Methane fermentation of selected lignocellulosic materials

Seven lignocellulosic materials: corn stover, napier grass, wood grass, newspaper, white fir and wheat straw from two different crops; two pure cellulosics: Solka Floc BW200 and Whatman No. 5 filter paper; and glucose, propionic and acetic acids were subjected to long-term batch methane fermentation. Ninety per cent of the original COD was recovered as methane gas from the two pure cellulosics and glucose. For the lignocellulosics, depending on the material, variations from over 80% conversion efficiency to methane for corn stover to less than 10% for white fir were observed. Generally, herbaceous materials were degraded faster and more extensively than woody biomass. A first-order rate model described well the methane fermentation process for the lignocellulosics tested, but was a poor model for the soluble substrates. It was not possible to predict either the biodegradability or the rate of methane fermentation with a reasonable degree of accuracy based solely on the lignin content of the lignocellulosic materials.     

Pretreatments to enhance the digestibility of lignocellulosic biomass

Lignocellulosic biomass represents a rather unused source for biogas and ethanol production. Many factors, like lignin content, crystallinity of cellulose, and particle size, limit the digestibility of the hemicellulose and cellulose present in the lignocellulosic biomass. Pretreatments have as a goal to improve the digestibility of the lignocellulosic biomass. Each pretreatment has its own effect(s) on the cellulose, hemicellulose and lignin; the three main components of lignocellulosic biomass. This paper reviews the different effect(s) of several pretreatments on the three main parts of the lignocellulosic biomass to improve its digestibility. Steam pretreatment, lime pretreatment, liquid hot water pretreatments and ammonia based pretreatments are concluded to be pretreatments with high potentials. The main effects are dissolving hemicellulose and alteration of lignin structure, providing an improved accessibility of the cellulose for hydrolytic enzymes.     

Comparison of different pretreatment methods for lignocellulosic materials. Part II: Influence of pretreatment on the properties of rye straw lignin

Several processes have been suggested to convert various types of lignocellulosic biomass into lignin products and saccharides. This paper evaluates the suitability of an organosolv process, a process using soda, a hydrothermal process and a process developed in this work, called the “Aquasolve process” for inclusion into a lignocellulosic biorefinery concept. Part II of this paper investigates the influence of the different pretreatment processes on the properties of rye straw lignin and evaluates their ability to produce high recoveries of high quality lignin.Specifications for high quality lignin products are defined and the isolated lignin fractions are analysed by Klason lignin, carbohydrate and ash content, elemental analysis, thermo-gravimetric analysis, ³¹P NMR, and size exclusion chromatography. The organosolv process shows the largest lignin recovery, followed by the soda and Aquasolve processes. Lignin products from the soda process, the Aquasolve process and with reservation the organosolv process show interesting properties for polymer applications.     

Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw

Wheat and rye straws were pretreated with ozone to increase the enzymatic hydrolysis extent of potentially fermentable sugars. Through a 2(5-1) factorial design, this work studies the influence of five operating parameters (moisture content, particle size, ozone concentration, type of biomass and air/ozone flow rate) on ozonization pretreatment of straw in a fixed bed reactor under room conditions. The acid insoluble lignin content of the biomass was reduced in all experiments involving hemicellulose degradation. Near negligible losses of cellulose were observed. Enzymatic hydrolysis yields of up to 88.6% and 57% were obtained compared to 29% and 16% in non-ozonated wheat and rye straw respectively. Moisture content and type of biomass showed the most significant effects on ozonolysis. Additionally, ozonolysis experiments in basic medium with sodium hydroxide evidenced a reduction in solubilization and/or degradation of lignin and reliable cellulose and hemicellulose degradation.     

Natural deep eutectic solvents for lignocellulosic biomass pretreatment: Recent developments,challenges and novel opportunities

Conversion of lignocellulosic biomass to fuels and chemicals has attracted immense research and development around the world. Lowering recalcitrance of biomass in a cost-effective manner is a challenge to commercialize biomass-based technologies. Deep eutectic solvents (DESs) are new ‘green' solvents that have a high potential for biomass processing because of their low cost, low toxicity, biodegradability, easy recycling and reuse. This article discusses the properties of DESs and recent advances in their application for lignocellulosic biomass processing. The effectiveness of DESs in hydrolyzing lignin-carbohydrate complexes, removing lignin/hemicellulose from biomass as well as their effect on biomass deconstruction, crystallinity and enzymatic digestibility have been discussed. Moreover, this review presents recent findings on the compatibility of natural DESs with enzymes and microorganisms.     

