls. 2.7. Anaerobic digestion Anaerobic digestion was studied using the Biochemical Methane Potentials (BMP) protocol (Angelidaki et al., 2009). For each substrate, before and after fungal pretreatment, a mass of dry solids calculated to contain 1 g volatile solids was transferred into 500 mL serum bottles containing 200 mL of a nutritive medium (ISO 11734, 1998), and 100 mL of anaerobic inoculum suspension cultivated in the laboratory from a seed initially taken from an anaerobic digester treating domestic sewage sludge. Under these conditions, the ratio between the mass of volatile solids (VS) in the samples and that of the inoculum was 0.5 in each flask. The flasks were then flushed with a gas mixture of 70% N2 and 30% CO2 for 5 min, sealed with air-tight rubber stoppers and plastic seals, and incubated at 35 ± 2 C in the dark. Biogas production was monitored using a Digitron® 2085P pressure transducer. Gas samples were regularly taken for immediate analysis using an Agilent® gas micro-chromatograph equipped with a thermal conductivity detector and a PoraPLOT U column for CO2 and a Molsieve column for O2, N2, and CH4. Biomethane potential was calculated from the cumulated methane production obtained in the tests, and finally expressed under 0 C and 105 Pa in L of methane kg1 of substrate VS. All experiments were triplicated. Experimental time course data of cumulative methane production in each test (expressed under 0 C and 105 Pa) was fitted to first order kinetics model of eqn. (2):
VðtÞ ¼ V max 1 ekt
(2)
where V(t) was the cumulative methane production at time t, 1 Þ the overall methane production (i.e. BMP), and k Vmax ðLCH4 $kgVS the first order kinetic constant (d1). Best fit values of parameters k and Vmax were determined by least squares fitting of the above equations to each data set using Microsoft Excel's solver function.
3. Results and discussion 3.1. Mass balance evaluations of substrates biodegradation in fungal pretreatment Control-corrected mass balance results are given in Fig. 1. It was observed that the biotic degradation induced a total solids mass reduction of 8e12% for hazel and acacia branches, and up to 15% for bagasse. The overall degradation was stronger with strain F1 except for hazel, which was better degraded by strain F2. Barley straw was less degraded, with mass reductions below 5% with either strain. Various performances were observed in the biopolymers degradation depending on the nature of the substrates (Table 1), their particle size, and the strain tested. Both strains were able to efficiently degrade lignin in hazel branches samples, F2 being slightly more efficient than F1 (close to 25% degradation vs 20%). Both strains were also very efficient to degrade lignin in 1-mm bagasse (more than 40% degradation). None of them however was efficient to degrade lignin in acacia branches (degradation less than about 5%) and barley straw (no degradation) regardless of particle size tested. These observations suggested that the structure of the lignin gangue in these 2 samples was probably quite different from that of the other 2 substrates, inducing a lower accessibility to fungal enzymes and therefore a lower degradation. Cellulose was at best very poorly degraded, usually much less than lignin, thereby confirming the good selectivity of the selected fungal strains. In acacia branches however, close to 10% and 20% cellulose degradations were observed with F2 and F1 respectively, regardless of particle size, whereas lignin degradation was below 5%. This observation may be attributed to a better accessibility of cellulose, which would be less protected by lignin in this sample as compared to the other. Finally, hemicelluloses were in most cases degraded more than cellulose but less than lignin. These results were consistent with those of Blanchette et al. (1985) who studied lignin degradation in decaying forest wood and reported that delignification was not occurring at the same extent in the different samples which were analyzed. Several white-rot fungi exhibited selective lignin removal only in certain woods and were much less selective, or not at all, in other woods. The extent of biopolymers degradation by white-rot fungi was concluded to depend on the characteristics of the woods and environmental factors that influenced fungal physiology (Blanchette et al., 1985). Both strains revealed relatively similar abilities to degrade the substrates biopolymers. Strain F2 was more efficient on lignin degradation and F1 on cellulose and other compounds. In addition, F2 was found to excrete more soluble compounds than F1 from the degradation of the biomass samples, whereas F1 performed a more complete mineralization into CO2. This may be an advantage of strain F2 since the soluble products of degradation would remain available for methane production in the subsequent anaerobic digestion. 3.2. Enzymatic activity induced by fungal incubations No extracellular cellulase activity was detected in any of the conditions tested, confirming previously published works (Wan and Li, 2010; Heidorne et al., 2006). Ligninolytic enzymes were on the contrary secreted by F1 and F2 strains (Fig. 2). Laccase activity was detected under all of the experimental conditions tested. Particle size reduction induced a stronger laccase activity on barley straw, but the opposite was observed with forestry residues and especially acacia. Results therefore suggested that laccase would be a basal enzyme for C. subvermispora. This finding is in contradiction with some previous works such as in Wan and Li (2011) where no
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Fig. 1. Total mass loss and different losses (in % of initial total solid) in cellulose, hemicellulose, lignin due to fungi F1 and F2 growing on grounded particles of 1 mm or 4 mm: (A) Hazel 1 mm, (B) Hazel 4 mm, (C) Acacia 1 mm, (D) Acacia 4 mm, (E) Barley straw 1 mm, (F) Barley straw 4 mm and (G) Bagasse 1 mm.
laccase activity was detected from Soybean stalk pretreated by the same F2 strain, and in Aguiar et al. (2010) who reported no laccase activity in 10 days of fungal pretreatment of Pinus taeda wood chips. Other authors however (Enoki et al., 1999; Wan and Li, 2010) reported laccase activities of 2 U/g solid which were much higher than those measured here. It can be concluded that laccase production is extremely dependent upon the nature of the substrates and experimental conditions. In our study, the lowest laccase activity (0.03 U/g solid) was measured with F1 on 1-mm acacia particles. The highest activity (0.21 U/g solid) was recorded with F1 on 1-mm particles of barley straw. It was close to the 0.25 U/g solid reported by Ferraz et al. (2003) after 30 days with C. subvermispora on Eucalyptus grandis wood chips. Laccase secretions by strain F1 were similar on 1-mm and 4-mm hazel particles, whereas laccase
secretions by strain F2 was 1.8-fold higher on 4-mm hazel particles as compared to 1-mm particles. Laccase activity of F1 was 2.3 and 1.2-fold higher than that of F2 for 1-mm and 4-mm particles of hazel respectively. Regarding acacia, laccase production was higher with F2 than F1 whatever the particle size. Barley straw revealed an opposite pattern, with laccase activities 1.5-fold higher on 1-mm samples depending on the strain. Regarding peroxidase, MnP was secreted by both strains on 1mm and 4-mm hazel and 1-mm bagasse. With barley straw however, MnP was secreted only by F1 from 1-mm sample. No MnP activity was detected with F1 on 4-mm barley straw nor on acacia and barley straw with F2. These observations, along with others in this article, indicated that the 2 strains exhibited different metabolic responses to culture conditions. The secretion of MnP and
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Table 1 Characterization of the four untreated biomasses.a Composition (%)
Hazel
Acacia
Barley straw
Bagasse
Water extractives Ethanol extractives Cellulose Hemicellulose Lignin Ash Others
4.8 ± 0.2 2.9 ± 0.1 30.8 ± 0.2 15.9 ± 1.4 19.9 ± 0.4 2.7 ± 0.03 23.0 ± 2.2
7.8 ± 0.7 3.8 ± 0.4 32.9 ± 0.4 11.6 ± 0.3 15.3 ± 0.4 4.3 ± 0.01 24.3 ± 2.1
8.1 ± 0.1 2.7 ± 0.4 35.4 ± 0.1 28.7 ± 1.2 13.1 ± 0.9 4.5 ± 0.5 7.6 ± 3.1
23.7 ± 0.5 2.6 ± 0.4 29.7 ± 0.1 18.3 ± 0.1 15.3 ± 0.2 3.2 ± 0.2 7.2 ± 1.5
Elemental analysis C/H C/O C/N
C1eH1.561eO0.673eN0.009 7.63 1.12 95.28
C1eH1.627eO0.712eN0.023 7.32 1.05 37.28
C1eH1.652eO0.761eN0.009 7.21 0.99 95.28
C1eH1.502eO0.741eN0.008 7.93 1.01 107.19
a
Data reported as mean ± S.D. of three measurements and based on total solid.
