Inhibition of cellulose enzymatic hydrolysis by laccase‐derived compounds from phenols

The presence of inhibitors compounds after pretreatment of lignocellulosic materials affects the saccharification and fermentation steps in bioethanol production processes. Even though, external addition of laccases selectively removes the phenolic compounds from lignocellulosic prehydrolysates, when it is coupled to saccharification step, lower hydrolysis yields are attained. Vanillin, syringaldehyde and ferulic acid are phenolic compounds commonly found in wheat‐straw prehydrolysate after steam‐explosion pretreatment. These three phenolic compounds were used in this study to elucidate the inhibitory mechanisms of laccase‐derived compounds after laccase treatment. Reaction products derived from laccase oxidation of vanillin and syringaldehyde showed to be the strongest inhibitors. The presence of these products causes a decrement on enzymatic hydrolysis yield of a model cellulosic substrate (Sigmacell) of 46.6 and 32.6%, respectively at 24 h. Moreover, a decrease in more than 50% of cellulase and β‐glucosidase activities was observed in presence of laccase and vanillin. This effect was attributed to coupling reactions between phenoxyl radicals and enzymes. On the other hand, when the hydrolysis of Sigmacell was performed in presence of prehydrolysate from steam‐exploded wheat straw a significant inhibition on enzymatic hydrolysis was observed independently of laccase treatment. This result pointed out that the other components of wheat‐straw prehydrolysate are affecting the enzymatic hydrolysis to a higher extent than the possible laccase‐derived products. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:700–706, 2015     

Progress in biopulping of non-woody materials: Chemical, enzymatic and ultrastructural aspects of wheat straw delignification with ligninolytic fungi from the genus Pleurotus

Abstract: During screening of basidiomycetes for wheat straw delignification, considerable lignin degradation with a limited attack to cellulose was attained with Pleurotus eryngii . Straw solid-state fermentation (SSF) was optimized, and the enzymatic mechanisms for lignin degradation were investigated. No lignin peroxidase was detected under liquid or SSF conditions, but high laccase and aryl-alcohol oxidase levels were found. The latter enzyme has been fully characterized in PI. eryngii and it seems to be involved in a cyclic redox system for H₂0₂ generation from aromatic compounds. Results obtained using homoveratric acid suggest that Pleurotus laccase could be involved in degradation of phenolic and non-phenolic lignin moieties. Histological and ultrastructural studies provided some general morphological characteristics of the fungal attack on wheat straw. Whereas a simultaneous degradation pattern was observed in straw treated with Phanerochaete chrysosporium , PI. eryngii caused partial degradation of middle lamella and separation of individual sclerenchymatic fibers. When these straw samples were subjected to refining tests, energy saving after biological treatment was the highest in the case of straw treated with PI. eryngii , which also produced the lowest substrate loss. From these results, a correlation between preferential removal of lignin, separation of sclerenchymatic fibers and pulping properties was provided during fungal treatment of wheat straw.     

Production of cellulases and xylanase by Trichoderma reesei F-522 on pretreated wheat straw

Autohydrolyzed and ethanol-alkali pulped wheat straw was examined as a candidate feedstock for both cellulase and xylanase production and enzymatic hydrolysis. Submerged cultures of Trichoderma reesei F-522 grown on hydrothermally modified straw provided culture supernatants of the highest enzymatic activities, whereas the maximal efficiency of enzymatic hydrolysis was recorded in straw treated with ethanol-NaOH mixture. Some culture conditions were optimized to improve the growth and cellulase production by T. reesei on autohydrolyzed wheat straw.     

Comparison of the efficiency of bacterial and fungal laccases in delignification and detoxification of steam-pretreated lignocellulosic biomass for bioethanol production

This study evaluates the potential of a bacterial laccase from Streptomyces ipomoeae (SilA) for delignification and detoxification of steam-exploded wheat straw, in comparison with a commercial fungal laccase from Trametes villosa. When alkali extraction followed by SilA laccase treatment was applied to the water insoluble solids fraction, a slight reduction in lignin content was detected, and after a saccharification step, an increase in both glucose and xylose production (16 and 6%, respectively) was observed. These effects were not produced with T. villosa laccase. Concerning to the fermentation process, the treatment of the steam-exploded whole slurry with both laccases produced a decrease in the phenol content by up to 35 and 71% with bacterial and fungal laccases, respectively. The phenols reduction resulted in an improved performance of Saccharomyces cerevisiae during a simultaneous saccharification and fermentation (SSF) process, improving ethanol production rate. This enhancement was more marked with a presaccharification step prior to the SSF process.     

