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Parameters of lignocellulosic biomass influencing its anaerobic degradability
Polymers composition and enzymatic accessibility
A predictive model for methane production allowed classifying some parameters of lignocellulosic biomass: there was a high negative influence of lignin content and a small negative influence of crystalline cellulose content, a positive influence of soluble sugars content (measured after mild acid hydrolysis) and, to a lesser extent, a positive influence of crude protein and amorphous holocelluloses contents (Monlau et al., 2012b).
In addition, Monlau et al. (2013) inventoried the main parameters influencing anaerobic digestion of lignocellulose. This latter is limited by the hydrolysis step. Thus, the accessibility of the substrate to enzymatic attacks is primordial. Pore volume influences the digestibility (must be high). G/S (guaiacyl/synapyl) ratio influences lignin degradability. G units lead to condensed structure (C-C bounds) with little biodegradability whereas lignin S units are easier to degrade anaerobically (Abdellatif Barakat et al., 2014b). Crystalline cellulose is more recalcitrant than the amorphous form during anaerobic digestion, so crystalline index must be low. The higher the ratio xylose/arabinose (X/A), the better the hemicelluloses digestibility (A Barakat et al., 2014). The substitution of xylose by arabinose makes the structure more complex and decreases the digestibility. Hemicelluloses and cellulose are linked by H bonds (Sun, 2010). Some hemicelluloses contain acetyl groups contributing to those linkages (in graminaceous, for example). Acetyl groups removal led to a better hydrolysis of cellulose and xylan (Kong et al., 1992; Zhang et al., 2011). Those groups are partly responsible for the restricted degradation of forages in the rumen. Xylan degradation is difficult prior to the removal of lateral substituents. Finally, the hydrolysis of the complex lignin-carbohydrates is the main obstacle to anaerobic degradation.
The lignin-carbohydrates complex (LCC): linkages and derived compounds
Major lignin linkages in cell-wall: ferulic acid (FA) and p-coumaric acid (pCA)
Cross-linkages (both between lignin and carbohydrates and within lignin) enhance the rigidity, complicate the structure and limit the availability of carbohydrates to anaerobic microorganisms (Jalč, 2002) (Figure 1-16). Major obstacles to the biodegradability of lignocellulosic biomass remain the cleaving of LCC bonds and the removal of lignin (Monlau et al., 2013). Complete removal of lignin is not necessary for important glucose released after enzymatic hydrolysis and cleavage of bonds between lignin and cellulose would be sufficient (Brudecki et al., 2013). The level of delignification required may depend on the lignocellulosic matrix considered (species, etc.). It exist two biosynthetic pathways to build LCC (nucleophilic attack or oxidative coupling), but even if they modify the structure, they have no effect on degradability (Boukari et al., 2009; Grabber, 2005; Monlau et al., 2013).
A. Schematic representation of cell-wall and its main polymers (Adapted from Ceres, 2007). B. Representation of linkages between lignin and carbohydrates via phenolic acids and arabinose in non-woody plants (Monlau et al., 2013).
Lignin monomers are linked together by β-O-4 aryl ether linkages in majority (Annex 1). Uncondensed units linked in this way are preferentially degraded. However, Agosin et al. (1985) failed to find a direct correlation between the cleavage of these bounds and digestibility improvement (IVDMD). Thus, other linkages are important to cleave like lignin-carbohydrates bounds Monlau et al. (2013). Indeed, lignin is linked to hemicelluloses, limiting their hydrolysis. These linkages are in majority phenolic acids ester-linked (1-2% of cell walls, Annex 1) to arabinose. Ferulic acid (FA) is one of them and is also ether-linked to lignin (hemicelluloses-ester-FA-ether-lignin bridges). In gramineae, hemicelluloses can also be bound to lignin via diferulic acids (Figure 1-16B). Recent studies have also concluded that FA is an intrinsic part of the lignin structure in grasses (Río et al., 2012) . Then, p-coumaric acids (pCA) can sometimes have a lignin/hemicelluloses cross-linking function but lots of pCA are also ester-linked inside lignin molecules (Sun, 2010).