Ultrasound-assisted fractionation of the lignocellulosic material

In the present work the effectiveness of different lignocellulosic biomass fractionation processes based on ultrasounds technology was evaluated. Organosolv (acetic acid 60% v/v), alkaline (sodium hydroxide 7.5% w/w) and autohydrolysis treatments were applied at low temperature and the fractionation effectiveness was measured at different sonication conditions of the raw material. The obtained solid fractions were characterized using TAPPI standard methods, and the liquid fractions main components were quantified with the purpose of studying the effect that the treatment conditions had on the obtained by-products quality. Therefore, obtained lignin samples were characterized by ATR-IR spectroscopy and their thermal behaviour by TGA technique. The results showed that ultrasounds application improved the yield and selectivity of the studied processes and that the obtained lignin did not suffer significant modifications in its physicochemical properties.     

Enhanced saccharification of wheat straw with the application of ultrasonic-assisted quaternary ammonium hydroxide pretreatment

Lignocelluloses featuring complicated structure and poor degradability usually require pretreatment before its utilization. In this study, an ultrasonic-assisted pretreatment by using quaternary ammonium hydroxide was introduced to enhance biodegradability of lignocellulosic biomass. The synergistic chemical and mechanical pretreatment were supposed to be responsible for both external surface destruction and internal structure disruption of lignocelluloses. High-efficient lignin removal accompanied with obvious structural (crystallinity) transformation was achieved in the pretreated straws. Process analysis indicated that factors of time, temperature, concentration of solvent, and ultrasound power intensity turned out to be significant for pretreatment, and a 4-fold increased saccharification yield of around 92.4% as compared to untreated straw was obtained from the wheat straw pretreated by 15% solvent at 50 °C for 0.5 h in power intensity 344 W/cm². All results suggest that the combined chemical and mechanical treatment can significantly improve the bio-accessibility of lignocelluloses, leading to the enhanced utilization efficiency.     

RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass

Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down‐regulation of the sugarcane caffeic acid O‐methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.     

Biological pretreatment with a cellobiose dehydrogenase-deficient strain of Trametes versicolor enhances the biofuel potential of canola straw

The use of Trametes versicolor as a biological pretreatment for canola straw was explored in the context of biofuel production. Specifically, the effects on the straw of a wild-type strain (52 J) and a cellobiose dehydrogenase (CDH)-deficient strain (m4D) were investigated. The xylose and glucose contents of the straw treated with 52 J were significantly reduced, while only the xylose content was reduced with m4D treatment. Lignin extractability was greatly improved with fungal treatments compared to untreated straw. Saccharification of the residue of the m4D-treated straw led to a significant increase in proportional glucose yield, which was partially attributed to the lack of cellulose catabolism by m4D. Overall, the results of this study indicate that CDH facilitates cellulose access by T. versicolor. Furthermore, treatment of lignocellulosic material with m4D offers improvements in lignin extractability and saccharification efficacy compared to untreated biomass without loss of substrate due to fungal catabolism.     

Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review

One of the major challenges faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pre-treatment step of biomass. These inhibitory compounds are toxic to fermenting micro-organisms. The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during thermo-chemical pre-treatment step such as acid and steam explosion. This review describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates. Inhibition of yeast fermentation by the inhibitor compounds in the lignocellulosic hydrolysates can be reduced by treatment with enzymes such as the lignolytic enzymes, for example, laccase and micro-organisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus. Microbial and enzymatic detoxifications of lignocellulosic hydrolysate are mild and more specific in their action. The efficiency of enzymatic process is quite comparable to other physical and chemical methods. Adaptation of the fermentation yeasts to the lignocellulosic hydrolysate prior to fermentation is suggested as an alternative approach to detoxification. Increases in fermentation rate and ethanol yield by adapted micro-organisms to acid pre-treated lignocellulosic hydrolysates have been reported in some studies. Another approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance by Saccharomyces cerevisiae, for example, by overexpressing genes encoding enzymes for resistance against specific inhibitors and altering co-factor balance. Cloning of the laccase gene followed by heterologous expression in yeasts was shown to provide higher enzyme yields and permit production of laccases with desired properties for detoxification of lignocellulose hydrolysates. A combination of more inhibitor-tolerant yeast strains with efficient feed strategies such as fed-batch will likely improve lignocellulose-to-ethanol process robustness.     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