Comparison of Hydrolysis Efficiency and Enzyme Adsorption of Three Different Cellulosic Materials in the Presence of Poly(ethylene Glycol)

The addition of non-ionic surfactants has recently been confirmed to positively affect the enzymatic hydrolysis of cellulosic materials. However, the functional mechanisms of these surfactants remain unclear. This work investigated the influence of poly(ethylene glycol) (PEG) on the enzymatic hydrolysis of three cellulosic materials, namely, acid steam-exploded corn straw, pure microcrystalline cellulose (Avicel PH101), and bagasse sulfite pulp (BSP). The results showed that PEG addition led to varied effects on the enzymatic hydrolysis of different cellulosic materials. Addition of PEG was most effective on the enzymatic hydrolysis of PH101 and weakly effective on the hydrolysis of BSP. We further investigated PEG concentrations and enzymatic activities in the supernatant during hydrolysis and found that the positive effects of PEG treatment might contribute to its influence on enzyme desorption from different substrates. We also found that the efficiency of PEG depended on its capacity to bind to different substrates. PEG exhibited stronger affinity to pure cellulose than to the two other lignocellulosic substrates. These findings are helpful in further revealing the mechanism of surfactants and improving the enzymatic hydrolysis process.     

Effect and aftereffect of gamma radiation pretreatment on enzymatic hydrolysis of wheat straw

Irradiation pretreatment of wheat straw was carried out at different doses by using Co-60 gamma radiation. The weight loss and fragility of wheat straw after irradiation, the combination effect of irradiation and mechanical crushing on enzymatic hydrolysis of wheat straw as well as the aftereffect of irradiation were examined. It is shown that irradiation can cause significant breakdown of the structure of wheat straw. The weight loss of wheat straw increased and the size distribution after crushing moved to fine particles at elevated irradiation doses. The glucose yield of enzymatic hydrolysis of wheat straw increased with increasing doses and achieved the maximum (13.40%) at 500 kGy. A synergistic effect between irradiation and crushing was observed, with a glucose yield of 10.24% at a dose of 500 kGy with powder of 140 mesh. The aftereffect of irradiation had important impact on enzymatic hydrolysis of wheat straw. The aftereffect (at 22nd day) of 400 kGy irradiation accounted for 20.0% of the initial effect for glucose production, and the aftereffects of 50, 100, 200 (at 9th day) and 300 kGy (at 20th day) accounted for 12.9%, 14.9%, 8.9% and 9.1%, respectively, for reducing sugar production.     

Cellulase production using different streams of wheat grain- and wheat straw-based ethanol processes

Pretreatment is a necessary step in the biomass-to-ethanol conversion process. The side stream of the pretreatment step is the liquid fraction, also referred to as the hydrolyzate, which arises after the separation of the pretreated solid and is composed of valuable carbohydrates along with compounds that are potentially toxic to microbes (mainly furfural, acetic acid, and formic acid). The aim of our study was to utilize the liquid fraction from steam-exploded wheat straw as a carbon source for cellulase production by Trichoderma reesei RUT C30. Results showed that without detoxification, the fungus failed to utilize any dilution of the hydrolyzate; however, after a two-step detoxification process, it was able to grow on a fourfold dilution of the treated liquid fraction. Supplementation of the fourfold-diluted, treated liquid fraction with washed pretreated wheat straw or ground wheat grain led to enhanced cellulase (filter paper) activity. Produced enzymes were tested in hydrolysis of washed pretreated wheat straw. Supplementation with ground wheat grain provided a more efficient enzyme mixture for the hydrolysis by means of the near-doubled β-glucosidase activity obtained.     

Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw

In order to defray the cost of biodiesel production, the ensuing work was to further investigate utilization of the crude glycerol (CG) from oleochemicals industry in the atmospheric autocatalytic organosolv pretreatment (AAOP) to enhance enzymatic hydrolysis.

