The Role of Product Inhibition as a Yield-Determining Factor in Enzymatic High-Solid Hydrolysis of Pretreated Corn Stover

Industrially, enzymatic hydrolysis of lignocellulose at high solid content is preferable over low solids due to a reduction in processing costs. Unfortunately, the economic benefits are counteracted by a linear decrease in yield with solid content, referred to as the “solid effect” in the literature. In the current study, we investigate the contribution of product inhibition to the solid effect (7–33 % solids). Product inhibition was measured directly by adding glucose to high-solid hydrolysis samples and indirectly through variation of water content and beta-glucosidase concentration. The results suggest that the solid effect is mainly controlled by product inhibition under the given experimental conditions (washed pretreated corn stover as substrate). Cellobiose was found to be approximately 15 times more inhibitory than glucose on a molar scale. However, considering that glucose concentrations are at least 100 times higher than cellobiose concentrations under industrial conditions, glucose inhibition of cellulases is suggested to be the main cause of the solid effect.     

Bio-detoxification Bacteria Isolated from Dye-Polluted Soils Promote Lactic Acid Production from Ammonia Pretreated Corn Stover

Agro-stovers are the most abundant substrates for producing lactic acid, which has great potential application in the production of biodegradable and biocompatible polylactic acid polymers. However, chemical pretreatments on agro-stovers generate inhibitors that repress the subsequent lactic acid fermentation. In this study, three bacterial strains (Enterococcus faecalis B101, Acinetobacter calcoaceticus C1, and Pseudomonas aeruginosa CS) isolated from dye-polluted soils could utilize phenolic inhibitor mimics (vanillin, 4- hydroxybenzaldehyde, or syringaldehyde) from alkaline pretreated corn stovers as a sole carbon source. Lactic acid titer increased from 27.42 g/L (Bacillus coagulans LA204 alone) to 44.76 g/L (CS and LA204) using 50 g/L glucose with 1 g/L 4-hydroxybenzaldehyde added. Lactic acid production from 50 g/L ammonia pretreated corn stover was increased nearly twofold by inoculating phenolic degradation bacteria and lactic acid bacteria (C1& Lactobacillus pentosus FL0421). In the control (FL0421 alone), only 16.98 g/L of lactic acid was produced. The isolated and identified strains degraded the phenolic compounds and increased the lactic acid production from glucose and ammonia pretreated corn stover. These characteristics of the strains support industrial application with efficient in situ detoxification of phenolic compounds during lactic acid production from agro-stovers using simultaneous saccharification and fermentation (SSF).

     

微波辅助DMSO/AmimCl复合溶剂预处理玉米秸秆的酶解影响

为了实现玉米秸秆纤维素的高效糖化, 设计利用微波加热辅助的离子液体1-烯丙基-3-甲基咪唑氯盐(AmimCl)/二甲基亚砜(DMSO)复合溶剂生物质预处理体系, 破坏玉米秸秆天然结构, 提高纤维素酶解效率. 研究发现, 15% (w)DMSO, 110℃, 60 min 及4 g 秸秆/100 g 复合溶剂为最适预处理条件. 在此条件下, 秸秆溶解率、提取率可分别达46.6%和22.9%; 提取物纤维素酶解率14 h 可达71.4%, 相较于天然玉米秸秆的20 h 酶解率12.5%有极大提高. 通过XRD,SEM及¹H NMR 分析发现:秸秆预处理后, 提取物纤维素晶型由Ⅰ 型变为Ⅱ 型, 残渣纤维素相对结晶度明显降低, 有利于纤维素酶解的进行, 达到了生物质预处理的目的; 预处理过程中使用的AmimCl 离子液体经简单回收再生, 结构及秸秆溶解性能未发生变化, 可循环使用. 为玉米秸秆生物质预处理提供了一个新的方案.     

