Pretreatment of Lignocellulosic Biomasses with Filamentous Fungi for the Production of Bioenergy

Abstract

The invention relates to the use of a strain of basidiomycete fungus belonging to the Polyporus brumalis species, for the fungal pretreatment of a lignocellulosic biomass in a solid medium.

Claims

1. (canceled)

2. A method for producing bioenergy comprising pretreatment of a lignocellulosic biomass in a solid medium, hydrolysis of the pretreated biomass and fermentation of the hydrolyzed biomass, wherein said pretreatment comprises contacting the lignocellulosic biomass with Polyporus brumalis CNCM I-4900 strain.

3. The method of claim 2, wherein-the lignocellulosic biomass is selected from the group consisting of agricultural residues, forest residues, wood processing by-products, ligneous or herbaceous plants, municipal green waste, wood waste and fermentable fractions of household waste.

4. The method of claim 3, wherein the lignocellulosic biomass is wheat straw.

5. The method of claim 2, wherein the Polyporus brumalis CNCM I-4900 strain is present in the form of miscanthus inoculated with said strain.

6. The method of claim 5, wherein the miscanthus is in the form of granules, chips, mulch or briquettes of miscanthus inoculated with said strain.

7. The method of claim 2, wherein the duration of the pretreatment is 5 to 100 days.

8. The method of claim 2, wherein the humidity of the lignocellulosic biomass is kept constant throughout the duration of the pretreatment.

9. The method of claim 2, wherein air is blown into the lignocellulosic biomass and/or occasional turnings of the lignocellulosic biomass are performed during the pretreatment.

10. The method of claim 2, wherein the temperature of the lignocellulosic biomass is maintained between 25 and 35° C. for the entire duration of the pretreatment.

Description

[0037] The present invention will be understood more clearly using the supplementary description hereinafter, which refers to the pretreatment of lignocellulosic biomass (wheat straw) with filamentous fungi for the production of bioenergy, along with the appended figures:

[0038] FIG. 1: Glycoside hydrolase activities on complex substrates secreted by filamentous fungi during pretreatment. CMC: Carboxymethyl cellulose; Birch X: Birch xylan; Wheat X: Wheat xylan; Wheat XI: Insoluble wheat xylan; Man: Mannan; GalMan: GalactoMannan.

[0039] FIG. 2: Lignolytic activities secreted by filamentous fungi during pretreatment. Lac: laccase; Mnp: Manganese peroxidase; Mip: manganese-independent peroxidase.

[0040] FIG. 3: Impact of fungal strains used for wheat straw pretreatment on quantities of reducing sugars (left bar) and glucose (right bar) released after 96 hours of enzymatic hydrolysis.

[0041] FIG. 4: Production kinetics of reducing sugars (A) and glucose (B) during enzymatic hydrolysis of wheat straw pretreated with the P. brumalis strain (BRFM 985). The control wheat straw (CTL_Straw) is non-inoculated wheat straw subjected to the same operating conditions (SSF for 21 days+post-treatments).

[0042] FIG. 5: Digestibility and conversion yields of cell wall polysaccharides of pretreated wheat straw. Cellulose: bottom bar; Holocellulose: top bar; Hemicellulose: middle bar.

[0043] FIG. 6: BMP (Biological Methane Potential) of wheat straw in NmL of methane per gram of pretreated VM (volatile matter).

EXAMPLE 1

Effect of Fungal Pretreatment on Wheat Straw Composition: Effective Delignification of Biomass

[0044] The impact of fungal pretreatments on the lignocellulosic biomass (wheat straw) conducted within the scope of our study was evaluated by determining the contents of the main cell wall constituents (cellulose, hemicelluloses and lignin) and the mass yields.