Do furanic and phenolic compounds of lignocellulosic and algae biomass hydrolyzate inhibit anaerobic mixed cultures? A comprehensive review

Nowadays there is a growing interest on the use of both lignocellulosic and algae biomass to produce biofuels (i.e. biohydrogen, ethanol and methane), as future alternatives to fossil fuels. In this purpose, thermal and thermo-chemical pretreatments have been widely investigated to overcome the natural physico-chemical barriers of such biomass and to enhance biofuel production from lignocellulosic residues and, more recently, marine biomass (i.e. macro and microalgae). However, the pretreatment technologies lead not only to the conversion of carbohydrate polymers (ie cellulose, hemicelluloses, starch, agar) to soluble monomeric sugar (ie glucose, xylose, arabinose, galactose), but also the generation of various by-products (i.e. furfural and 5-HMF). In the case of lignocellulosic residues, part of the lignin can also be degraded in lignin derived by-products, mainly composed of phenolic compounds. Although the negative impact of such by-products on ethanol production has been widely described in literature, studies on their impact on biohydrogen and methane production operated with mixed cultures are still very limited.     

Comparison of Feedstock Pretreatment Performance and Its Effect on Soluble Sugar Availability

Cellulosic feedstocks for bioenergy differ in composition and processing requirements for efficient conversion to chemicals and fuels. This study discusses and compares the processing requirements for three lignocellulosic feedstocks??soybean hulls, wheat straw, and de-starched wheat bran. They were ground with a hammer mill to investigate how differences in composition and particle size affect the hydrolysis process. Enzyme hydrolysis was conducted using cellulase from Trichoderma reesei at 50°C and pH 5. Ground fractions were also subjected to dilute sulfuric acid treatment at 125°C, 15 psi for 30 min prior to cellulase treatment. Reducing particle size of biomass resulted in segregated components of feedstock. Grinding wheat straw to particle size <132 ??m resulted in measured lignin content from 20% to ??5% and reduced hemicellulose content. Reducing lignin content increased the effectiveness of enzyme hydrolysis of wheat straw. Particles sized <132 ??m exhibited the highest soluble sugar release upon hydrolysis for all three feedstocks studied. Hemicellulose digestion improved with dilute sulfuric acid treatment with residual hemicellulose content <5% in all three feedstocks after acid treatment. This enhanced the cellulase action and resulted in approximately 1.6-fold increase in sugar availability in de-starched wheat bran and ??1.5-fold for wheat straw and soybean hulls. Higher sugar availability in wheat bran after acid-mediated enzyme treatment correlated to higher ethanol yields during yeast fermentation compared with soybean hulls and wheat straw.     

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.     

Characterization of degradation products from alkaline wet oxidation of wheat straw

Alkaline wet oxidation pre-treatment (water, sodium carbonate, oxygen, high temperature and pressure) of wheat straw was performed as a 2(4-1) fractional factorial design with the process parameters: temperature, reaction time, sodium carbonate and oxygen. Alkaline wet oxidation was an efficient pre-treatment of wheat straw that resulted in solid fractions with high cellulose recovery (96%) and high enzymatic convertibility to glucose (67%). Carbonate and temperature were the most important factors for fractionation of wheat straw by wet oxidation. Optimal conditions were 10 min at 195 degrees C with addition of 12 bar oxygen and 6.5 g l(-1) Na2CO3. At these conditions the hemicellulose fraction from 100 g straw consisted of soluble hemicellulose (16 g), low molecular weight carboxylic acids (11 g), monomeric phenols (0.48 g) and 2-furoic acid (0.01 g). Formic acid and acetic acid constituted the majority of degradation products (8.5 g). The main phenol monomers were 4-hydroxybenzaldehyde, vanillin, syringaldehyde. acetosyringone (4-hydroxy-3,5-dimethoxy-acetophenone), vanillic acid and syringic acid, occurring in 0.04-0.12 g per 100 g straw concentrations. High lignin removal from the solid fraction (62%) did not provide a corresponding increase in the phenol monomer content but was correlated to high carboxylic acid concentrations. The degradation products in the hemicellulose fractions co-varied with the pre-treatment conditions in the principal component analysis according to their chemical structure, e.g. diacids (oxalic and succinic acids), furan aldehydes, phenol aldehydes, phenol ketones and phenol acids. Aromatic aldehyde formation was correlated to severe conditions with high temperatures and low pH. Apart from CO2 and water, carboxylic acids were the main degradation products from hemicellulose and lignin.     