Fungal Pretreatment by Phanerochaete chrysosporium for Enhancement of Biogas Production from Corn Stover Silage

Corn stover silage (CSS) was pretreated by Phanerochaete chrysosporium in solid-state fermentation (SSF), to enhance methane production via subsequent anaerobic digestion (AD). Effects of washing of corn stover silage (WCSS) on the lignocellulosic biodegradability in the fungal pretreatment step and on methane production in the AD step were investigated with comparison to the CSS. It was found that P. chrysosporium had the degradation of cellulose, hemicellulose, and lignin of CSS up to 19.9, 32.4, and 22.6 %, respectively. Consequently, CSS pretreated by 25 days achieved the highest methane yield of 265.1 mL/g volatile solid (VS), which was 23.0 % higher than the untreated CSS. However, the degradation of cellulose, hemicellulose, and lignin in WCSS after 30 days of SSF increased to 45.9, 48.4, and 39.0 %, respectively. Surface morphology and Fourier-transform infrared spectroscopy analyses also demonstrated that the WCSS improved degradation of cell wall components during SSF. Correspondingly, the pretreatment of WCSS improved methane production by 19.6 to 32.6 %, as compared with untreated CSS. Hence, washing and reducing organic acids (such as lactic acid, acetic acid, propionic acid, and butyric acid) present in CSS has been proven to further improve biodegradability in SSF and methane production in the AD step.     

Enhancement of Enzymatic Hydrolysis and Klason Lignin Removal of Corn Stover Using Photocatalyst-Assisted Ammonia Pretreatment

Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0–12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO₂ and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20–1:50) were also tested. Ammonia pretreatment assisted by TiO₂-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid?=?1:20, photocatalyst/biomass?=?1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH₃-TiO₂-treated solid and 82 % for glucan and 77 % for xylan with NH₃-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2–13 % improvement over ammonia pretreatment alone.     

Catalytic Performance of Corn Stover Hydrolysis by a New Isolate <Emphasis Type="Italic">Penicillium</Emphasis> sp. ECU0913 Producing both Cellulase and Xylanase

A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml) and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding any accessory enzyme.     

Effect of Pretreatment Severity on Accumulation of Major Degradation Products from Dilute Acid Pretreated Corn Stover and Subsequent Inhibition of Enzymatic Hydrolysis of Cellulose

The concept of reaction severity, which combines residence time and temperature, is often used in the pulp and paper and biorefining industries. The influence of corn stover pretreatment severity on yield of sugar and major degradation products and subsequent effects on enzymatic cellulose hydrolysis was investigated. The pretreatment residence time and temperature, combined into the severity factor (Log R _o), were varied with constant acid concentration. With increasing severity, increasing concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) coincided with decreasing yields of oligosaccharides. With further increase in severity factor, the concentrations of furans decreased, while the formation of formic acid and lactic acid increased. For example, from severity 3.87 to 4.32, xylose decreased from 6.39 to 5.26?mg/mL, while furfural increased from 1.04 to 1.33?mg/mL; as the severity was further increased to 4.42, furfural diminished to 1.23?mg/mL as formate rose from 0.62 to 1.83?mg/mL. The effects of dilute acid hydrolyzate, acetic acid, and lignin, in particular, on enzymatic hydrolysis were investigated with a rapid microassay method. The microplate method gave considerable time and cost savings compared to the traditional assay protocol, and it is applicable to a broad range of lignocellulosic substrates.     

Optimization of Cellulase and Xylanase Productions by Streptomyces thermocoprophilus Strain TC13W Using Oil Palm Empty Fruit Bunch and Tuna Condensate as Substrates