Methodologies

Fungal Strains

[0045] Five BRFM (Marseille fungal resource bank) supplied by CIRM (International Center for Microbial Resources, Marseille) were selected on the basis of screening for the potential thereof to be used as a biological pretreatment agent (SSF cultures) for lignocellulosic substrates (wheat straw and miscanthus) in order to improve the subsequent enzymatic hydrolysis thereof:

[0046] BRFM 957: Trametes ljubarskii

[0047] BRFM 985: Polyporus brumalis CNCM I-4900

[0048] BRFM 1048: Leiotrametes sp

[0049] BRFM 1369: Trametes menziesii

[0050] BRFM 1554: Trametes pavonia

[0051] These strains belong to the group of ligninolytic filamentous wood-decay fungi. They belong to the basidiomycetes class, to the Polyporales order and to the Polyporaceae family.

Culture Conditions

[0052] The SSF cultures are produced in glass columns with an effective volume of 250 mL (20 cm in height and 4 cm in diameter). A column contains 20 g of dry matter (DM) of wheat straw, 0.5 g of glucose and 50 mg of diammonium tartrate. Adding a source of carbon and nitrogen in minimum quantities is intended to ensure the initiation of growth of the fungus. Each column is inoculated with 120 mg of DM of mycelium homogenate. The columns are immersed in a thermostatically-controlled tank at 28° C. for 21 days. The initial water retention of the straw is 90%. A humidity-saturated upward air flow is controlled at 120 ml/min by a ball flow meter.

[0053] For each of the strains, the SSF cultures were tripled and the pretreated wheat straw obtained pooled and homogenized to supply batches of substrates in sufficient quantities for the analyses thereof. Non-inoculated wheat straw is treated under the same conditions and as such represents the control wheat straw.

Determination of Dry Matter Controls and Mass Yields of Pretreated Wheat Straw

[0054] The dry matter contents and mass yields of the pretreated wheat straw are determined using the pretreated wheat straw with or without washing with water.

[0055] The dry matter contents are determined by gravimetry by measuring the weight loss obtained after drying the solid in an oven at 105° C. to a constant weight (after 48 hours). Based on the dry matter, mass yields (%) are calculated according to the following expression:

Mass yield (%)=Dry matter after treatment/Dry matter before treatment×100

Determination of Cell Wall Polysaccharide and Lignin Composition of Pretreated Wheat Straw

[0056] The determination of the cell wall polysaccharide and lignin composition of the biomass is based on a two-step acid hydrolysis according to the operating protocol described by the NREL (National Renewable Energy Laboratory). The first step consists of hydrolysis at 30° C. in the presence of 72% sulfuric acid for 1 hour. The acid is then diluted to 4% and the second acid hydrolysis step is conducted in an autoclave at 120° C. for 1 hour. The hydrolysate is then filtered and the supernatant is analyzed by enzyme assay (RTU glucose kit, Biomérieux) and colorimetric assay (dinitrosalicylic acid method) in order to the cellulose and hemicellulose contents. The lignin is partially solubilized during hydrolysis (acid-soluble lignin). The solid residue obtained after hydrolysis thereby contains acid-insoluble lignin and ash.

Results

[0057] The filamentous fungi cultured on wheat straw draw the metabolites and energy required for the growth thereof from this substrate. Furthermore, the fungal metabolism results in the release of saccharide fractions (oligosaccharides and monomeric sugars) and non-assimilated lignin fragments and which are found in the washing water of the pretreated wheat straw.

[0058] This results in dry matter losses, the values of which are given in table 1 below.

TABLE-US-00001 TABLE 1 Mass yields of pretreated wheat straw BRFM No. 957 985 1048 1369 1554 Control.sup.c Strain T. ljubarskii P. brumalis Leiotrametes sp T. menziesii T. pavonia — Without Mass yield .sup.a 68.7 ± 2.5 83.3 ± 1.2 82.1 ± 2.4 76.5 ± 2.6 81.8 ± 1.0 96.5 ± 0.7 wash (%) Overall mass 71.2 86.3 85.1 79.3 84.8 100.0 yield .sup.b (%) With Mass yield .sup.a 52.9 ± 1.2 68.9 ± 1.7 71.5 ± 1.4 66.1 ± 1.7 70.2 ± 1.0 88.0 ± 1.0 wash (%) Overall mass 60.0 78.2 81.2 75.0 79.7 100.0 yield .sup.b (%) .sup.a The final mass of wheat straw determined after SSF is referenced to the initial wheat straw mass before SSF. .sup.b Corresponds to the mass yield with respect to the control corresponding to 100%. .sup.cNon-inoculated wheat straw treated with SSF.