The AAOP–CG enabled wheat straw to achieve with reasonable enzymatic hydrolysis yields, reaching 75% for the wet substrate and 63% for the dried. Lipophilic compounds from the CG formed pitch deposition on the fiber, which was responsible for low delignification (30%) and also troublesome in practical operation. Pitch deposits itself had no significant role on enzymatic hydrolysis. A striking finding of the lignin recondensation and/or lignin–carbohydrate complex helped explain why dried pretreated wheat straw had a low enzymatic hydrolysis yield. The CG was suitable for the AAOP to enhance enzymatic hydrolysis of lignocellulosic biomass. But it was advisable to remove lipophilic compounds from crude glycerol before utilization.     

Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification

Agricultural by-products such as wheat straw are attractive feedstocks for the production of second-generation bioethanol due to their high abundance. However, the presence of lignin in these lignocellulosic materials hinders the enzymatic hydrolysis of cellulose. The purposes of this work are to study the ability of a laccase-mediator system to remove lignin improving saccharification, as a pretreatment of wheat straw, and to analyze the chemical modifications produced in the remaining lignin moiety. Up to 48 % lignin removal from ground wheat straw was attained by pretreatment with Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as mediator, followed by alkaline peroxide extraction. The lignin removal directly correlated with increases (～60 %) in glucose yields after enzymatic saccharification. The pretreatment using laccase alone (without mediator) removed up to 18 % of lignin from wheat straw. Substantial lignin removal (37 %) was also produced when the enzyme-mediator pretreatment was not combined with the alkaline peroxide extraction. Two-dimensional nuclear magnetic resonance (2D NMR) analysis of the whole pretreated wheat straw material swollen in dimethylsulfoxide-d ₆ revealed modifications of the lignin polymer, including the lower number of aliphatic side chains involved in main β-O-4′ and β-5′ inter-unit linkages per aromatic lignin unit. Simultaneously, the removal of p-hydroxyphenyl, guaiacyl, and syringyl lignin units and of p-coumaric and ferulic acids, as well as a moderate decrease of tricin units, was observed without a substantial change in the wood polysaccharide signals. Especially noteworthy was the formation of Cα-oxidized lignin units during the enzymatic treatment.     

纤维素酶降解小麦秸秆最适条件的研究及其动力学分析

以小麦秸秆为原料,通过正交实验对纤维素酶降解秸秆纤维的影响因素进行了研究。结果表明,影响小麦秸秆降解的因素依次为:酶量>酶解时间>料液比>反应温度,其最适条件是:加酶量为40u/g,酶解时间为10h,反应温度为40℃,料液比为1∶3,总糖含量达到43.24%。以米氏方程为基础,建立起最适酶解条件下总纤维素降解的动力学模型。     

Improving the fermentation performance of saccharomyces cerevisiae by laccase during ethanol production from steam‐exploded wheat straw at high‐substrate loadings

Operating the saccharification and fermentation processes at high‐substrate loadings is a key factor for making ethanol production from lignocellulosic biomass economically viable. However, increasing the substrate loading presents some disadvantages, including a higher concentration of inhibitors (furan derivatives, weak acids, and phenolic compounds) in the media, which negatively affect the fermentation performance. One strategy to eliminate soluble inhibitors is filtering and washing the pretreated material. In this study, it was observed that even if the material was previously washed, inhibitory compounds were released during the enzymatic hydrolysis step. Laccase enzymatic treatment was evaluated as a method to reduce these inhibitory effects. The laccase efficiency was analyzed in a presaccharification and simultaneous saccharification and fermentation process at high‐substrate loadings. Water‐insoluble solids fraction from steam‐exploded wheat straw was used as substrate and Saccharomyces cerevisiae as fermenting microorganism. Laccase supplementation reduced strongly the phenolic content in the media, without affecting weak acids and furan derivatives. This strategy resulted in an improved yeast performance during simultaneous saccharification and fermentation process, increasing significantly ethanol productivity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Superfine grinding of steam-exploded rice straw and its enzymatic hydrolysis

