Interactions between fungal growth,substrate utilization,and enzyme production during solid substrate cultivation of Phanerochaete chrysosporium on cotton stalks

Fungal pretreatment, using lignin-degrading microorganisms to improve lignocellulosic feedstocks with minimal energy input, is a potential alternative to physiochemical pretreatment methods. Identifying the kinetics for fungal pretreatment during solid substrate cultivation is needed to help establish the processing conditions for effective scale up of this technology. In this study, a set of mathematical models were proposed for describing the interactions between holocellulose consumption, lignin degradation, cellulase, ligninolytic enzyme, and the growth of Phanerochaete chrysosporium during a 14 day fungal pretreatment process. Model parameters were estimated and validated by the System Biology Toolbox in MatLab. Developed models provided sufficiently accurate predictions for fungal growth (R ² = 0.97), holocellulose consumption (R ² = 0.97), lignin degradation (R ² = 0.93) and ligninolytic enzyme production (R ² = 0.92), and fair prediction for cellulase production (R ² = 0.61). The models provide valuable information for understanding the interactive mechanisms in biological systems as well as for fungal pretreatment process scale up and improvement.     

Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover

In this study, a newly isolated Trametes hirsuta yj9 was used to pretreat corn stover in order to enhance enzymatic digestibility. T. hirsuta yj9 preferentially degraded lignin to be as high as 71.49% after 42-day pretreatment. Laccase and xylanase was the major ligninolytic and hydrolytic enzyme, respectively and filter paper activity (FPA) increased gradually with prolonged pretreatment time. Sugar yields increased significantly after pretreatment with T. hirsuta yj9, reaching an enzymatic digestibility of 73.99% after 42 days of pretreatment. Scanning electron microscopy (SEM) showed significant structural changes in pretreated corn stover, the surface of pretreated corn stover became increasingly coarse, the gaps between cellulose fibers were visible, and many pores were developed. Correlation analysis showed that sugar yields were inversely proportional to the lignin contents, less related to cellulose and hemicellulose contents.     

Carbohydrate derived‐pseudo‐lignin can retard cellulose biological conversion

Dilute acid as well as water only (hydrothermal) pretreatments often lead to a significant hemicellulose loss to soluble furans and insoluble degradation products, collectively termed as chars and/or pseudo‐lignin. In order to understand the factors contributing to reducing sugar yields from pretreated biomass and the possible influence of hemicellulose derived pseudo‐lignin on cellulose conversion at the moderate to low enzyme loadings necessary for favorable economics, dilute acid pretreatment of Avicel cellulose alone and mixed with beechwood xylan or xylose was performed at various severities. Following pretreatment, the solids were enzymatically hydrolyzed and characterized for chemical composition and physical properties by NMR, FT‐IR, and SEM imaging. It was found that hemicelluloses (xylan) derived‐pseudo‐lignin was formed at even moderate severities and that these insoluble degradation products can significantly retard cellulose hydrolysis. Furthermore, although low severity (CSF ～ 1.94) dilute acid pretreatment of a xylan–Avicel mixture hydrolyzed most of the xylan (98%) and produced negligible amounts of pseudo‐lignin, enzymatic conversion of cellulose dropped significantly (>25%) compared to cellulose pretreated alone at the same conditions. The drop in cellulose conversion was higher than realized for cellulase inhibition by xylooligomers reported previously. Plausible mechanisms are discussed to explain the observed reductions in cellulose conversions. Biotechnol. Bioeng. 2013; 110: 737–753. © 2012 Wiley Periodicals, Inc.     

Aromatic compound degradation by the wood-feeding termite Coptotermes formosanus (Shiraki)

Wood-feeding termites (WFT) have proven to be highly efficient for wood digestion. There is evidence to support the hypothesis that there are ligninolytic enzymes existing in the gut of WFT responsible for wood pretreatment toward cellulose utilization. Elucidating the mechanism of biomass pretreatment through lignin modification in termites will help to develop more efficient lignocellulosic biofuel production processes. The in-vivo degradation of aromatic compounds with different substructures, including dyes, lignin model monomers and dimers, and lignin sulfonate, by Coptotermes formosanus (Shiraki) was investigated. The degradation of aromatic compounds was determined using pyrolysis-gas chromatography/mass spectrometry. The results revealed that WFT were able to metabolize the conjugated aromatic structures and that the degradation efficiency is higher in the foregut and midgut regions than in the hindgut. This is the first time that evidence has been provided to show different aromatic compound degradation in the separate gut segments of a termite. This study provides information on the C. formosanus (Shiraki) lignin modification phenomenon, and it demonstrates that phenomenon’s potential in the breakdown of the plant cell wall. Understanding this lignin modification could contribute to technology that will supplant current harsh pretreatment protocols for plant cell walls and thereby better facilitate the conversion of cellulose and hemicellulose.     

Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding

Enzymatic saccharification of cellulose is a key step in conversion of plant biomass to advanced biofuel and chemicals. Many substrate-related factors affect saccharification. Rather than examining the role of each individual factor on overall saccharification efficiency, this study examined how each factor affects the three basic processes of a heterogeneous biochemistry reaction: (1) substrate accessibility to cellulose—the roles of component removal and size reduction by pretreatments, (2) substrate and cellulase reactivity limited by component inhibition, and (3) reaction conditions—substrate-specific optimization. Our in-depth analysis of published literature work, especially those published in the last 5 years, explained and reconciled some of the conflicting results in literature, especially the relative importance of hemicellulose vs. lignin removal and substrate size reduction on enzymatic saccharification of lignocelluloses. We concluded that hemicellulose removal is more important than lignin removal for creating cellulase accessible pores. Lignin removal is important when alkaline-based pretreatment is used with limited hemicellulose removal. Partial delignification is needed to achieve satisfactory saccharification of lignocelluloses with high lignin content, such as softwood species. Rather than using passive approaches, such as washing and additives, controlling pretreatment or hydrolysis conditions, such as pH, to modify lignin surface properties can be more efficient for reducing or eliminating lignin inhibition to cellulase, leading to improved lignocellulose saccharification.     

Comparative ligninolytic and polysaccharolytic potentials of an alkaliphilic basidiomycete on native ligninocellulose

A comparison of the ligninolytic, cellulolytic and hemicellulolytic abilities of an alkaliphilic white-rot fungus. Coprinus fimetarius, on wheat straw under varying conditions of solid-substrate fermentation is presented. The extent of fractional degradation (percentage of the original dry weight of the fraction) of straw under an optimized set of cultural conditions (pH 9·0, moisture 65%, temperature 37°C, period 21 days) was in the following order: lignin (45%), cellulose (42%), hemicellulose (27%). Urea nitrogen favoured the degradation of lignin as well as cellulose and hemicellulose up to a certain level (1·5% sterile urea or 3% unsterile urea on a dry weight basis) beyond which the degradation of lignin was relatively more adversely affected than cellulose. The addition of phosphorus and sulphur was found essential for selective lignin removal. Increasing the C:N ratio by addition of free carbohydrates resulted in an overall decrease in the degradation wherein cellulose utilization was the most affected event. The pre-treatment (physical or chemical) of the substrate caused a general increase in biodegradation of lignin, cellulose and hemicellulose. The degrading activity of the fungus declined with the scaling-up of the fermentation particularly under non-sterile conditions.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in in vitro culture

The fibrolytic activities of rumen fungi were studied in terms of dry matter loss, plant cell wall degradation and enzyme (cellulase and xylanase) activities, when grown in vitro on either untreated or sodium hydroxide treated stems of barley straw over a 12 day period. Changes in fungal growth, development and overall biomass were followed using chitin assay and scanning electron microscopy. Treatment with sodium hydroxide resulted in a decrease in the NDF content together with the disruption of cuticle and the loosening and separation of the plant cells within the straw fragments. The enzyme activities of the anaerobic fungi have a high positive correlation (R(2)=0.99) with their biomass concentration assessed by chitin assay indicating that chitin is a valuable index for the estimation of the fungal biomass in vitro. The anaerobic fungi produced very extensive rhizoidal systems in these in vitro cultures. After incubation with rumen fungi, dry matter losses were, respectively, 35% and 38% for the untreated and treated straw samples and the overall fungal biomass, determined by chitin assay, was significantly higher in the treated samples. In vitro degradation of cellulose and hemicellulose was also higher in the treated than that of untreated cultures. Although, comparatively, xylanase activity was higher than that of cellulase, the cellulose fraction of the straw was degraded more than hemicellulose in both treated and untreated straw.     