[0059] As a general rule, mass yields of approximately 80% are obtained after 21 days of SSF. The greatest loss of matter was caused by T. ljubarskii (40%).

[0060] The composition of the wheat straw after fungal treatment was analyzed (see table 2 hereinafter). The specific degradations of each of the main cell wall constituents, expressed as a percentage of the initial content thereof were determined (see table 3 hereinafter).

TABLE-US-00002 TABLE 2 Cellulose, hemicellulose and lignin compositions of the various samples as a % (g of constituent/100 g Dry Matter of pretreated wheat straw) BRFM Cellulose % Hemicelluloses % Lignin % Sum %  957 38.5 ± 0.1 29.8 ± 0.3 18.1 ± 0.2 86.4  985 41.0 ± 0.2 29.2 ± 0.3 17.1 ± 0.1 87.3 1048 38.6 ± 0.4 31.4 ± 0.4 18.2 ± 1.1 88.2 1369 37.3 ± 0.5 30.5 ± 0.3 19.3 ± 0.2 87.1 1554 36.8 ± 0.4 31.0 ± 0.8 18.6 ± 0.4 86.4 Control .sup.a 37.5 ± 0.9 31.4 ± 0.3 21.9 ± 1.0 90.8 .sup.a Non-inoculated wheat straw treated with SSF.

TABLE-US-00003 TABLE 3 Cellulose, hemicelluloses and lignin yields of the various samples Mass Cellulose Hemicelluloses Lignin BRFM yield .sup.a % % .sup.b % loss .sup.c % .sup.b % loss .sup.c % .sup.b % loss .sup.c 957 60.0 23.1 38.4 17.9 43.1 10.8 50.5 985 78.2 32.1 14.5 22.9 27.3 13.4 39.0 1048 81.2 31.4 16.3 25.5 18.9 14.8 32.5 1369 75.0 28.0 25.2 22.9 27.2 14.5 33.9 1554 79.7 29.3 21.8 24.7 21.5 14.8 32.4 Control .sup.d 100.0  37.5 — 31.4  0.0 21.9 — .sup.a Overall mass yields (see Table 1). .sup.b Composition expressed as a % (g of constituent/100 g Dry Matter of pretreated wheat straw). It accounts for the mass yield of the fungal pretreatment specified in the preceding column. For the calculation thereof: mass yield × % constituent.sub.table 2 .sup.c The loss percentages were determined with respect to the composition of non-inoculated control straw (the values determined for each constituent of the control straw make up 100%) .sup.d Non-inoculated wheat straw

[0061] All the filamentous fungi studied delignify wheat straw effectively. Of these, P. brumalis_BRFM 985 and Leiotrametes sp_BRFM 1048 are the most effective for selective straw delignification (33-39% delignification) while preserving cellulose (84-85% preservation). Moreover, it is interesting to note that T. menziesii_BRFM 1369, T. pavonia_BRFM 1554 and Leiotrametes sp_BRFM 1048 degrade cellulose and hemicelluloses with similar loss rates (22-25% and 21-27% respectively, for BRFM 1369 and BRFM 1554; 16% and 19% respectively, for BRFM 1048) whereas P. brumalis_BRFM 985 preferentially use hemicelluloses as a carbon source thereby preserving most of the cellulose (27% degradation of hemicelluloses and 14% degradation of cellulose). The T. ljubarskii_BRFM 957 strain appears to simultaneously degrade cellulose, hemicelluloses and lignin (38%, 43% and 50% respectively).