Fig. 2. Production of Laccase and MnP after 28 d pretreatment: (A) Hazel, (B) Acacia, (C) Barley straw and (D) Bagasse.
laccase by the strains was found to depend both on the nature of the substrate and its particle size. Wheat straw and Soybean stalk were reported by other authors to induce no MnP activity by strain F2 (Wan and Li, 2011). Similar results were reported by Wang et al. (2002) who did not detect MnP activity in four out of ten strains of another white-rot fungus, Bjerkandera adusta, incubated with 3% rice bran medium. MnP activities measured in our study were in the same order of magnitude as those reported on wood chips by de Souza-Cruz et al. (2004). The highest activity, recorded with strain F2 on 4-mm hazel particles, was 2.6 times higher than that measured with strain F1 under the same conditions. Moreover, MnP production was higher on 4-mm samples rather than 1-mm, and higher with strain F2 than strain F1 on both hazel and bagasse samples. The significant MnP activity recorded on hazel, barley straw and bagasse may be related to their high C/N ratios (Table 1) as suggested by other authors (Kluczek-Turpeinen, 2007; Tian et al., 2012). When both MnP and laccase were secreted, laccase was generally the dominant enzyme except with F2 on 4-mm hazel and 1mm bagasse particles. Previous studies reported that MnP should be the dominant enzyme during the first 20 days of incubation, and then laccase activity should increase and become dominant (Vicentim and Ferraz, 2007; Wan and Li, 2010). Since incubations
were done over 28 days in our study, the observed predominance of laccase was consistent. No clear relationship could be observed between ligninolytic enzyme activities and lignin degradation in the assays. For instance, with strain F2 on hazel branches samples, MnP and laccase activities were found to be much lower on < 1-mm as compared to < 4mm particles, but almost no difference was observed in lignin degradation (Fig. 2). Similar observations were made by Aguiar and Ferraz (2012), who reported that although spiking the cultures with oxalic acid increased MnP secretion, lignin degradation was not stimulated. The selectivity of lignin degradation observed especially on hazel and bagasse (Fig. 1), could be related however to the synergistic effects of MnP and laccase (Wan and Li, 2011). 3.3. Effects of pretreatments on cellulose availability to enzymatic hydrolysis The ECD measured on the 4 samples before fungal incubations were slightly higher with <1-mm than <4-mm particles, indicating that the availability of cellulose to enzymatic hydrolysis was in a logical manner slightly increased by reducing the particle size (Fig. 3). Hazel and bagasse were the substrates in which cellulose was least available to hydrolysis, with as little as 8.8%, 8.4% and
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Fig. 3. Results of enzymatic hydrolysis (diamonds) and BMP (columns): (A) Hazel 1 mm, (B) Hazel 4 mm, (C) Acacia 1 mm, (D) Acacia 4 mm, (E) Barley straw 1 mm, (F) Barley straw 4 mm and (G) Bagasse 1 mm. UT: untreated; A: autoclaved; CTL: control.
11.7% of their cellulose contents hydrolyzed in 1-mm and 4-mm hazel particles and 1-mm bagasse, respectively. On the contrary, acacia and barley straw contained much more available cellulose since approximately 20% of their cellulose contents were hydrolyzed in the ECD protocol. Fig. 3 showed that the ECD of all samples was increased by autoclaving. Other authors have already suggested that thermal pretreatments of lignocellulosic substrates would affect the structural integrity of the complex and make cellulose more accessible (Martín-Sampedro et al., 2012). The highest effect (57%) was observed on bagasse and the smallest effect (24%) on 1-mm acacia. Fig. 3 also showed that fungal treatment with either strains increased cellulose availability in hazel and bagasse samples by around 2 to more than 4-fold compared to their respective controls, regardless of particle size. This result was attributed to the higher degradation of lignin than cellulose (less than 7% of the initial content) observed on these samples over the 28 days of fungal incubation. With acacia samples however, fungal treatment induced an apparent reduction of cellulose accessibility, particularly with strain F1. This observation was probably related to the strong degradation of cellulose observed in this sample (up to 17% with F1), which affected the most readily accessible part of cellulose. Similar trend was observed with the barley straw, which also exhibited a relatively strong rate of cellulose biodegradation.
3.4. Effect of fungal incubation on subsequent anaerobic digestion of the substrates The experimental time course data of cumulated methane production obtained from anaerobic digestion of each substrate before and after fungal pretreatment were fitted to first order kinetics law to determine the Vmax (corresponding to the BMP value, Fig. 3) and kinetic constant k (Table 2). Results are shown in Table 2. It can firstly be observed that the first-order kinetic constants were in the same order of magnitude as those reported by other authors (Gunaseelan, 2004). Table 2 also showed that the BMP values as well as the kinetic constants obtained with 1-mm samples where at most only slightly higher than with 4-mm samples in the cases of hazel and acacia, and even slightly smaller in the case of barley straw. This observation was explained by the fact that the incubations were long enough to allow the overall cumulated methane production not to be timelimited, and the surface area of the particles was not kinetically limiting their biodegradation under our experimental conditions. Table 2 also showed that forestry samples (hazel and acacia branches) exhibited lower methane potentials (ca. 90e150 L methane per kg VS) than agricultural samples (ca. 250e330 L methane per kg agricultural samples VS). This observation was explained by the lower lignin contents and higher holocellulose contents and availability of cellulose in agricultural samples as
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Table 2 1 ) and k (d1 ). BMP kinetic parameters Vmax (L,kgVS Sample Hazel
1 mm 4 mm
Acacia
1 mm 4 mm
Barley straw
1 mm 4 mm
Bagasse
1 mm
Kinetic parameters
UT
A
CTL
F1
F2
Vmax k Vmax k Vmax k Vmax k
109.8 0.049 93.3 0.043 149.6 0.107 135.9 0.120
131.9 0.048 98.6 0.054 173.2 0.100 172.4 0.080
131.5 0.042 119.9 0.042 169.9 0.088 164.5 0.083
173.9 0.070 164.2 0.089 138.9 0.041 109.4 0.054
219.6 0.084 195.9 0.087 204.5 0.054 185.0 0.044
Vmax k Vmax k Vmax k
293.7 0.087 330.8 0.086 247.9 0.114
260.0 0.125 251.1 0.104 216.4 0.183
258.1 0.121 245.4 0.112 233.1 0.160
246.5 0.093 248.3 0.097 238.9 0.202
227.2 0.124 250.1 0.108 239.4 0.208