The combination of low severity steam explosion and superfine grinding has been studied with respect to side products generation and enzymatic hydrolysis efficiency. Chemical compositions, fiber characteristics and composed cells contents in the superfine ground product and the ground residue particles produced by superfine grinding were also studied. At the determined parameters using FJM-200 fluidized bed opposed jet mill, 78% superfine ground steam-exploded rice straw (SERS) products with the mean fiber length of 60 μm were obtained, the particles yield was 179% higher than that from the native rice straw (RS). Enzymatic hydrolysis, chemical composition, fiber characteristics and composed cells proportion of the superfine ground SERS product and the ground residue all show great differences. The difference in enzymatic hydrolysis and structural properties indicates that superfine grinding is a good way to fractionate SERS into easily bio-converted part and difficultly hydrolysed part.     

Solid-state production of biopulp by Phanerochaete chrysosporium using steam-exploded wheat straw as substrate.

A novel material for biopulp-making, steam-exploded wheat straw (SEWS), was studied. During the steam explosion process, the hemicellulose was partly degraded and became water-soluble sugar as the carbon resource of the chosen microbe growth, and compared with non-SEWS, the degradation amount of cellulose decreased and the degradation amount of lignin increased for the fermented steam-exploded wheat straw (FSEWS) cultured with Phanerochaete chrysosporium ME-446. Under the optimum conditions of solid-state ferrmentation (SSF), the degradation amount of lignin reached 60% on the 5th day and the fermented straw residue could be used directly as the material for pulp making.     

Kinetic model of enzymatic hydrolysis of steam-exploded wheat straw

The hydrolysis kinetics of steam-exploded wheat straw treated with cellulase NS 50013 enzyme complex in combination with β-glucosidase NS 50010 is studied. The time dependence of the reducing sugars amount is followed at varying the temperature value and the amount of the enzyme introduced. The activation energy determined on the ground of the rate temperature dependence stays unchanged in the course of the process. The preexponential factor decreases with the increase of the degree of hydrolysis and is responsible for the process rate decrease. A new expression for the dependence of degree of hydrolysis of one of carbohydrate polymers (cellulose) in wheat straw on the time, the enzyme concentration and the temperature is obtained. It is of practical importance as well because it provides estimation of the degree of hydrolysis required at predetermined values of the temperature, the enzyme concentration and the time used. The expression can be used for control of the enzyme hydrolysis of cellulose in the wheat straw.     

Optimization of Ethanol Production From Microfluidized Wheat Straw by Response Surface Methodology

In this study, wheat straw was pretreated with a microfluidizer to improve its enzymatic hydrolysis and ethanol yields. The pretreatment was performed at various pressures (500, 1000, and 1500 bar) and solid loadings (1, 2, and 3%). The microfluidized biomass was then subjected to hydrolysis and simultaneous saccharification and co-fermentation (SSCF) experiments at different enzyme loadings (5, 10, and 15 FPU/g dry wheat straw) using a mutant yeast. The results indicated that the microfluidization method alters the structure of biomass and leads to a reduction in lignin content. The samples pretreated at 1% solid loading contained the minimum lignin concentration and provided the maximum sugar and ethanol yields. These results signified that the microfluidization method is more effective on biomass at low solid loadings. The process conditions were optimized for higher ethanol and sugar yields using response surface methodology (RSM). The optimum pressure and solid and enzyme loadings were found as 1500 bar, 1%, and 15 FPU/g dry wheat straw, respectively. The yields obtained at this condition were 82%, 94%, and 65% for glucose, xylose, and ethanol, respectively. High sugar yields implied that microfluidization is an effective pretreatment method for cellulosic ethanol production. On the other hand, low ethanol yield may indicate that the microorganism was sensitive to inhibitory compounds present in the fermentation medium.     