[0062] These data on the composition of the plant cell walls associated with the mass yields of fungal pretreatments provide information on the quantity of glucose and xylose (main compound of wheat straw hemicelluloses) that can theoretically be released during enzymatic hydrolysis. The duration of the fungal pretreatment of the wheat straw set at 21 days appears to be suitable for the majority of strains since the holocellulose and dry matter losses remain reasonable and compatible with bioenergy production methods. For T. ljubarskii_BRFM 957, the significant degradation of lignocellulose associated with a high dry matter loss is suggestive of a probably excessively long pretreatment time under the culture conditions used.

[0063] Each pretreated wheat straw was subjected to enzymatic hydrolysis and the conversion yield of polysaccharides to fermentable sugars (digestibility) was calculated.

EXAMPLE 2

Biological Characterization of Fungal Pretreatment: Lignocelluloytic Enzymes Secreted

[0064] The breakdown of lignocelluloses is the result of the secretion by the filamentous fungi of complex lignocellulolytic enzyme systems including hydrolytic and oxidative activities acting synergistically on the different cell wall polymers.

Methodologies

[0065] The different enzyme activities were determined using aqueous extracts (5% (m of wheat straw/v of water) consistency; 1 hr; 4° C.; under stirring) of pretreated wheat straw at an SSF column outlet. These activities were expressed in Enzyme units per gram of dry matter of pretreated wheat straw.

Glycoside Hydrolase (GH) Activities

[0066] The enzyme activities on carboxymethylcellulose (CMC), microcrystalline cellulose (avicel PH-101), xylans, pectins, mannan, galactomannan, arabinan and arabinogalactan were measured using the dinitrosalicylic acid method using glucose as a standard.

[0067] A unit of enzyme activity was defined as the quantity of enzyme releasing 1 μmol of reducing sugars per minute.

Lignolytic Activities

[0068] The lignolytic activities measured are laccase, peroxidases and cellobiose dehydrogenase.

[0069] The laccase activity is determined by monitoring the oxidation of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)).

[0070] The overall peroxidase activity including manganese peroxidase (MnP) and the manganese-independent peroxidase (MiP) activity is determined by monitoring the oxidation of a solution of DMP (2,6-dimethyloxyphenol) in the presence of MnSO4, H.sub.2O.sub.2 and sodium fluoride. On the basis of this activity, it is possible to distinguish: [0071] the manganese-independent peroxidase (MiP) activity which is determined by monitoring the oxidation of a solution of DMP (2,6-dimethyloxyphenol) in the presence of only H.sub.2O.sub.2; [0072] the manganese peroxidase (MnP) activity which is determined by monitoring the oxidation of a solution of DMP (2,6-dimethyloxyphenol) in the presence of MnSO4 and H.sub.2O.sub.2. This activity is calculated by subtracting the MiP activity from the overall peroxidase activity.

[0073] The cellobiose dehydrogenase activity corresponds to the rate of reduction of 2,6-dichlorophenol-indophenol (DCPIP) in the presence of cellobiose and sodium fluoride.

[0074] The ligninolytic activities are expressed in U/g DM where a unit of enzyme activity has been defined as the quantity of enzyme oxidizing 1 μmol of substrate per minute.

Results

[0075] The filamentous fungi studied produce a diversity of hydrolases (FIG. 1) and ligninolytic enzymes (FIG. 2). It is interesting to note that none of the strains produces CDH (cellobiose dehydrogenase).

[0076] Of the strains tested, P. brumalis_BRFM 985 is the strain which produces the lowest GH activities and the greatest ligninolytic activities. This enzymatic profile corroborates selective delignification of wheat straw by this fungus. On the other hand, T. ljubarskii_BRFM 957 secretes the most GH activities. This result is consistent with the significant degradation of holocellulose observed for this fungus.

EXAMPLE 3

Effect of Fungal Pretreatments on Enzymatic Hydrolysis: Improvement of Hydrolysis Kinetics and Yields of Pretreated Straw

[0077] The purpose of the fungal pretreatment is to break down lignocellulose in order to render the cell wall polysaccharides more accessible to hydrolysis and thereby increase the fermentable sugar yields. The effectiveness of the fungal pretreatment was evaluated by determining the digestibilities and conversion yields of the cell wall polysaccharides of the pretreated wheat straw. These were calculated on the basis of the measurements of the quantities of reducing sugars and glucose released upon the hydrolysis thereof by commercial mixtures of GC220 cellulases of Trichoderma reesei and SP188 of Aspergillus niger.