A new algorithm to characterize biodegradability of biomass during anaerobic digestion: influence of lignin concentration on methane production potential

We examined the influence of fibrous fractions of biomass on biochemical methane potential (BMP) with the objective of developing an economical and easy-to-use statistical model to predict BMP, and hence the biodegradability of organic material (BD) for biogas production. The model was developed either for energy crops (grass, maize, and straw) or for animal manures, or as a combined model for these two biomass groups. It was found that lignin concentration in volatile solids (VS) was the strongest predictor of BMP for all the biomass samples. The square of the sample correlation coefficient (R(2)) from the BMP versus lignin was 0.908 (p<0.0001), 0.763 (p<0.001) and 0.883 (p<0.001) for animal manure, energy crops and the combined model, respectively. Validation of the combined model was carried out using 65 datasets from the literature.     

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose-to-glucose conversion of maize stover. Here, we analyzed a field-grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD-deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose-to-glucose conversion efficiency was determined after 10 different thermochemical pre-treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre-treatment. However, the relative increase in glucose yields after alkaline pre-treatment was not higher than the relative increase when no pre-treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p-coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD-deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar-platform biorefineries.     

耐高温木质素降解菌筛选及降解效果研究

为提高育苗基质中废弃物木质素降解速率,在废弃物堆腐生产育苗基质高温阶段取样,筛选耐高温木质素降解菌,并对菌种进行鉴定,同时测定其对秸秆木质素和菌糠木质素的降解效果。获得了1株较好的木质素高温降解菌HZ11,鉴定为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）,结果显示,该菌株对秸秆木质素和菌糠木质素降解效果较好,50 ℃条件下,20 d木质素降解率分别为46.7%和42.4%。菌株HZ11在降解秸秆和菌糠方面具有很好的应用潜力,为利用农业废弃物生产育苗基质提供更加丰富的菌种资源,具有重要的参考价值。     

Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw

ABSTRACT: BACKGROUND: Lipids produced from filamentous fungi show great promise for biofuel production, but a major limiting factor is the high production cost attributed to feedstock. Lignocellulosic biomass is a suitable feedstock for biofuel production due to its abundance and low value. However, very limited study has been performed on lipid production by culturing oleaginous fungi with lignocellulosic materials. Thus, identification of filamentous fungal strains capable of utilizing lignocellulosic hydrolysates for lipid accumulation is critical to improve the process and reduce the production cost. RESULTS: The growth performances of eleven filamentous fungi were investigated when cultured on glucose and xylose. Their dry cell weights, lipid contents and fatty acid profiles were determined. Six fungal strains with high lipid contents were selected to culture with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. The results showed that all the selected fungal strains were able to grow on both detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH). The highest lipid content of 39.4% was obtained by Mortierella isabellina on NDLH. In addition, NDLH with some precipitate could help M. isabellina form pellets with an average diameter of 0.11 mm. CONCLUSION: This study demonstrated the possibility of fungal lipid production from lignocellulosic biomass. M. isabellina was the best lipid producer grown on lignocellulosic hydrolysates among the tested filamentous fungi, because it could not only accumulate oils with a high content by directly utilizing NDLH to simplify the fermentation process, but also form proper pellets to benefit the downstream harvesting. Considering the yield and cost, fungal lipids from lignocellulosic biomass are promising alternative sources for biodiesel production.