compared to forestry samples (Table 1, Fig. 3). Hazel, which contained the highest lignin content (19.9%, Table 1), was identified as the least biodegradable of the four substrates, with a kinetic constant being by far the lowest of all (Table 2). These results can be clearly explained by the poor availability of cellulose in its structure (Fig. 3). Acacia exhibited the second lowest BMP due to its high lignin content (15.3%, Table 1), but its kinetic constant k was high, probably because of the good availability of cellulose in its structure (Fig. 3). Autoclaving showed a positive effect on the BMP of forestry samples, but not on the kinetics of methane production (which was even reduced in acacia). Conversely with agricultural samples, a negative effect of autoclaving was observed on BMPs whereas the kinetics was increased. With bagasse, the kinetic constant was strongly increased by autoclaving. Abiotic incubation was found not to affect the BMPs nor the kinetic constant. It is suggested that different effects of autoclaving could be due to the differences in the lignin contents and nature in forest and agricultural samples. Forest samples contained more S-rich lignin than agricultural samples (data not shown). However,, although changes in BMP and/or kinetics were observed in autoclaved forest and agricultural samples as compared to untreated samples, no significant differences was observed in the BMP curves of autocalved and untreated samples (curves not shown). Fungal treatment revealed variable effects on methane potential of forestry waste, which were related to the variable yields of lignin biodegradation recorded in these samples (Fig. 1). Fungal treatment of hazel branches with strains F1 or F2 strongly increased both the methane potential per unit mass of volatile solids (BMP) and the kinetic constant. BMP was increased by ca. 60% with F1 and was nearly doubled with F2 as compared to their corresponding controls. The effect of fungal treatment was so positive that the overall methane production of F1 and F2 was higher than their corresponding controls when taking mass loss into account (Fig. 4). With acacia branches however, fungal pretreatment induced a significant reduction of BMP with strain F1 (34%) whereas strain F2 only induce a slightly positive effect on 1-mm sample. A positive effect of the same strains F1 and F2 on the BMP of Japanese cedar has also been reported by Amirta et al. (2006). Unlike forestry samples, anaerobic digestion of agricultural samples was not improved by fungal pretreatment. A reduction of their BMPs and kinetic constants of methane production was even observed, except with bagasse where the kinetic constant was almost doubled with strain F2 as compared to the untreated sample. This result was in opposition with the study by Tuyen et al. (2012), who reported a 50% increase of BMP on wheat straw in 49 days of incubation with C. subvermispora. Our data combined to
results from other authors therefore showed that the effects of fungal pretreatment of lignocellulosic substrates on their methane production by anaerobic digestion depends both on the nature of the substrates, the fungal strain used, and the experimental conditions. It can be seen in Fig. 3 that the trends of ECD curves obtained with hazel and acacia samples pretreated with strains F1 or F2 compared to untreated samples were in accordance with the curves of methane production from these samples regardless of particle size in the range studied here. Rodriguez et al. (2005) reported similar relationships between ECD and BMP as observed here. Additionally, the accessibility to cellulose and hemicelluloses was improved in similar proportions (Suppl. Table 1). It should be noted that while there were no significant differences in lignin degradation between strains F1 and F2 on hazel samples, the accessibility to cellulose in hazel after pretreatment by F2 was much higher than after pretreatment by F1. This explains why methane production from hazel branches was higher by the pretreatment with F2 than F1. Regarding acacia, the reduction of methane production by fungal pretreatment with strain F1 may be explained by the reduction of cellulose accessibility induced by F1 in the considered sample. This decrease of cellulose accessibility in acacia may be related to the degradation by F1 of 17% cellulose (Fig. 3). On the other hand, less variable methane productions were observed on agricultural samples and they were found less fluctuant than those of ECD according to the different treatments. However regarding the bagasse samples, the significant evolution of ECD results after fungal treatment compared to the similar BMP achieved with or without C. subvermispora activity could be related to probably significant sucrose amounts that were initially trapped in cellulose fibers and were released after lignin degradation. This hypothesis should also be related to the 3 to 5 times higher water extracts initially measured in bagasse (after extraction carried out at high temperature enabling major sucrose to solubilize in water) compared to the other studied biomasses. Therefore, since the ECD test and the associated HPLC measurements could not consider separately the glucose released from sucrose or cellulosic compounds, it can be assumed that the about 60% apparent cellulose degradation recorded after both successive bagasse fungal treatment and enzymatic activity of ECD test (both carried out at temperature not enabling fast sucrose solubilizing, confirmed by the progressively higher ECD results after autoclave and CTL conditions) would have considered some release of the sucrose as degraded cellulose. As a consequence, a potential “false recalcitrant” biomass (i.e. leading to only 11.7 ± 2% cellulose degradation in untreated bagasse sample) has been assumed for this agricultural sample. This confirm the need to always consider some
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Fig. 4. Results of BMP taking mass loss into account, ( positive and negative difference of BMP of A, CTL, F1 and F2 sample compared to corresponding UT sample): (A) Hazel 1 mm, (B) Hazel 4 mm, (C) Acacia 1 mm, (D) Acacia 4 mm, (E) Barley straw 1 mm, (F) Barley straw 4 mm and (G) Bagasse 1 mm.
complementary assessments, e.g. with the classical long term BMP tests, when carrying out rapid tests such as the ECD test on a new biomass. Indeed, although the accessibility to cellulose as estimated by ECD test in untreated hazel sample was similar to that of the untreated bagasse sample, it is obvious that low accessibility to cellulose was caused by different reasons. In the case of hazel, it is suggested that the recalcitrant structure principally results from an effectively low accessibility to cellulose due to a strong lignin gangue that is only degraded to a 2-fold lower extent than that from bagasse. Therefore, for this kind of “real recalcitrant” biomass, it is proposed that improving cellulose accessibility of lignocellulose is more important for promoting methane fermentation than lignin degradation, which was similar to the results reported by Rollin et al. (2011).
4. Conclusion The present study showed that the effects of fungal pretreatment and the secretion of ligninolytic enzyme strongly depend on the nature of the considered lignocellulosic substrate. Fungal pretreatment with C. subvermispora increased biomethane potentials and kinetics of forestry sample hazel significantly. However, regarding agricultural sample bagasse, the positive effect of fungal pretreatment was not observed on methane potential, but the kinetics of production was increased. Acknowledgements This work was supported by a doctoral fellowship from the China Scholarship Council (CSC Grant No. 2011008074) awarded to