Methodologies

[0078] The enzymatic hydrolyses are carried out using wheat straw pretreated and treated with sodium hydroxide in a TORNADO reactor (Tornado™ Overhead Stirring System, Radleys Discovery Technologies, United Kingdom). It was demonstrated that the alkaline treatment conducted under mild conditions improves the enzymatic digestibility of the pretreated substrate. It is suggested that this treatment may help remove the mycelium from the fiber surface which could have a negative effect on the subsequent enzymatic step. The lack of washing of the lignocellulosic biomass between the alkaline treatment and enzymatic hydrolysis steps makes it possible to limit losses and ensure the reliability of results.

Mild Alkaline Treatment

[0079] The alkaline treatment conditions are as follows: [0080] Duration: 1 hour [0081] Temperature: 50° C. [0082] Pretreated wheat straw consistency: 6% (m/m) [0083] Sodium hydroxide concentration: 0.12% (m/V) 30 mM equivalent, 2% (m/m) [0084] pH=12

Hydrolysis Tests

[0085] The pretreated wheat straw, washed with water and subjected to the sodium hydroxide treatment, are hydrolyzed with commercial mixtures of cellulases of T. reesei and A. niger. The hydrolysis conditions are as follows: [0086] Duration: 96 hours [0087] Temperature: 50° C. [0088] Consistency: 3% (m/m) in 50 mM Citrate Phosphate buffer, pH 4.4

[0089] The decrease in the consistency of the wheat straw suspension, from 6% (alkaline treatment) to 3% by adding buffer makes it possible to attain and maintain the reaction pH at 4.8. [0090] GC220 cellulases: 12 U/g DM [0091] β-glucosidase SP188: 60 U/mg DM [0092] Antibiotics: 0.15 ml/ml tetracycline and 0.04 mg/ml cycloheximide

[0093] The addition of antibiotics makes it possible to prevent any microbial contamination. [0094] Stirring: 500 rpm.

Assay of Sugars Released

[0095] The measurement of the quantities of glucose released during hydrolysis was conducted by enzyme assay (RTU glucose kit, Biomérieux), using a glucose standard curve (2 to 10 mM). The reducing sugars released were measured by the dinitrosalicylic acid method using glucose as a standard (2 to 10 mM).

Determination of Digestibilities, Crystallinity of Cellulose and Conversion Yields of Cell Wall Polysaccharides of Pretreated Straw

[0096] The digestibilities of cellulose and hemicelluloses were determined according to the following expression:

[00001]$\mathrm{Digestibility}.Math..Math.\left(\%\right)=\frac{g.Math..Math.\mathrm{of}.Math..Math.\mathrm{glc}.Math..Math.\mathrm{or}.Math..Math.\mathrm{xyl}.Math..Math.\mathrm{released}/g.Math..Math.\mathrm{of}.Math..Math.\mathrm{pretreated}.Math..Math.\mathrm{straw}}{g.Math..Math.\mathrm{of}.Math..Math.\mathrm{glc}.Math..Math.\mathrm{or}.Math..Math.\mathrm{xyl}.Math..Math.\mathrm{released}/g.Math..Math.\mathrm{of}.Math..Math.\mathrm{pretreated}.Math..Math.\mathrm{straw}}\times 100$

[0097] This data item expresses the yield of the enzymatic hydrolysis step.