生物质生物预处理研究进展与展望

木质纤维素生物质分布广、产量大、可再生,用于制备生物基能源、生物基材料和生物基化学品。木质纤维素生物质组成复杂,包含纤维素、半纤维素和木质素等,木质素与半纤维素通过共价键、氢键交联形成独特的“包裹结构”,纤维素含有复杂的分子内与分子间氢键,上述因素制约着其资源化利用。生物预处理以其独特优越性成为生物质研究的重要方面。系统阐述了生物预处理过程中木质素降解和基团修饰对纤维素酶解的影响,纤维素含量及结晶区变化,半纤维素五碳糖利用,微观物理结构的改变。进一步提出了以生物预处理为核心的组合预处理、基于不同功能的多酶协同催化体系、木质纤维素组分分级利用和新型高效细菌预处理工艺是生物预处理未来发展的重要趋势。     

玉蕈降解木质纤维素的生理生化基础

本文研究了玉蕈在纤维废弃物上生长期间,培养基中主要成分的降解规律及有关的酶学分析。实验结果表明:1.玉蕈分解纤维素和半纤维素的能力较强,分解木素的能力很弱。因此,玉蕈是褐腐型木腐菌。2.纤维素是玉蕈子实体生长阶段的主要碳源。3.玉蕈生长期间可向培养基中释放羧甲基纤维素酶、滤纸纤维素酶、半纤维素酶、淀粉酶和蛋白酶。酶活性在子实体生长阶段显著增加。进一步证明了子实体阶段酶活性增加与培养温度和子实体形成有密切关系。     

Microbial cultures in the utilization of cellulosic materials

This review elaborates on the most recent microbial development in saccharification of cellulose and cellulase formation. A particular highlight is a new genetic-immunochemical approach investigating the mechanism of adhesion of bacterial cellulase to cellulose during cellulose conversion. New developments and recent reviews in hemicellulose and lignin degradation are also covered.     

Fractionating recalcitrant lignocellulose at modest reaction conditions

Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose biorefineries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (50 degrees C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ( approximately 97% in 24 h) during the hydrolysis step at the enzyme loading of 15 filter paper units of cellulase and 60 IU of beta-glucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorefinery.     

Fungal biodegradation and enzymatic modification of lignin

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H₂O₂-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.     

白腐真菌血红密孔菌漆酶复合酶预处理烟梗丝的响应面法优化

烟梗是烟草工业的重要副产物,也是宝贵的自然资源。本研究首先利用白腐菌漆酶对烟梗丝进行预处理,提升了添加烟梗丝的卷烟品质;然后分别以木质素、纤维素、半纤维素和果胶的降解率为响应值,采用Box-Behnken设计建立方程模型,对漆酶、纤维素酶、半纤维素酶和果胶酶组成的复合酶预处理烟梗丝条件进行了优化。结果表明：每100g烟梗丝加入30U漆酶,在料液比为35%、温度为30℃、酶解pH为5处理48h的条件下预处理的烟梗丝对提升卷烟品吸效果最佳,烟梗丝中木质素、纤维素、半纤维素和果胶的降解率分别为20.16%、15.10%、7.20%和12.40%;为获得与之相同的各组分降解率,响应面法优化漆酶复合酶最佳处理条件为：每100g烟梗丝加入漆酶14.72U、纤维素酶1.00U、半纤维素酶1.00U、果胶酶8.45U。验证发现烟梗丝各组分降解率实测值与理论值无显著性差异,且显微结构观察显示复合酶处理后的烟梗丝表面致密结构被破坏,孔洞数量明显增加。本研究获得的白腐菌漆酶预处理后的烟梗丝在卷烟中的添加能有效改善卷烟品质,且漆酶复合酶的使用大幅减少了漆酶的用量,降低了漆酶预处理烟梗丝的成本,为废弃烟梗生物质的资源化利用提供了重要依据。     

Inhibition of cellulase, xylanase and beta-glucosidase activities by softwood lignin preparations

The conversion of lignocellulosic biomass to fuel ethanol typically involves a disruptive pretreatment process followed by enzyme-catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. Attempts to improve process economics include protein engineering of cellulases, xylanases and related hydrolases to improve their specific activity or stability. However, it is recognized that enzyme performance is reduced during lignocellulose hydrolysis by interaction with lignin or lignin-carbohydrate complex (LCC), so the selection or engineering of enzymes with reduced lignin interaction offers an alternative means of enzyme improvement. This study examines the inhibition of seven cellulase preparations, three xylanase preparations and a beta-glucosidase preparation by two purified, particulate lignin preparations derived from softwood using an organosolv pretreatment process followed by enzymatic hydrolysis. The two lignin preparations had similar particle sizes and surface areas but differed significantly in other physical properties and in their chemical compositions determined by a 2D correlation HSQC NMR technique and quantitative 13C NMR spectroscopy. The various cellulases differed by up to 3.5-fold in their inhibition by lignin, while the xylanases showed less variability (< or = 1.7-fold). Of all the enzymes tested, beta-glucosidase was least affected by lignin.     