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Xun Liu and an invited lecturer-researcher fellowship from INSA awarded to Serge Hiligsmann. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jenvman.2017.01.075. References Aguiar, A., Ferraz, A., 2012. Effects of exogenous calcium or oxalic acid on Pinus taeda treatment with the white-rot fungus Ceriporiopsis subvermispora. Int. Biodeter. Biodegr. 72, 88e93. Aguiar, A., Mendonça, R., Rodriguez, J., Ferraz, A., 2010. Behavior of Ceriporiopsis subvermispora during Pinus taeda biotreatment in soybean-oil-amended cultures. Int. Biodeter. Biodegr. 64 (7), 588e593. Alexandropoulou, M., Antonopoulou, G., Fragkou, E., Ntaikou, I., Lyberatos, G., 2016. Fungal pretreatment of willow sawdust and its combination with alkaline treatment for enhancing biogas production. J. Environ. Manag. http:// dx.doi.org/10.1016/j.jenvman.2016.04.006. Amirta, R., Tanabe, T., Watanabe, T., Honda, Y., Kuwahara, M., 2006. Methane fermentation of Japanese cedar wood pretreated with a white rot fungus, Ceriporiopsis subvermispora. J. Biotechnol. 123 (1), 71e77. Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J., Guwy, A., Kalyuzhnyi, S., Jenicek, P., Van Lier, J., 2009. Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Sci. Technol. 59 (5), 927e934. Bertrand, V., Dequiedt, B., Le Cadre, E., 2014. Biomass for electricity in the EU-27: potential demand, CO2 abatements and breakeven prices for co-firing. Energy Policy 73, 631e644. Blanchette, R., Otjen, L., Effland, M., Eslyn, W., 1985. Changes in structural and chemical components of wood delignified by fungi. Wood Sci. Technol. 19 (1), 35e46. Bourbonnais, R., Paice, M.G., 1990. Oxidation of non-phenolic substrates: an expanded role for laccase in lignin biodegradation. FEBS Lett. 267 (1), 99e102. Cardona, C.A., Quintero, J.A., Paz, I.C., 2010. Production of bioethanol from sugarcane bagasse: status and perspectives. Bioresour. Technol. 101, 4754e4766. de Souza-Cruz, P.B., Freer, J., Siika-Aho, M., Ferraz, A., 2004. Extraction and determination of enzymes produced by Ceriporiopsis subvermispora during biopulping of Pinus taeda wood chips. Enzyme Microb. Technol. 34 (3e4), 228e234. Enoki, M., Watanabe, T., Nakagame, S., Koller, K., Messner, K., Honda, Y., Kuwahara, M., 1999. Extracellular lipid peroxidation of selective white-rot fungus, Ceriporiopsis subvermispora. FEMS Microbiol. Lett. 180 (2), 205e211. Fernandez-Fueyo, E., Ruiz-Duenas, F.J., Ferreira, P., et al., 2012. Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis. Proc. Natl. Acad. Sci. U. S. A. 109 (14), 5458e5463. rdova, A.M., Machuca, A., 2003. Wood biodegradation and enzyme Ferraz, A., Co production by Ceriporiopsis subvermispora during solid-state fermentation of Eucalyptus grandis. Enzyme Microb. Technol. 32 (1), 59e65. Floudas, D., Riley, M., Binder, R., et al., 2012. The paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336 (6089), 1715e1719. Ghose, T., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59 (2), 257e268. Goshadrou, A., Karimi, K., Taherzadeh, M.J., 2013. Ethanol and biogas production from birch by NMMO pretreatment. Biomass Bioenerg. 49, 95e101. Gunaseelan, V.N., 2004. Biochemical methane potential of fruits and vegetable solid waste feedstocks. Biomass Bioenerg. 26 (4), 389e399. Heidorne, F.O., Magalh~ aes, P.O., Ferraz, A.L., Milagres, A.M.F., 2006. Characterization of hemicellulases and cellulases produced by Ceriporiopsis subvermispora grown on wood under biopulping conditions. Enzyme Microb. Technol. 38 (3e4), 436e442. ISO, E. 1998. 11734, 1998. Water QualityeEvaluation of the “ultimate” Anaerobic Biodegradability of Organic Compounds in Digested SludgeeMethod by
Measurement of the Biogas Production (ISO 11734: 1995). German version: DIN EN ISO, 11734. Itoh, H., Wada, M., Honda, Y., Kuwahara, M., Watanabe, T., 2003. Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. J. Biotechnol. 103 (3), 273e280. Klein, M., Griess, O., Pulidindi, I.N., Perkas, N., Gedanken, A., 2016. Bioethanol production from Ficus religiosa leaves using microwave irradiation. J. Environ. Manage. 177, 20e25. Kluczek-Turpeinen, B., 2007. Lignocellulose degradation and humus modification by the fungus. Paecilomyces Inflatus 1e84. Martín-Sampedro, R., Eugenio, M.E., García, J.C., Lopez, F., Villar, J.C., Diaz, M.J., 2012. Steam explosion and enzymatic pre-treatments as an approach to improve the enzymatic hydrolysis of Eucalyptus globulus. Biomass Bioenerg. 42, 97e106. Monlau, F., Kaparaju, P., Trably, E., Steyer, J.P., Carrere, H., 2015. Alkaline pretreatment to enhance one-stage CH4 and two-stage H2/CH4 production from sunflower stalks: mass, energy and economical balances. Chem. Eng. J. 260, 377e385. rrez, A., 2014. Pretreatment Rico, A., Rencoret, J.C., del Río, J.T., Martínez, A., Gutie with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol. Biofuels 7, 6. Rodriguez, C., Hiligsmann, S., Ongena, M., Charlier, R., Thonart, P., 2005. Development of an enzymatic assay for the determination of cellulose bioavailability in municipal solid waste. Biodegradation 16 (5), 415e422. Rollin, J.A., Zhu, Z., Sathitsuksanoh, N., Zhang, Y.H., 2011. Increasing cellulose accessibility is more important than removing lignin: a comparison of cellulose solvent-based lignocellulose fractionation and soaking in aqueous ammonia. Biotechnol. Bioeng. 108 (1), 22e30. Sasaki, C., Takada, R., Watanabe, T., Honda, Y., Karita, S., Nakamura, Y., 2011. Surface carbohydrate analysis and bioethanol production of sugarcane bagasse pretreated with the white rot fungus, Ceriporiopsis subvermispora and microwave hydrothermolysis. Bioresour. Technol. 102 (21), 9942e9946. Selig, M., Weiss, N., Ji, Y., 2008. Enzymatic Saccharification of Lignocellulosic Biomass. Laboratory analytical procedure. Technical report: NREL/TP510e42629. National Renewable Energy Laboratory, Golden, CO, USA. Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2005. Determination of Extractives in Biomass. Laboratory Analytical Procedure. Technical report: NREL/TP-510e42619. National Renewable Energy Laboratory, Golden, CO, USA. Tanaka, H., Koike, K., Itakura, S., Enoki, A., 2009. Degradation of wood and enzyme production by Ceriporiopsis subvermispora. Enzyme Microb. Technol. 45 (5), 384e390. ri Horva th, I., Taherzadeh, M.J., 2014. TechnoTeghammar, A., Forg acs, G., S arva economic study of NMMO pretreatment and biogas production from forest residues. Appl. Energy 116, 125e133. Tian, X.-f., Fang, Z., Guo, F., 2012. Impact and prospective of fungal pre-treatment of lignocellulosic biomass for enzymatic hydrolysis. Biofuel. Bioprod. Bior. 6 (3), 335e350. Tuyen, V.D., Cone, J.W., Baars, J.J., Sonnenberg, A.S., Hendriks, W.H., 2012. Fungal strain and incubation period affect chemical composition and nutrient availability of wheat straw for rumen fermentation. Bioresour. Technol. 111, 336e342. Vicentim, M.P., Ferraz, A., 2007. Enzyme production and chemical alterations of Eucalyptus grandis wood during biodegradation by Ceriporiopsis subvermispora in cultures supplemented with Mn2þ, corn steep liquor and glucose. Enzyme Microb. Technol. 40 (4), 645e652. Wan, C., Li, Y., 2010. Microbial delignification of corn stover by Ceriporiopsis subvermispora for improving cellulose digestibility. Enzyme Microb. Technol. 47 (1e2), 31e36. Wan, C., Li, Y., 2011. Effectiveness of microbial pretreatment by Ceriporiopsis subvermispora on different biomass feedstocks. Bioresour. Technol. 102 (16), 7507e7512. Wan, C., Li, Y., 2012. Fungal pretreatment of lignocellulosic biomass. Biotechnol. Adv. 30 (6), 1447e1457. Wang, Y., Vazquez-Duhalt, R., Pickard, M.A., 2002. Purification, characterization, and chemical modification of manganese peroxidase from Bjerkandera adusta UAMH 8258. Curr. Microbiol. 45 (2), 77e87. Zheng, Y., Zhao, J., Xu, F., Li, Y., 2014. Pretreatment of lignocellulosic biomass for enhanced biogas production. Prog. Energy Combust. 42, 35e53.