Anaerobic degradability of FA and pCA
The impact of FA and pCA on anaerobic digestion is not very clear (Yosef and Ben-Ghedalia, 2000) but they can be inhibitory depending on their concentrations (Table 1-6). If added individually, those phenolic acids (also called hydroxycinnamic acids) can decrease in vitro fermentation of cellulose (Borneman et al., 1986; Jung et al., 1992). pCA added to wheat straw did not lead to limitation of degradation but lignin-linked pCA can potentially limit straw hydrolysis (Besle et al., 1986). Borneman et al. (1986) observed in vitro that 1mM pCA is generally inhibitory for rumen bacteria studied and 10 mM FA is only inhibitory for one out of four strains. In addition, pCA was less degraded than FA in the rumen (Varel and Jung, 1986). With synthetically esterified phenolic acids to oat spelts xylan fractions, Jung et al. (1991 cited by Yosef and Ben-Ghedalia 2000) observed a negative correlation between concentration of esterified phenolic acids and IVDMD. In this study, esterified FA was slightly more inhibitory to IVDMD than esterified pCA (Yosef and Ben-Ghedalia, 2000). Akin et al. (1993a) tested the effect of esterified pCA and FA on the digestion of linked arabinoxylan by three rumen strains. They concluded that ester bonds can limit the availability of carbohydrates in a species dependent way; perhaps due to different enzymatic material especially in terms of esterases. Finally, it seems that a lot of rumen organisms have high levels of cinnamoyl esterases (responsible for hydrolysis of hydroxycinnamic acids) (McSweeney et al., 1998) and thus can cut those links.
Other cell-wall linkages in anaerobic digestion
Pectins bind to cellulose and are covalently and non-covalently linked to xyloglucan (Cosgrove, 2005). Pectins are principally formed by galacturonic acids that are linked together with carbohydrates and α-1,4 bounds (Sun, 2010). They constitute an amorphous structure (Le Troedec et al., 2008). To a lesser extent, other molecules have a role as lignin inter-bound or lignin/hemicelluloses cross-linkage: sinapic, cinnamic, ρ-hydroxybenzoic, galacturonic and glucuronic acids (Bidlack et al., 1992; Sun, 2010). Some of them (including FA and pCA) can be covalently bound to cell wall pectins (Dinis et al., 2009; Sun, 2010).
Uronic acids are pretty well degraded in the rumen (Ben-Ghedalia and Miron, 1984). Monlau et al. (2012b) did not observe an impact of uronic acids content on methane production of various lignocellulosic residues. Thus, they may be easily digested. During anaerobic digestion, breaking bounds of pectin occurred during hydrolysis step and allowed anaerobic microorganisms to use linked fermentable carbohydrates. Pretreatment with pectinases can improve hydrolysis and subsequent methane production even if pectin is a minor compounds. Generally, activities used are high (more than 2000 U) (Pakarinen et al., 2012). However, Frigon et al. (2012) obtained 72% methane more with only 50 U of pectinase but with an incubation of 78 days with sodium acetate.
Lignin-derived compounds
Lignin-derived compounds can bind proteins rendering them unavailable for anaerobic microbial utilization (Han et al., 1975; Karunanandaa and Varga, 1996a; Velez et al., 1985) and/or inactivating anaerobic digestive enzymes (Gamble et al., 1996; Han et al., 1975). Rumen cellulolytic bacteria can be inhibited by small phenolic compounds. The lignin compound syringaldehyde was well digested anaerobically, alone or in combination with xylose. With vanillin, even if methane was produced, some inhibitions are possible (Barakat et al., 2012). Anaerobic digestion processes are pretty tolerant to phenolic compounds, depending on their concentrations (Monlau et al., 2014) and their structures (Hernandez and Edyvean, 2008). Reactor mode (batch or continuous) and inoculum adaptation influence the inhibitory capacity of such molecules (Monlau et al., 2014).