Biomass photochemistry: XI. Photochemical pretreatment of cellulose and its fungal degradation

In order to study the single-cell proteins and cellulase production, fungal degradation of alpha-cellulose was tested. Photochemical pretreatment of alpha-cellulose at lambda > 254 nm increases by over 100% the cellulase production by Chrysonilia sitophila (TFB strain). This is reflected in a larger production of soluble proteins in the filtrate with higher cellulase activity when compared with unirradiated cellulose. Single-cell protein production (insoluble proteins) was also improved after preirradiation of cellulose or even on rice hull as the only carbon source after inoculation with the spore of C. sitophila. This pretreatment is a valuable method for increasing the digestibility of lignocellulosic materials.     

Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification

Approximately one-half of the lignin and most of the hemicellulose present in agricultural residues such as wheat straw and corn stover are solubilized when the residue is treated at 25 degrees C in an alkaline solution of hydrogen peroxide. The delignification reaction is most efficient when the ratio of hydrogen peroxide to substrate is at least 0.25 (w/w) and the pH is 11.5. The supernatant fraction from a given pretreatment, after addition of makeup peroxide and readjustment of the pH, can be recycled to treat at least six additional batches of substrate, resulting in a substantial concentration of hemicellulose and soluble lignin degradation products. Hydrolysis of the insoluble fraction with Trichoderma reesei cellulase after alkaline peroxide treatment yields glucose with almost 100% efficiency, based upon the cellulose content of the residue before treatment. These data indicate that alkaline peroxide pretreatment is a simple and efficient method for enhancing the enzymatic digestibility of lignocellulosic crop residues to levels approaching the theoretical maximum.     

Bioconversion of sugar cane crop residues with white-rot fungiPleurotus sp.

Four mushroom strains ofPleurotus spp. were cultivated on sugar cane crop residues for 30 days at 26°C. Biochemical changes affected the substrate as a result of fungal growth, in terms of nitrogen, lignin, cellulose and hemicellulose contents. All strains showed a strong ligninolytic activity together with variable cellulolytic and xylanolytic action.Pleurotus sajor-caju attacked lignin and cellulose at the same rate, showing a degradation of 47% and 55%, respectively. A better balance was shown by theP. ostreatus-P. pulmonarius hybrid, which exhibited the poorest cellulolytic action (39%) and the highest ligninolytic activity (67%). The average composition of mushroom fruit bodies, in terms of nitrogen, carbohydrates, fats and amino acid profiles, was determined. Crude protein and total carbohydrate varied from 23% to 33% and 36% to 68% of dry matter, respectively. Fat ranged from 3.3% to 4.7% and amino acid content from 12.2% to 22.2%. Slight evidence for a nitrogen fixing capability was encountered in the substrate to fruit body balance.     

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.     

Potential of selected fungal species to degrade wheat straw,the most abundant plant raw material in Europe

Background

Structural component of plant biomass, lignocellulose, is the most abundant renewable resource in nature. Lignin is the most recalcitrant natural aromatic polymer and its degradation presents great challenge. Nowadays, the special attention is given to biological delignification, the process where white-rot fungi take the crucial place owing to strong ligninolytic enzyme system. However, fungal species, even strains, differ in potential to produce high active ligninolytic enzymes and consequently to delignify plant biomass. Therefore, the goals of the study were characterization of Mn-oxidizing peroxidases and laccases of numerous mushrooms as well as determination of their potential to delignify wheat straw, the plant raw material that, according to annual yield, takes the first place in Europe and the second one in the world.

Results

During wheat straw fermentation, Lentinus edodes HAI 858 produced the most active Mn-dependent and Mn-independent peroxidases (1443.2 U L⁻¹ and 1045.5 U L⁻¹, respectively), while Pleurotus eryngii HAI 711 was the best laccase producer (7804.3 U L⁻¹). Visualized bends on zymogram confirmed these activities and demonstrated that laccases were the dominant ligninolytic enzymes in the studied species. Ganoderma lucidum BEOFB 435 showed considerable ability to degrade lignin (58.5%) and especially hemicellulose (74.8%), while the cellulose remained almost intact (0.7%). Remarkable selectivity in lignocellulose degradation was also noted in Pleurotus pulmonarius HAI 573 where degraded amounts of lignin, hemicellulose and cellulose were in ratio of 50.4%:15.3%:3.8%.

Conclusions

According to the presented results, it can be concluded that white-rot fungi, due to ligninolytic enzymes features and degradation potential, could be important participants in various biotechnological processes including biotransformation of lignocellulose residues/wastes in food, feed, paper and biofuels.