Hydrothermal treatment of wheat straw at pilot plant scale using a three-step reactor system aiming at high hemicellulose recovery, high cellulose digestibility and low lignin hydrolysis

A pilot plant (IBUS) consisting of three reactors was used for hydrothermal treatment of wheat straw (120-150 kg/h) aiming at co-production of bioethanol (from sugars) and electricity (from lignin). The first reactor step was pre-soaking at 80 degrees C, the second extraction of hemicellulose at 170-180 degrees C and the third improvement of the enzymatic cellulose convertibility at 195 degrees C. Water added to the third reactor passed countercurrent to straw. The highest water addition (600 kg/h) gave the highest hemicellulose recovery (83%). With no water addition xylose degradation occurred resulting in low hemicellulose recovery (33%) but also in high glucose yield in the enzymatic hydrolysis (72 g/100g glucose in straw). Under these conditions most of the lignin was retained in the fibre fraction, which resulted in a lignin rich residue with high combustion energy (up to 31 MJ/kg) after enzymatic hydrolysis of cellulose and hemicellulose.     

Effect of compounds released during pretreatment of wheat straw on microbial growth and enzymatic hydrolysis rates

In the process of producing ethanol from lignocellulosic materials such as wheat straw, compounds that can act inhibitory to enzymatic hydrolysis and to cellular growth may be generated during the pretreatment. Ethanol production was evaluated on pretreated wheat straw hydrolysate using four different recombinant Saccharomyces cerevisiae strains, CPB.CR4, CPB.CB4, F12, and FLX. The fermentation performance of the four S. cerevisiae strains was tested in hydrolysate of wheat straw that has been pretreated at high dry matter content (220 g/L dry matter). The results clearly showed that F12 was the most robust strain, whereas the other three strains were strongly inhibited when the fraction of hydrolysate in the fermentation medium was higher than 60% (v/v). Furthermore, the impact of different lignin derivatives commonly found in the hydrolysate of pretreated wheat straw, was tested on two different enzyme mixtures, a mixture of Celluclast 1.5 L FG and Novozym 188 (3:1) and one crude enzyme preparation produced from Penicillium brasilianum IBT 20888. From all the potential inhibiting compounds that were tested, formic acid had the most severe influence on the hydrolysis rate resulting in a complete inactivation of the two enzyme mixtures.     

Effectiveness of microbial pretreatment by Ceriporiopsis subvermispora on different biomass feedstocks

Different types of feedstocks, including corn stover, wheat straw, soybean straw, switchgrass, and hardwood, were tested to evaluate the effectiveness of fungal pretreatment by Ceriporiopsis subvermispora. After 18-d pretreatment, corn stover, switchgrass, and hardwood were effectively delignified by the fungus through manganese peroxidase and laccase. Correspondingly, glucose yields during enzymatic hydrolysis reached 56.50%, 37.15%, and 24.21%, respectively, which were a 2 to 3-fold increase over those of the raw materials. A further 10-30% increase in glucose yields was observed when pretreatment time extended to 35 d. In contrast, cellulose digestibility of wheat straw and soybean straw was not significantly improved by fungal pretreatment. When external carbon sources and enzyme inducers were added during fungal pretreatment of wheat straw and soybean straw, only glucose and malt extract addition improved cellulose digestibility of wheat straw. The cellulose digestibility of soybean straw was not improved.     

Solid state fermentation of a Mycelia Sterilia laccase using steam-exploded wheat straw

Mycelia Sterilia YY-5, an endophytic fungus isolated from Rhus Chinensis Mill, was used in SSF for laccase production using steam-exploded wheat straw (SEWS). The fermentation period of YY-5 in solid state fermentation (SSF) shortened to 4 days compared with 5 days of submerged liquid fermentation (SmF) and the maximum laccase activity was 678.1 IU g⁻¹ substrate. The steam-explosion intensity (Log₁₀ R ₀) of SEWS had a significant effect on the growth of YY-5 and laccase activity, since SEWS could provide enough carbon source for YY-5 and inducers for laccase. The optimum SSF conditions using SEWS with Log₁₀ R ₀ = 3.597 as substrate were: inoculating with liquid inocula, keeping the solid-to-liquid ratio (S/L) for 1:4 and cultivating at 26°C. Under the optimum fermentation condition the laccase activity of YY-5 reached 849.5 ± 42.5 IU g⁻¹ substrate. The enzyme composition analysis indicated that laccase was the dominant enzyme of YY-5. Assayed with SDS-PAGE and active PAGE electrophoresis, the molecular weight of YY-5 laccase was approximately 45 kDa.