[0098] X-ray measurements were made with an analytical X-ray diffractometer (Philips Analytical), using Cu Ka radiation at k=0.1540 nm (40 kV, 40 mA). The measurements were made on powder compacted into small sheets. The X-ray diffraction (XRD) data were collected for an angle range 2θ between 5° and 50° with an interval of 0.02°. The crystallinity index (CrI) of cellulose was expressed as a percentage. The equation used for calculating the CrI is:

[00002]$\mathrm{CrI}=\frac{I.Math..Math.002-\mathrm{Iam}}{I.Math..Math.002}\times 100$

[0099] where I.sub.002 corresponds to the peak intensity read on the meter, for an angle 2θ of 22° and I.sub.am corresponds to the peak intensity for an angle 2θ of 16°. I.sub.002-I.sub.am corresponds to the peak intensity of crystalline cellulose and I.sub.002 is the total peak intensity of cellulose after subtracting the background noise measured without cellulose. Crystalline cellulose was determined using the equation:

Crystalline cellulose.sub.XRD(% DM)=CrI×Cellulose NREL (% DM)

[0100] The conversion yields of cellulose and hemicelluloses make it possible to account for the dry matter losses caused by the fungal pretreatments. They are expressed as a percentage and are calculated according to the following expression:

[00003]$\mathrm{Conversion}.Math..Math.\mathrm{yield}.Math..Math.\left(\%\right)=\frac{\left(g.Math..Math.\mathrm{of}.Math..Math.\mathrm{glc}.Math..Math.\mathrm{or}.Math..Math.\mathrm{xyl}.Math..Math.\mathrm{released}/g.Math..Math.\mathrm{of}.Math..Math.\mathrm{pretreated}.Math..Math.\mathrm{straw}\right)*\mathrm{mass}.Math..Math.\mathrm{yield}}{g.Math..Math.\mathrm{of}.Math..Math.\mathrm{glc}.Math..Math.\mathrm{or}.Math..Math.\mathrm{xyl}.Math..Math.\mathrm{released}/g.Math..Math.\mathrm{of}.Math..Math.\mathrm{non}.Math.\text{-}.Math.\mathrm{inoculated}.Math..Math.\mathrm{control}.Math..Math.\mathrm{straw}}\times 100$

[0101] This data item expresses the yield of the method (pretreatment+enzymatic hydrolysis).

Results

[0102] All the pretreated wheat straw hydrolyzed by cellulases of T. reesei and A. niger made it possible to obtain quantities of reducing sugars released greater than that of the control straw with increases of up to 85%. Increases of 12% to 83% of the quantity of glucose released from pretreated wheat straw were observed (FIG. 3). The pretreatment of wheat straw with the P. brumalis_BRFM 985 strain proved to be the most effective for improving the enzymatic hydrolysis giving rise to increases of 85% and 83% of reducing sugars and glucose released, respectively.

[0103] The hydrolysis kinetics of the wheat straw pretreated with the most effective strain, P. brumalis_BRFM 985, are shown in FIG. 4.

[0104] The digestibilities and conversion yields of the different cell wall polysaccharides of the pretreated wheat straw into fermentable sugars were determined. The results are represented in FIG. 5.

[0105] For the majority of the strains tested, the enzymatic digestibilities of the pretreated wheat straw compared to that of the control straw increased, in particular with Leotrametes_BRFM 1048, P. brumalis_BRFM 985 and T. ljubarskii_BRFM 957 (FIG. 5).

[0106] Cellulose crystallinity is recognized as being a parameter limiting enzymatic attack (Hendriks & Zeeman, 2009). The crystallinity measurements of the straw pretreated with the strains which give rise to improvements in enzymatic hydrolysis (T. ljubarskii BRFM 957 and P. brumalis BRFM 985) as well as the control straw are reported in table 4 hereinafter. The results show a significant reduction in crystallinity during the pretreatment with these two strains.