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Research article
Anaerobic digestion of lignocellulosic biomasses pretreated with Ceriporiopsis subvermispora X. Liu a, S. Hiligsmann b, R. Gourdon a, R. Bayard a, * a b
Univ. Lyon, INSA-Lyon, DEEP Laboratory, EA4126, Bldg. S. Carnot, 20 Avenue A. Einstein, F-69621 Villeurbanne, France 3BIO-BioTech, Universit e Libre de Bruxelles, Av. F. Roosevelt 50, CP 165/61, Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2016 Received in revised form 26 January 2017 Accepted 28 January 2017
Fungal pretreatment by Ceriporiopsis subvermispora of two forest residues (hazel and acacia branches) and two agricultural lignocellulosic residues (barley straw and sugarcane bagasse) were studied as a pretreatment to improve their subsequent anaerobic digestion for methane production. Biomass samples were grinded to 2 ranges of particle sizes (<4 or 1 mm), autoclaved, inoculated with two strains of C. subvermispora (ATCC 90467 and ATCC 96608) and incubated at 28 C for 28 days. The effects of fungal pretreatment were assessed by analyzing the samples before and after incubations for dry solids mass, biochemical composition, bio-methane production (BMP) and availability of cellulose to hydrolysis. The production of ligninolytic enzymes MnP and/or laccase was observed with both strains during incubation on most of the samples tested. It almost doubled the hazel branches BMP per unit mass of dry solids but did not improve however the BMP of the agricultural residues and acacia branches. These observations were explained by the fact that although both strains were able to degrade 20e25% of lignin in <1 mm and <4 mm hazel branches samples, none of them was successful however to significantly degrade lignin in the other samples, except for sugarcane bagasse. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Ceriporiopsis subvermispora White-rot fungi Lignocellulosic biomass Biomethane potential Enzymatic hydrolysis Fungal pretreatment
1. Introduction Lignocellulosic biomass is the predominant organic resource on the surface of the planet and as such is considered as one of the most promising renewable energy resources (Bertrand et al., 2014; Monlau et al., 2015). It is not digestible by human beings and for a great part neither by animals. Therefore using it for energy recovery purposes does not raise competition with alimentary usages unlike first generation energy crops such as sugar beet, wheat or maize. In addition, available lignocellulosic biomass can be obtained in large amounts from forestry or agricultural residues, causing no pressure on arable land unlike energy crops (Bertrand et al., 2014; Monlau et al., 2015). Lignocellulosic biomass is mainly composed of cellulose, hemicelluloses and lignin. Although potentially biodegradable, cellulose and hemicelluloses are protected within a gangue of lignin, which is a 3D hydrophobic polymer of great chemical stability (Klein et al., 2016; Floudas et al., 2012). Bioconversion of lignocellulosic materials is therefore limited by the protective structure of recalcitrant lignin (Goshadrou et al., 2013; Teghammar et al., 2014). * Corresponding author. E-mail address: [email protected] (R. Bayard). http://dx.doi.org/10.1016/j.jenvman.2017.01.075 0301-4797/© 2017 Elsevier Ltd. All rights reserved.
A variety of physical, chemical and biological pretreatment methods have been evaluated in the literature with the objective to break down the lignin gangue and increase the bioavailability of cellulose and hemicelluloses which bear the biomethane (or bioethanol) potential of the material (Zheng et al., 2014). Biological pretreatments seem particularly attractive because they require mild operating conditions, involve relatively low operational costs and are often environmentally friendly. However their main drawbacks are the relatively slow action and the lack of specificity (Rico et al., 2014). Fungal pretreatment with selective white-rot fungi is one of the most promising processes of pretreatment (Alexandropoulou et al., 2016; Cardona et al., 2010). White-rot fungi are known to produce ligninolytic enzymes (predominantly lignin peroxidase - LiP, laccase or manganese-peroxidase eMnP) that break down the network of lignin with relatively little degradation of cellulose and hemicelluloses (Amirta et al., 2006; Tanaka et al., 2009). Among the various selective white-rot fungi studied in the literature (Amirta et al., 2006; Tian et al., 2012; Tuyen et al., 2012), Ceriporiopsis subvermispora was reported to exhibit a high lignin-degrading selectivity (Fernandez-Fueyo et al., 2012; Itoh et al., 2003). Wan and Li (2010), reported close to 40% lignin biodegradation over 42 days of cultivation on corn stover while cellulose degradation was
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less than 5%. Other authors reported similar trends (Sasaki et al., 2011; Tuyen et al., 2012). Some studies however reported variable efficiencies depending on the considered strain of C. subvermispora. For example, Amirta et al. (2006), observed a better efficiency of C. subvermispora ATCC 90467 as compared to ATCC 96608 in the capacity to increase methane potential of Japanese cedar wood. Based on these considerations, the objective of the present study was to investigate the efficiency of the 2 strains on lignocellulosic biomasses which were not yet investigated. Two ranges of particle sizes were studied (<1 and < 4 -mm) as this parameter is known to affect the performance of microbial pretreatment (Wan and Li, 2012). The pretreatment with each strain was evaluated by monitoring ligninolytic and cellulolytic enzyme activities, substrates’ mass reductions, the cellulose availability to hydrolysis in the substrates, and the efficiency of subsequent anaerobic digestion in terms of biomethane potential (BMP) and methane production kinetics. 2. Materials and methods 2.1. Samples collection and preparation Two forest residues and two agricultural residues with currently low added value were selected. Hazel and acacia branches were gny, Rho ^ne-Alpes, France. collected from a private forest in Re Barley straw and sugar cane bagasse were selected for their abundant production and their distinctive biochemical characteristics. They were collected respectively from a private farm located in Picardie, France and from an artisanal distillery in Plaine de Cul-deSac, Port-au-Prince, Haiti. About 10 kg of each biomass material were sampled in autumn, coarsely crushed on site and carefully homogenized. They were then immediately taken to the laboratory where they were dried in an oven at 60 C for 3 days, shredded three times with a low-speed shredder (Blik® monorotor M420, ^t, France) and sieved at 10 mm to obtain homogenous Milly-la-Fore stock powders. The stock powders were then further grinded using a cutting mill (Retsch® SM 200, Haan, Germany) into 2 subfractions of particle sizes below 4-mm and below 1-mm, respectively. All samples thus prepared were stored at 2 C before analysis or experiments. Autoclaved samples were also prepared in the same manner but not incubated in order to investigate the effect of the thermal treatment. 2.2. Fungal strains and inoculum preparation Cultures of Ceriporiopsis subvermispora ATCC 90467 (hereafter named F1) and Ceriporiopsis subvermispora ATCC 96608 (hereafter named F2) were grown on 33.6 g L1 malt extract agar at 28 C for 7 days. Ten discs of 10 mm diameter of the agar medium of each culture were then transferred into 500 mL sterile Erlenmeyer flasks containing 50 mL of 33.6 g L1 malt-extract medium, and the flasks were incubated (unshaken) at 28 C. After 7 days, each culture was aseptically blended in three cycles of 15 s and the mycelium suspensions were used as an inoculum (Wan and Li, 2010).
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substrate and strain. After incubation, three of them were used for the determination of ligninolytic and cellulolytic enzymatic activities. Three others were used for chemical and biochemical analyses of the solid samples, cellulose enzymatic hydrolysis and BMP determinations. Four control flasks were followed. Two of them served as controls for enzymatic activities, and the other two were used for the other tests and analyses. After 28 days of incubation, the residual solids were collected and dried at 60 C for 72 h prior to analyses. The dry samples were weighed and the mass compared to initial mass to determine the extent of total solids biodegradation. In the assays however, the masses of the samples after incubation included the associated fungal biomass which could not be separated from the samples. This caused an overestimation of the residual masses of substrates. 2.4. Measurements of fungal enzymatic activities The residual solids collected after the incubations (assays and controls) were suspended in 120 mL of sodium acetate buffer (4.1 g L1, pH 4.5) and stirred in an orbital shaker at 150 r min1 at 28 C (Wan and Li, 2010). After for 4 h, the suspensions were filtered through 0.45 mm pore size cellulose acetate membranes and the solutions analyzed for enzymatic activities. Manganese-dependent peroxidase activity (MnP) was determined by phenol red oxidation using ε ¼ 22,000 M1 cm1 as the molar absorbance coefficient (Vicentim and Ferraz, 2007). Laccase activity was determined by oxidation of 2,2'-azino-bis-3-ethyl benzothiazoline-6-sulphonate (ABTS), using ε ¼ 36,000 M1 cm1 (Bourbonnais and Paice, 1990; Wan and Li, 2010). Cellulase activity was measured according to IUPAC guidelines (Ghose, 1987) using filter paper as a substrate. For these analyses, absorbance was measured with a SHIMADZU UV-2450 spectrophotometer. All analyses were triplicated. The digestibility of cellulose was determined as follows:
% cellulose degradation ðECD %Cell Þ ¼
cellulose digested ðgÞ 100 cellulose added ðgÞ (1)
The effect of fungal incubation on cellulose accessibility within the substrates was evaluated by comparing the efficiency of cellulose enzymatic hydrolysis before and after incubation following the NREL procedure (Selig et al., 2008). The suspensions were filtered through 0.22 mm pore size cellulose acetate membranes and the solutions analyzed for glucose by HPLC using a Waters® chromatograph equipped with a Bio-Rad HPX-87H column and a refractive index detector (RID) maintained at 45 C and 30 C, respectively. An aqueous H2SO4 solution at 0.49 g$L1was used as the mobile phase at a constant flow rate of 0.6 mL min1. 2.5. Chemical and biochemical analyses
2.3. Fungal pretreatment
2.5.1. Elemental analyses Total carbon (C), hydrogen (H) and nitrogen (N) contents were determined following standard procedures at CNRS analytical center SCA of Villeurbanne, France.