Table of contents :
Chapter 1. Literature review
1.1 ANAEROBIC DIGESTION
1.1.1 A four steps biological reaction
1.1.2 Anaerobic digestion of lignocellulosic crop residues
1.1.2.1 Crop residues: an important resource
1.1.2.2 Main constituents of lignocellulosic biomass
1.1.2.3 Other possibilities for energy recovery from crop residues
1.1.2.4 A solid substrate adapted to Solid-State anerobic digestion
1.1.3 Solid-State Anaerobic Digestion: definition, advantages and limits
1.1.4 Main parameters influencing anaerobic digestion
1.1.4.1 Temperature
1.1.4.2 Mixing
1.1.4.3 pH and alkalinity
1.1.4.4 Carbon/Nitrogen (C/N) ratio
1.1.4.5 Nutrient supplementation
1.1.4.6 Inhibitors
1.1.4.7 Particle size of the substrate
1.1.4.8 Inoculum
1.1.5 Anaerobic digestion processes
1.1.5.1 Biochemical Methane potential (BMP)
1.1.5.2 SSAD processes
1.2 PRETREATMENTS FOR ANAEROBIC DIGESTION
1.2.1 Non-biological pretreatments
1.2.2 Biological treatments
1.3 ENZYMATIC MECHANISMS OF WRF AND CULTURE PARAMETERS
1.3.1 Enzymatic degradation by WRF and Solid-State Fermentation (SSF)
1.3.2 Cultivation parameters for Solid-State Fermentation (SSF)
1.4 QUANTIFICATION OF ANAEROBIC DIGESTIBILITY IMPROVEMENT AFTER WRF RETREATMENT
1.4.1 Anaerobic digestion studies with WRF pretreatment
1.4.1.1 Nitrogen metabolism for anaerobic digestion of fungal pretreated substrates
1.4.1.2 Slight acidification of pretreated substrates
1.4.2 In Vitro Dry Matter Digestibility (IVDMD) studies
1.5 PARAMETERS OF LIGNOCELLULOSIC BIOMASS INFLUENCING ITS ANAEROBIC DEGRADABILITY
1.5.1 Polymers composition and enzymatic accessibility
1.5.2 The lignin-carbohydrates complex (LCC): linkages and derived compounds
1.5.2.1 Major lignin linkages in cell-wall: ferulic acid (FA) and p-coumaric acid (pCA)
1.5.2.2 Anaerobic degradability of FA and pCA
1.5.2.3 Other cell-wall linkages in anaerobic digestion
1.5.2.4 Lignin-derived compounds
1.5.3 Influence of histology
1.5.3.1 Epidermis role and composition
1.5.3.2 Anaerobic digestion at cell scale
1.6 QUALITATIVE EFFECTS OF FUNGAL PRETREATMENT ON ANAEROBIC DIGESTIBILITY
1.6.1 Modifications of lignin: G/S (guaiacyl/synapyl) ratio
1.6.2 Cellulose digestibility improvement
1.6.3 Changes in hemicelluloses X/A (xylose/arabinose) ratio and acetate
1.6.4 Increase of porosity
1.6.5 Actions of WRF on lignin-carbohydrates complex (LCC) linkages
1.6.6 Release of lignocellulosic polymers main constituents
1.6.6.1 Lignin-derived compounds
1.6.6.2 Hemicelluloses and cellulose derived compounds
1.6.7 Influence of histology: fungal degradation at cell scale
1.7 PYROLYSIS-GAZ CHROMATOGRAPHY-MASS SPECTROPHOTOMETRY (PY-GC-MS): A POWERFUL TOOL FOR STRUCTURE ANALYSIS
1.7.1 Definition and interest
1.7.2 Pyrolysis products (pyrolysates)
1.7.2.1 Formation mechanisms
1.7.2.2 Parameters influencing pyrolysates yield and nature
1.7.3 Various existing technologies for Py-GC-MS
1.7.4 Py-GC-MS compared to chemical methods to study lignin structure
1.8 CONCLUSION
Chapter 2. Main experimental strategy