TABLE-US-00004 TABLE 4 Measurement of crystallinity by XRD and crystalline cellulose content Crystallinity Crystalline index CrI cellulose (% DM Cellulose) (% DM) T. ljubarskii 26.85 14.45 BRFM 957 P. brumalis BRFM 36.27 10.28 985 Control.sup.a 51.09 20.84 .sup.anon-inoculated wheat straw

[0107] Moreover, two of these fungi, Leotrametes_BRFM 1048 and P. brumalis_BRFM 985, gave rise to significant increases in the conversion yields of the different cell wall compounds. The significant delignification rate of the wheat straw pretreated with T. ljubarskii_BRFM 957 (approximately 50%) is in correlation with the improvement in the digestibility thereof. However, the significant loss of dry matter caused by this pretreatment has an adverse effect on the conversion yields of lignocellulose. The best conversion yields obtained from wheat straw pretreated with P. brumalis_BRFM 985 attain values of 46%, 43.7% and 45% conversion of cellulose, hemicelluloses and holocellulose, respectively.

EXAMPLE 4

Effect of Fungal Pretreatments on Methanization: Improvement of Biochemical Methane Potential of Pretreated Straw

[0108] The methanization reaction induces the conversion of the organic matter into biogas mainly consisting of methane and carbon dioxide. The use of complex microbial consortia enables the conversion of the majority of organic compounds (sugars, proteins, fats) into biogas with the exception of lignin. Under certain conditions, aromatic compounds from the degradation of lignin can be converted into biogas. In the case of the pretreated straw, the holocelluloses accessible to microorganisms as well as the fungal biomass are converted into biogas. The pretreated samples were methanized without washing so as not to lose the biogas obtained from labile sugars and the fungal biomass.

Methodologies

Determination of Volatile Matter (VM) Contents

[0109] The volatile matter contents (representing the organic matter) are determined by gravimetry (APHA standard method, 1998). This consists of the weight loss obtained by combustion (4 hrs at 550° C.) on a previously dried sample (passed for 48 hrs at 105° C.)

Measurement of Biochemical Methane Potential (BMP)

[0110] The BMP tests are conducted under favorable conditions for methanization and indicate the maximum quantity of methane that can be obtained for these substrates. They were conducted with the control straw and the pretreated strew, unwashed and freeze-dried, as well as on a fungal biomass (BRFM 1554) cultured in liquid medium, washed with water (to remove traces of culture medium) and freeze-dried. It is noted that two samples pretreated with the BRFM 985 strain were measured for BMP.

[0111] The 600 mL BMP flasks have an effective volume of 400 mL, which contains the substrate (1.3 g DM/flask), a methanizer inoculum (3 g VM/L), water, macro- and micro-elements along with a bicarbonate buffer in order to ensure optimal methanization conditions. The flasks are placed at 36° C. under stirring and the biogas production was monitored by measuring the pressure (pressure gauge), until the end of production (plateau phase), in triplicate. The measurement of the biogas composition was performed for each pressure reading with a micro-gas chromatography analysis: Varian GC-CP4900 (injector at 100° C., capillary columns at 30° C., carrier gas: helium). The volumes of gas are expressed in Normal milliliters (NmL). They consist of the volumes obtained under normal temperature and pressure conditions (0° C., 1 atm).

Results

[0112] The pretreated samples have a BMP greater than that of the control, which reflects the improvement in sugar accessibility particularly due to delignification (see FIG. 6 and table 5 hereinafter). In particular, the BRFM 957 and BRFM 985 strains resulting in the greatest delignification rates also results in the highest methane potentials (approximately 230 NmL CH4/g pretreated VM). Moreover, it is interesting to note that white rot is readily biodegraded: the BRFM 1554 strain has a BMP of 256±60 NmL CH4/g VM, which is greater than that of the control straw (192±9 NmL CH4/g VM). As such, even if there are material losses, a portion of the lignocellulosic biomass is converted into fungal biomass. In other words, the straw is converted into more readily biodegradable biomass.