10 g of powdered samples of the required particle size were transferred into 250 mL Erlenmeyer flasks containing 30 mL deionized water. The flasks were then autoclaved at 121 C for 30 min, cooled to room temperature, and inoculated with 2 mL F1 or F2 mycelium suspension. Control flasks were prepared in the same manner but water was added instead of inoculum. All flasks were incubated at 28 C. Six replicates were incubated for each
2.5.2. Cellulose, hemicelluloses and lignin contents Analyses were done in triplicates following NREL Laboratory Analytical Procedure (Sluiter et al., 2005). The samples were firstly extracted successively with HPLC-grade water and with ethanol using the Dionex ASE 350 system to remove soluble components, then hydrolyzed with a concentrated sulphuric acid aqueous solution (72% w/w) at 30 C for 1 h. The suspensions were diluted to
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4% with de-ionized water, autoclaved at 121 C for 1 h, cooled to room temperature and filtered. The solutions were analyzed for acid-soluble lignin by measuring UV absorbance at 205 nm and for monomeric sugars (glucose, xylose, galactose, arabinose and mannose) using a Waters® HPLC equipped with a Bio-Rad Aminex HPX-87P column and a refractive index detector (RID) operated at 85 C and 49 C respectively, with HPLC-grade water as the mobile phase at a flow rate of 0.6 mL min1. Cellulose and hemicelluloses contents of the samples were calculated from the concentrations of the corresponding monomers analyzed in the solutions. The acid-insoluble solid residues were dried at 105 C overnight, weighed, calcinated at 575 C for 4 h, and the ashes were weighed after cooling. The mass loss was considered as acid-insoluble lignin. 2.6. Mass balance on total solids and biochemical fractions All substrates were weighed and analyzed for their biochemical compositions (i) in their powdered initial form as prepared for the experiments, (ii) after autoclaving, (iii) after autoclaving and abiotic incubation (controls), and (iv) after autoclaving and fungal incubation (assays). In order to evaluate the extent of degradation of the substrates specifically due to the action of the fungus, the mass reductions observed in the assays on the dry solids and on their biochemical fractions were corrected with the respective variations observed on the same parameters in the autoclaving and/or the abiotic controls. 2.7. Anaerobic digestion Anaerobic digestion was studied using the Biochemical Methane Potentials (BMP) protocol (Angelidaki et al., 2009). For each substrate, before and after fungal pretreatment, a mass of dry solids calculated to contain 1 g volatile solids was transferred into 500 mL serum bottles containing 200 mL of a nutritive medium (ISO 11734, 1998), and 100 mL of anaerobic inoculum suspension cultivated in the laboratory from a seed initially taken from an anaerobic digester treating domestic sewage sludge. Under these conditions, the ratio between the mass of volatile solids (VS) in the samples and that of the inoculum was 0.5 in each flask. The flasks were then flushed with a gas mixture of 70% N2 and 30% CO2 for 5 min, sealed with air-tight rubber stoppers and plastic seals, and incubated at 35 ± 2 C in the dark. Biogas production was monitored using a Digitron® 2085P pressure transducer. Gas samples were regularly taken for immediate analysis using an Agilent® gas micro-chromatograph equipped with a thermal conductivity detector and a PoraPLOT U column for CO2 and a Molsieve column for O2, N2, and CH4. Biomethane potential was calculated from the cumulated methane production obtained in the tests, and finally expressed under 0 C and 105 Pa in L of methane kg1 of substrate VS. All experiments were triplicated. Experimental time course data of cumulative methane production in each test (expressed under 0 C and 105 Pa) was fitted to first order kinetics model of eqn. (2):
VðtÞ ¼ V max 1 ekt
(2)
where V(t) was the cumulative methane production at time t, 1 Þ the overall methane production (i.e. BMP), and k Vmax ðLCH4 $kgVS the first order kinetic constant (d1). Best fit values of parameters k and Vmax were determined by least squares fitting of the above equations to each data set using Microsoft Excel's solver function.
3. Results and discussion 3.1. Mass balance evaluations of substrates biodegradation in fungal pretreatment Control-corrected mass balance results are given in Fig. 1. It was observed that the biotic degradation induced a total solids mass reduction of 8e12% for hazel and acacia branches, and up to 15% for bagasse. The overall degradation was stronger with strain F1 except for hazel, which was better degraded by strain F2. Barley straw was less degraded, with mass reductions below 5% with either strain. Various performances were observed in the biopolymers degradation depending on the nature of the substrates (Table 1), their particle size, and the strain tested. Both strains were able to efficiently degrade lignin in hazel branches samples, F2 being slightly more efficient than F1 (close to 25% degradation vs 20%). Both strains were also very efficient to degrade lignin in 1-mm bagasse (more than 40% degradation). None of them however was efficient to degrade lignin in acacia branches (degradation less than about 5%) and barley straw (no degradation) regardless of particle size tested. These observations suggested that the structure of the lignin gangue in these 2 samples was probably quite different from that of the other 2 substrates, inducing a lower accessibility to fungal enzymes and therefore a lower degradation. Cellulose was at best very poorly degraded, usually much less than lignin, thereby confirming the good selectivity of the selected fungal strains. In acacia branches however, close to 10% and 20% cellulose degradations were observed with F2 and F1 respectively, regardless of particle size, whereas lignin degradation was below 5%. This observation may be attributed to a better accessibility of cellulose, which would be less protected by lignin in this sample as compared to the other. Finally, hemicelluloses were in most cases degraded more than cellulose but less than lignin. These results were consistent with those of Blanchette et al. (1985) who studied lignin degradation in decaying forest wood and reported that delignification was not occurring at the same extent in the different samples which were analyzed. Several white-rot fungi exhibited selective lignin removal only in certain woods and were much less selective, or not at all, in other woods. The extent of biopolymers degradation by white-rot fungi was concluded to depend on the characteristics of the woods and environmental factors that influenced fungal physiology (Blanchette et al., 1985). Both strains revealed relatively similar abilities to degrade the substrates biopolymers. Strain F2 was more efficient on lignin degradation and F1 on cellulose and other compounds. In addition, F2 was found to excrete more soluble compounds than F1 from the degradation of the biomass samples, whereas F1 performed a more complete mineralization into CO2. This may be an advantage of strain F2 since the soluble products of degradation would remain available for methane production in the subsequent anaerobic digestion. 3.2. Enzymatic activity induced by fungal incubations No extracellular cellulase activity was detected in any of the conditions tested, confirming previously published works (Wan and Li, 2010; Heidorne et al., 2006). Ligninolytic enzymes were on the contrary secreted by F1 and F2 strains (Fig. 2). Laccase activity was detected under all of the experimental conditions tested. Particle size reduction induced a stronger laccase activity on barley straw, but the opposite was observed with forestry residues and especially acacia. Results therefore suggested that laccase would be a basal enzyme for C. subvermispora. This finding is in contradiction with some previous works such as in Wan and Li (2011) where no
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Fig. 1. Total mass loss and different losses (in % of initial total solid) in cellulose, hemicellulose, lignin due to fungi F1 and F2 growing on grounded particles of 1 mm or 4 mm: (A) Hazel 1 mm, (B) Hazel 4 mm, (C) Acacia 1 mm, (D) Acacia 4 mm, (E) Barley straw 1 mm, (F) Barley straw 4 mm and (G) Bagasse 1 mm.