2.1 MATERIAL AND METHOD COMMON TO ALL RESULTS CHAPTERS: MATERIAL ORIGIN AND BMP TESTS
2.1.1 Fungal strains
2.1.2 Wheat straw
2.1.3 Cultivation of Polyporus brumalis BRFM 985 in liquid medium
2.1.4 Total Solids and Volatile Solids determination
2.1.5 Biochemical Methane Potential (BMP) measurements: general case
2.2 OVERVIEW OF ANALYSED SAMPLES
2.3 MATERIAL AND METHODS SPECIFIC TO RESULTS OVERVIEW (CHAPTER 7)
2.3.1 Contamination problem
2.3.1.1 Pilot-reactor cleaning verification
2.3.1.2 Wheat straw microflora
2.3.1.3 BMP-tests
2.3.2 Enzyme assays
2.3.2.1 Glycoside Hydrolases (GH) activities
2.3.2.2 Ligninolytic activities
2.3.3 Wheat straw compositional analyses
2.3.3.1 Data obtained for ethanol production study and PCA
2.3.3.2 Fourier Transform InfraRed (FTIR) spectroscopy
2.3.3.3 Crystallinity measurement with X-Ray Diffractometry (XRD)
Chapter 3. White-rot Fungi selection for pretreatment of lignocellulosic biomass for anaerobic digestion and impact of glucose supplementation
3.1 INTRODUCTION
3.2 MATERIAL AND METHODS SPECIFIC TO SELECTION STEP
3.2.1 Fungal pretreatment
3.2.1.1 SSF in 24-well plates
3.2.1.2 SSF in columns
3.2.2 Anaerobic digestion: samples characterization and normalization of BMP results
3.2.2.1 Total Carbon (TC) measurements for pretreated samples in columns
3.2.2.2 BMP-tests
3.2.2.3 Normalization of BMP results for samples pretreated in deep-well
3.2.3 Impact of nutrient solution amount
3.2.3.1 SSF in deep well
3.2.3.2 Acid hydrolysis (Klason lignin, cellulose and hemicelluloses)
3.2.3.3 Determination of residual glucose
3.3 BMP IMPROVEMENT
3.3.1 Straw pretreated in 24-well plates
3.3.2 Straw pretreated in SSF columns
3.3.3 Pretreatments comparison
3.4 INFLUENCE OF STARTER SOLUTION ON PRETREATMENT
3.4.1 Fate of the nutrient solution during the pretreatment
3.4.2 Nutrient solution and wheat straw composition
3.4.2.1 Limited delignification
3.4.2.2 Carbohydrates consumption
3.5 CONCLUSION
Chapter 4. Influence of Polyporus brumalis culture parameters for the fungal pretreatment: optimization step
4.1 INTRODUCTION
4.2 MATERIAL AND METHODS SPECIFIC TO OPTIMIZATION STEP
4.2.1 Fungal pretreatment for anaerobic digestion
4.2.1.1 SSF in columns
4.2.1.2 BMP-tests
4.2.2 Experimental design
4.2.2.1 Building of the experimental design
4.2.2.2 Analyze of the experimental design and response variables
4.2.3 Substrate characterization
4.2.3.1 Dry matter losses after pretreatment
4.2.3.2 Klason lignin, cellulose and hemicelluloses determination by acid hydrolysis
4.2.3.3 Lignin losses in pretreated samples compared to Non inoculated Controls (NIC)
4.2.3.4 Soluble sugars
4.2.3.5 Enzymatic hydrolysis of cellulose: glucose yield
4.2.3.6 Real Time Quantitative Polymerase Chain Reaction (qPCR)
4.3 RELATIONSHIPS BETWEEN ANAEROBIC DIGESTION AND SUBSTRATE CHARACTERISTICS
4.3.1 Methane production results during BMP tests
4.3.2 Parameters influencing anaerobic degradability
4.3.3 Negative impact of lignin amount on methane production