TABLE-US-00005 TABLE 5 BMP and standard deviation of the different samples BMP BMP (NmL (NmL CH4/g CH4/g pretreated Standard initial Standard BRFM VM) deviation DM) deviation  985a .sup.a 242 15 198 12  985b 220 2 181 1  957 .sup.a 230 9 153 6 1048 .sup.a 217 12 185 10 1369 .sup.a 202 6 166 5 1554 .sup.a 198 1 159 7 Control 192 9 184 9 .sup.a Non-definitive results (end-of-product plateau not reached)

[0113] The improvement in methane production is to be compared to the loss of matter caused. As such, the results were expressed in NmL/g initial DM (Table 5), i.e. per g DM before SSF (determined by means of the mass yield). Accounting for the loss of matter obtained for the culture conditions used, only a few strains can increase the BMP of the straw (BRFM 1048 and BRFM 985). The BRFM 957 strain makes it possible to obtain a significant BMP when this is expressed with respect to the VM, which reflects the significant delignification. On the other hand, it is the most mediocre when the result is expressed with respect to the initial DM, which reflects the significant DM losses occurring during SSF.

EXAMPLE 5

Pretreatment of 2 Metric Tons of Wheat Straw with the Polyporus Brumalis CNCM I-4900 Strain

Preparation of Inocula

[0114] Primary Inoculum (Production of Fungal Biomass with Liquid Fermentation)

[0115] The P. brumalis CNCM I-4900 strain is inoculated on malt-based agar medium and incubated for 5 to 10 days at 25-35° C.

[0116] A preculture is produced in Roux flasks containing 200 ml of malt-based medium. Each flask is inoculated with 5 disks of mycelium (5 mm in diameter) sampled on agar. After 7-10 days of growth at 25-35° C. and protected from light, the mycelium is harvested and ground using a mill (Ultra Turrax).

[0117] The homogenate is used to inoculate a bioreactor having an effective volume of 50 liters in order to produce the primary inoculum. The inoculation rate is between 0.5 and 5% m/v. The liquid medium for biomass production medium in a fermenter consists of malt extract and yeast extract. The aeration is between 0.1 and 3 vvm and the stirring between 50 and 200 rpm. The temperature of the culture is maintained between 25 and 35° C.

[0118] After 2 to 10 days, a maximum biomass production is attained. It is harvested and then used as a primary inoculum for the production of miscanthus colonized by the P. brumalis CNCM I-4900 fungus forming the secondary inoculum. Miscanthus can thereby be produced in different forms such as granules, chips, mulch or briquettes of miscanthus.

Secondary Inoculum (Production of Colonized Miscanthus by SSF).

[0119] Miscanthus, for example in the form of granules, chips, straw or briquettes, serves as a nutrient substrate for the P. brumalis CNCM I-4900 fungal strain.

[0120] The miscanthus is packaged in bags or trays and humidified to a level of 50 to 300%.

[0121] This miscanthus is inoculated between 0.1% and 2% m/m with primary inoculum.

[0122] The aeration of the fungal culture is promoted by packaging the inoculated miscanthus in containers below the maximum filling capacity thereof and by the closure thereof with a filtering device.

[0123] The bags or trays are periodically stirred so as to promote the homogeneity of the colonization of the substrate.

[0124] The incubation lasts from 4 to 15 days, preferably between 4 and 7 days, at a temperature between 25 and 35° C.

[0125] The inoculated miscanthus can be used extemporaneously, after storage between 4° C. and 15°, after freeze-drying or any other preservation means.

[0126] If necessary, this step is repeated before the inoculation of the straw, in order to increase the quantity of inoculum available.

Fungal Pretreatment of Wheat Straw

[0127] The straw is inoculated with the secondary inoculum at an inoculation rate between 5 and 50%, preferentially between 20 and 40% and humidified to a level of 50 to 100%.

[0128] The inoculated wheat straw is stored in trays equipped with forced aeration equipment. This device enables the air to be blown between 0.1 and 3 vvm. Occasional turnings of the lignocellulosic matter promote the aeration of the culture and consequently the fungal growth.

[0129] The temperature is maintained between 25 and 35° C.

[0130] The humidity is kept constant throughout the duration of the pretreatment.

[0131] The duration of the pretreatment is between 7 and 90 days.

REFERENCES

[0132] Chandler, J. A., & Jewell, W. J. (1980). Predicting methane fermentation biodegradability (p. 234). Solar Energy Research Institute.

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