laccase activity was detected from Soybean stalk pretreated by the same F2 strain, and in Aguiar et al. (2010) who reported no laccase activity in 10 days of fungal pretreatment of Pinus taeda wood chips. Other authors however (Enoki et al., 1999; Wan and Li, 2010) reported laccase activities of 2 U/g solid which were much higher than those measured here. It can be concluded that laccase production is extremely dependent upon the nature of the substrates and experimental conditions. In our study, the lowest laccase activity (0.03 U/g solid) was measured with F1 on 1-mm acacia particles. The highest activity (0.21 U/g solid) was recorded with F1 on 1-mm particles of barley straw. It was close to the 0.25 U/g solid reported by Ferraz et al. (2003) after 30 days with C. subvermispora on Eucalyptus grandis wood chips. Laccase secretions by strain F1 were similar on 1-mm and 4-mm hazel particles, whereas laccase
secretions by strain F2 was 1.8-fold higher on 4-mm hazel particles as compared to 1-mm particles. Laccase activity of F1 was 2.3 and 1.2-fold higher than that of F2 for 1-mm and 4-mm particles of hazel respectively. Regarding acacia, laccase production was higher with F2 than F1 whatever the particle size. Barley straw revealed an opposite pattern, with laccase activities 1.5-fold higher on 1-mm samples depending on the strain. Regarding peroxidase, MnP was secreted by both strains on 1mm and 4-mm hazel and 1-mm bagasse. With barley straw however, MnP was secreted only by F1 from 1-mm sample. No MnP activity was detected with F1 on 4-mm barley straw nor on acacia and barley straw with F2. These observations, along with others in this article, indicated that the 2 strains exhibited different metabolic responses to culture conditions. The secretion of MnP and
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Table 1 Characterization of the four untreated biomasses.a Composition (%)
Hazel
Acacia
Barley straw
Bagasse
Water extractives Ethanol extractives Cellulose Hemicellulose Lignin Ash Others
4.8 ± 0.2 2.9 ± 0.1 30.8 ± 0.2 15.9 ± 1.4 19.9 ± 0.4 2.7 ± 0.03 23.0 ± 2.2
7.8 ± 0.7 3.8 ± 0.4 32.9 ± 0.4 11.6 ± 0.3 15.3 ± 0.4 4.3 ± 0.01 24.3 ± 2.1
8.1 ± 0.1 2.7 ± 0.4 35.4 ± 0.1 28.7 ± 1.2 13.1 ± 0.9 4.5 ± 0.5 7.6 ± 3.1
23.7 ± 0.5 2.6 ± 0.4 29.7 ± 0.1 18.3 ± 0.1 15.3 ± 0.2 3.2 ± 0.2 7.2 ± 1.5
Elemental analysis C/H C/O C/N
C1eH1.561eO0.673eN0.009 7.63 1.12 95.28
C1eH1.627eO0.712eN0.023 7.32 1.05 37.28
C1eH1.652eO0.761eN0.009 7.21 0.99 95.28
C1eH1.502eO0.741eN0.008 7.93 1.01 107.19
a
Data reported as mean ± S.D. of three measurements and based on total solid.
Fig. 2. Production of Laccase and MnP after 28 d pretreatment: (A) Hazel, (B) Acacia, (C) Barley straw and (D) Bagasse.
laccase by the strains was found to depend both on the nature of the substrate and its particle size. Wheat straw and Soybean stalk were reported by other authors to induce no MnP activity by strain F2 (Wan and Li, 2011). Similar results were reported by Wang et al. (2002) who did not detect MnP activity in four out of ten strains of another white-rot fungus, Bjerkandera adusta, incubated with 3% rice bran medium. MnP activities measured in our study were in the same order of magnitude as those reported on wood chips by de Souza-Cruz et al. (2004). The highest activity, recorded with strain F2 on 4-mm hazel particles, was 2.6 times higher than that measured with strain F1 under the same conditions. Moreover, MnP production was higher on 4-mm samples rather than 1-mm, and higher with strain F2 than strain F1 on both hazel and bagasse samples. The significant MnP activity recorded on hazel, barley straw and bagasse may be related to their high C/N ratios (Table 1) as suggested by other authors (Kluczek-Turpeinen, 2007; Tian et al., 2012). When both MnP and laccase were secreted, laccase was generally the dominant enzyme except with F2 on 4-mm hazel and 1mm bagasse particles. Previous studies reported that MnP should be the dominant enzyme during the first 20 days of incubation, and then laccase activity should increase and become dominant (Vicentim and Ferraz, 2007; Wan and Li, 2010). Since incubations
were done over 28 days in our study, the observed predominance of laccase was consistent. No clear relationship could be observed between ligninolytic enzyme activities and lignin degradation in the assays. For instance, with strain F2 on hazel branches samples, MnP and laccase activities were found to be much lower on < 1-mm as compared to < 4mm particles, but almost no difference was observed in lignin degradation (Fig. 2). Similar observations were made by Aguiar and Ferraz (2012), who reported that although spiking the cultures with oxalic acid increased MnP secretion, lignin degradation was not stimulated. The selectivity of lignin degradation observed especially on hazel and bagasse (Fig. 1), could be related however to the synergistic effects of MnP and laccase (Wan and Li, 2011). 3.3. Effects of pretreatments on cellulose availability to enzymatic hydrolysis The ECD measured on the 4 samples before fungal incubations were slightly higher with <1-mm than <4-mm particles, indicating that the availability of cellulose to enzymatic hydrolysis was in a logical manner slightly increased by reducing the particle size (Fig. 3). Hazel and bagasse were the substrates in which cellulose was least available to hydrolysis, with as little as 8.8%, 8.4% and
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Fig. 3. Results of enzymatic hydrolysis (diamonds) and BMP (columns): (A) Hazel 1 mm, (B) Hazel 4 mm, (C) Acacia 1 mm, (D) Acacia 4 mm, (E) Barley straw 1 mm, (F) Barley straw 4 mm and (G) Bagasse 1 mm. UT: untreated; A: autoclaved; CTL: control.
11.7% of their cellulose contents hydrolyzed in 1-mm and 4-mm hazel particles and 1-mm bagasse, respectively. On the contrary, acacia and barley straw contained much more available cellulose since approximately 20% of their cellulose contents were hydrolyzed in the ECD protocol. Fig. 3 showed that the ECD of all samples was increased by autoclaving. Other authors have already suggested that thermal pretreatments of lignocellulosic substrates would affect the structural integrity of the complex and make cellulose more accessible (Martín-Sampedro et al., 2012). The highest effect (57%) was observed on bagasse and the smallest effect (24%) on 1-mm acacia. Fig. 3 also showed that fungal treatment with either strains increased cellulose availability in hazel and bagasse samples by around 2 to more than 4-fold compared to their respective controls, regardless of particle size. This result was attributed to the higher degradation of lignin than cellulose (less than 7% of the initial content) observed on these samples over the 28 days of fungal incubation. With acacia samples however, fungal treatment induced an apparent reduction of cellulose accessibility, particularly with strain F1. This observation was probably related to the strong degradation of cellulose observed in this sample (up to 17% with F1), which affected the most readily accessible part of cellulose. Similar trend was observed with the barley straw, which also exhibited a relatively strong rate of cellulose biodegradation.