4.3.4 Influence of fungal mycelium on CH4 production
4.3.5 Contribution of cellulose degradation to methane production
4.4 RESPONSE SURFACES FOR METHANE PRODUCTION
4.4.1 Methane production after 6 days
4.4.2 Methane production after 57 days
4.4.2.1 Samples pretreated with metals addition
4.4.2.2 Samples pretreated without metals addition
4.4.3 BMP
4.5 CONCLUSION
Chapter 5. Pyrolysis-GC-MS study of White-Rot Fungi pretreated wheat straws for anaerobic digestion
5.1 INTRODUCTION
5.2 MATERIAL AND METHODS SPECIFIC TO PY-GC-MS STUDY OF PRETREATED STRAWS
5.2.1 Fungal pretreated samples
5.2.2 Pyrolysis-GC-MS
5.2.2.1 Spectra obtaining
5.2.2.2 Spectrum analysis and interpretation
5.2.3 Characterization of fungal pretreated straws for anaerobic digestion
5.2.3.1 Acid hydrolysis (Klason lignin, cellulose and hemicelluloses) with NREL method
5.2.3.2 Qpcr
5.2.3.3 BMP-tests
5.2.4 Statistical analysis
5.2.4.1 Analysis of Variances (ANOVA)
5.2.4.2 Principal Component Analysis (PCA)
5.3 ORIGIN OF THE MAIN COMPOUNDS FOUND IN PYROLYSATES
5.3.1 Fungus characterization
5.3.1.1 N-containing (N) and unspecific compounds (UN)
5.3.1.2 Polysaccharide-derived products (PS)
5.3.1.3 Possible presence of small lignin amount
5.3.2 Straws characterization
5.3.2.1 Lignin derived pyrolysis products
5.3.2.2 Polysaccharide-derived products
5.3.2.3 N-containing compounds
5.4 COMPARISON BETWEEN PY-GC-MS DATA, OTHER CHARACTERIZATION TECHNICS AND ANAEROBIC DIGESTIBILITY
5.4.1 Variations of the S/G ratio
5.4.2 PCA to study relationships between samples characteristics
5.4.2.1 qPCR variable opposed to straw composition variables in the plan 1-2
5.4.2.2 Correlation between BMP and PS/LIG-pyr or PS/LIG NREL in the plan 1-3
5.4.3 Fungal biomass (qPCR variable)
5.4.4 Polysaccharides (PS)/Lignin (LIG) ratio determined by py-GC-MS
5.5 CONCLUSION
Chapter 6. Solid-State Anaerobic Digestion of wheat straw: impact of substrate/inoculum ratio and of fungal pretreatment
6.1 INTRODUCTION
6.2 MATERIAL AND METHODS SPECIFIC TO SSAD IN BATCH REACTORS (CHAPTER 6)
6.2.1 Wheat straw pretreatment in a pilot-reactor
6.2.2 Solid state Anerobic Digestion in batch
6.2.2.1 SSAD reactors design
6.2.2.2 Experiment I: tests of several S/I
6.2.3 Experiment II: fungal pretreatment for SSAD
6.2.3.1 Substrates and inoculum
6.2.3.2 Experimental configuration
6.2.4 Analytical methods
6.2.4.1 SSAD monitoring parameters: leachate pH, VFA, alkalinity and biogas composition
6.2.4.2 BMP-tests
6.2.4.3 Total Kjeldahl Nitrogen (TKN)
6.2.4.4 Ammonium concentration in final leachate
6.2.4.5 Final Total Carbon (TC) in digestate
6.2.4.6 Analysis of Variances (ANOVA)
6.3 EXPERIMENT I: TESTS OF SEVERAL S/I
6.3.1 Anaerobic digestion of wheat straw: start-up phase progress
6.3.2 TVFA/alkalinity as process stability indicator
6.4 EXPERIMENT II: FUNGAL PRETREATMENT FOR SSAD
6.4.1 Substrates anaerobic digestibility
6.4.2 SSAD start-up phase
6.4.3 Methane production of fungal pretreated straw in SSAD
6.5 CONCLUSION