3.4. Effect of fungal incubation on subsequent anaerobic digestion of the substrates The experimental time course data of cumulated methane production obtained from anaerobic digestion of each substrate before and after fungal pretreatment were fitted to first order kinetics law to determine the Vmax (corresponding to the BMP value, Fig. 3) and kinetic constant k (Table 2). Results are shown in Table 2. It can firstly be observed that the first-order kinetic constants were in the same order of magnitude as those reported by other authors (Gunaseelan, 2004). Table 2 also showed that the BMP values as well as the kinetic constants obtained with 1-mm samples where at most only slightly higher than with 4-mm samples in the cases of hazel and acacia, and even slightly smaller in the case of barley straw. This observation was explained by the fact that the incubations were long enough to allow the overall cumulated methane production not to be timelimited, and the surface area of the particles was not kinetically limiting their biodegradation under our experimental conditions. Table 2 also showed that forestry samples (hazel and acacia branches) exhibited lower methane potentials (ca. 90e150 L methane per kg VS) than agricultural samples (ca. 250e330 L methane per kg agricultural samples VS). This observation was explained by the lower lignin contents and higher holocellulose contents and availability of cellulose in agricultural samples as
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Table 2 1 ) and k (d1 ). BMP kinetic parameters Vmax (L,kgVS Sample Hazel
1 mm 4 mm
Acacia
1 mm 4 mm
Barley straw
1 mm 4 mm
Bagasse
1 mm
Kinetic parameters
UT
A
CTL
F1
F2
Vmax k Vmax k Vmax k Vmax k
109.8 0.049 93.3 0.043 149.6 0.107 135.9 0.120
131.9 0.048 98.6 0.054 173.2 0.100 172.4 0.080
131.5 0.042 119.9 0.042 169.9 0.088 164.5 0.083
173.9 0.070 164.2 0.089 138.9 0.041 109.4 0.054
219.6 0.084 195.9 0.087 204.5 0.054 185.0 0.044
Vmax k Vmax k Vmax k
293.7 0.087 330.8 0.086 247.9 0.114
260.0 0.125 251.1 0.104 216.4 0.183
258.1 0.121 245.4 0.112 233.1 0.160
246.5 0.093 248.3 0.097 238.9 0.202
227.2 0.124 250.1 0.108 239.4 0.208
compared to forestry samples (Table 1, Fig. 3). Hazel, which contained the highest lignin content (19.9%, Table 1), was identified as the least biodegradable of the four substrates, with a kinetic constant being by far the lowest of all (Table 2). These results can be clearly explained by the poor availability of cellulose in its structure (Fig. 3). Acacia exhibited the second lowest BMP due to its high lignin content (15.3%, Table 1), but its kinetic constant k was high, probably because of the good availability of cellulose in its structure (Fig. 3). Autoclaving showed a positive effect on the BMP of forestry samples, but not on the kinetics of methane production (which was even reduced in acacia). Conversely with agricultural samples, a negative effect of autoclaving was observed on BMPs whereas the kinetics was increased. With bagasse, the kinetic constant was strongly increased by autoclaving. Abiotic incubation was found not to affect the BMPs nor the kinetic constant. It is suggested that different effects of autoclaving could be due to the differences in the lignin contents and nature in forest and agricultural samples. Forest samples contained more S-rich lignin than agricultural samples (data not shown). However,, although changes in BMP and/or kinetics were observed in autoclaved forest and agricultural samples as compared to untreated samples, no significant differences was observed in the BMP curves of autocalved and untreated samples (curves not shown). Fungal treatment revealed variable effects on methane potential of forestry waste, which were related to the variable yields of lignin biodegradation recorded in these samples (Fig. 1). Fungal treatment of hazel branches with strains F1 or F2 strongly increased both the methane potential per unit mass of volatile solids (BMP) and the kinetic constant. BMP was increased by ca. 60% with F1 and was nearly doubled with F2 as compared to their corresponding controls. The effect of fungal treatment was so positive that the overall methane production of F1 and F2 was higher than their corresponding controls when taking mass loss into account (Fig. 4). With acacia branches however, fungal pretreatment induced a significant reduction of BMP with strain F1 (34%) whereas strain F2 only induce a slightly positive effect on 1-mm sample. A positive effect of the same strains F1 and F2 on the BMP of Japanese cedar has also been reported by Amirta et al. (2006). Unlike forestry samples, anaerobic digestion of agricultural samples was not improved by fungal pretreatment. A reduction of their BMPs and kinetic constants of methane production was even observed, except with bagasse where the kinetic constant was almost doubled with strain F2 as compared to the untreated sample. This result was in opposition with the study by Tuyen et al. (2012), who reported a 50% increase of BMP on wheat straw in 49 days of incubation with C. subvermispora. Our data combined to
results from other authors therefore showed that the effects of fungal pretreatment of lignocellulosic substrates on their methane production by anaerobic digestion depends both on the nature of the substrates, the fungal strain used, and the experimental conditions. It can be seen in Fig. 3 that the trends of ECD curves obtained with hazel and acacia samples pretreated with strains F1 or F2 compared to untreated samples were in accordance with the curves of methane production from these samples regardless of particle size in the range studied here. Rodriguez et al. (2005) reported similar relationships between ECD and BMP as observed here. Additionally, the accessibility to cellulose and hemicelluloses was improved in similar proportions (Suppl. Table 1). It should be noted that while there were no significant differences in lignin degradation between strains F1 and F2 on hazel samples, the accessibility to cellulose in hazel after pretreatment by F2 was much higher than after pretreatment by F1. This explains why methane production from hazel branches was higher by the pretreatment with F2 than F1. Regarding acacia, the reduction of methane production by fungal pretreatment with strain F1 may be explained by the reduction of cellulose accessibility induced by F1 in the considered sample. This decrease of cellulose accessibility in acacia may be related to the degradation by F1 of 17% cellulose (Fig. 3). On the other hand, less variable methane productions were observed on agricultural samples and they were found less fluctuant than those of ECD according to the different treatments. However regarding the bagasse samples, the significant evolution of ECD results after fungal treatment compared to the similar BMP achieved with or without C. subvermispora activity could be related to probably significant sucrose amounts that were initially trapped in cellulose fibers and were released after lignin degradation. This hypothesis should also be related to the 3 to 5 times higher water extracts initially measured in bagasse (after extraction carried out at high temperature enabling major sucrose to solubilize in water) compared to the other studied biomasses. Therefore, since the ECD test and the associated HPLC measurements could not consider separately the glucose released from sucrose or cellulosic compounds, it can be assumed that the about 60% apparent cellulose degradation recorded after both successive bagasse fungal treatment and enzymatic activity of ECD test (both carried out at temperature not enabling fast sucrose solubilizing, confirmed by the progressively higher ECD results after autoclave and CTL conditions) would have considered some release of the sucrose as degraded cellulose. As a consequence, a potential “false recalcitrant” biomass (i.e. leading to only 11.7 ± 2% cellulose degradation in untreated bagasse sample) has been assumed for this agricultural sample. This confirm the need to always consider some
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Fig. 4. Results of BMP taking mass loss into account, ( positive and negative difference of BMP of A, CTL, F1 and F2 sample compared to corresponding UT sample): (A) Hazel 1 mm, (B) Hazel 4 mm, (C) Acacia 1 mm, (D) Acacia 4 mm, (E) Barley straw 1 mm, (F) Barley straw 4 mm and (G) Bagasse 1 mm.
complementary assessments, e.g. with the classical long term BMP tests, when carrying out rapid tests such as the ECD test on a new biomass. Indeed, although the accessibility to cellulose as estimated by ECD test in untreated hazel sample was similar to that of the untreated bagasse sample, it is obvious that low accessibility to cellulose was caused by different reasons. In the case of hazel, it is suggested that the recalcitrant structure principally results from an effectively low accessibility to cellulose due to a strong lignin gangue that is only degraded to a 2-fold lower extent than that from bagasse. Therefore, for this kind of “real recalcitrant” biomass, it is proposed that improving cellulose accessibility of lignocellulose is more important for promoting methane fermentation than lignin degradation, which was similar to the results reported by Rollin et al. (2011).
4. Conclusion The present study showed that the effects of fungal pretreatment and the secretion of ligninolytic enzyme strongly depend on the nature of the considered lignocellulosic substrate. Fungal pretreatment with C. subvermispora increased biomethane potentials and kinetics of forestry sample hazel significantly. However, regarding agricultural sample bagasse, the positive effect of fungal pretreatment was not observed on methane potential, but the kinetics of production was increased. Acknowledgements This work was supported by a doctoral fellowship from the China Scholarship Council (CSC Grant No. 2011008074) awarded to
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