The modified medium composed of the alkaline-pretreated oil palm empty fruit bunch (APEFB) and tuna condensate powder was used for cellulase and xylanase productions by Streptomyces thermocoprophilus strain TC13W. The APEFB contained 74.46% (w/w) cellulose, 15.72% (w/w) hemicellulose, and 6.40% (w/w) lignin. The tuna condensate powder contained 55.49% (w/w) protein and 11.05% (w/w) salt. In the modified medium with only 6.75 g/l tuna condensate powder, 10 g/l APEFB, and 0.5 g/l Tween 80, S. thermocoprophilus strain TC13W produced cellulase 4.9 U/ml and xylanase 9.0 U/ml. The enzyme productions in the modified medium were lower than cellulase (6.0 U/ml) and xylanase (12.0 U/ml) productions in the complex medium (CaCl₂ 0.1, MgSO₄·7H₂O 0.1, KH₂PO₄ 0.5, K₂HPO₄ 1.0, NaCl 0.2, yeast extract 5.0, NH₄NO₃ 1.0, Tween 80 0.5). When tuna condensate powder in the modified medium was reduced to 5.0 g/l and Tween 80 was increased to 1.5 g/l, S. thermocoprophilus strain TC13W produced cellulase and xylanase activities of 9.1 and 12.1 U/ml, respectively. This study shows that the cost of enzyme production could be reduced by using pretreated EFB and tuna condensate as a carbon and a nitrogen source, respectively.

     

Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment

An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.     

Isolation and Identification of Two New Fungal Strains for Xylanase Production

Fungi are well known for their ability to excrete enzymes into the environment. The aim of this work was to evaluate xylanase production by fungi isolated from soil. One hundred and thirty-six fungal isolates were screened for xylanase production. Two xylanase producing isolates, FSS117 and FSS129, were identified on the basis of analyses of 5,8S gene sequencing. The closest phylogenetic neighbors according to 5,8S gene sequence data for the two isolates were Aspergillus tubingensis and Aspergillus terreus, respectively. When birchwood xylan or corn cob hulls was used as a substrate for 5 days under submerged culture cultivation, xylanase production from A. terreus FSS129 was 113 and 174 IU ml^?1, respectively. The pH and temperature for optimum xylanase activity were 8 and 65?ºC.     

Two-Stage Fractionation of Corn Stover Using Aqueous Ammonia and Hot Water

Hot water and aqueous ammonia fractionation of corn stover were used to separate hemicellulose and lignin and improve enzymatic digestibility of cellulose. A two-stage approach was used: The first stage was designed to recover soluble lignin using aqueous ammonia at low temperature, while the second stage was designed to recover xylan using hot water at high temperature. Specifically, the first stage employed a batch reaction using 15 wt.% ammonia at 60 °C, in a 1:10 solid:liquid ratio for 8 h, while the second stage employed a percolation reaction using hot water, 190–210 °C, at a 20 ml/min flow rate for 10 min. After fractionation, the remaining solids were nearly pure cellulose. The two-stage fractionation process achieved 68% lignin purity with 47% lignin recovery in the first stage, and 78% xylan purity, with 65% xylan recovery in the second stage. Two-stage treatment enhanced the enzymatic hydrolysis of remaining cellulose to 96% with 15 FPU/g of glucan using commercial cellulase enzymes. Enzyme hydrolyses were nearly completed within 12–24 h with the remaining solids fraction.     

Influence of High Solid Concentration on Enzymatic Hydrolysis and Fermentation of Steam-Exploded Corn Stover Biomass

Steam-exploded corn stover biomass was used as the substrate for fed-batch separate enzymatic hydrolysis and fermentation (SHF) to investigate the solid concentration ranging from 10% to 30% (w/w) on the lignocellulose enzymatic hydrolysis and fermentation. The treatment of washing the steam-exploded material was also evaluated by experiments. The results showed that cellulose conversion changed little with increasing solid concentration, and fermentation by Saccharomyces cerevisiae revealed a nearly same ethanol yield with the water-washed steam-exploded corn stover. For the washed material at 30% substrate concentration, i.e., 30% water insoluble solids (WIS), enzymatic hydrolysis yielded 103.3 g/l glucose solution and a cellulose conversion of 72.5%, thus a high ethanol level up to 49.5 g/l. With the unwashed steam-exploded corn stover, though a cellulose conversion of 70.9% was obtained in hydrolysis at 30% solid concentration (27.9% WIS), its hydrolysate did not ferment at all, and the hydrolysate of 20% solid loading containing 3.3 g/l acetic acid and 145 mg/l furfural already exerted a strong inhibition on the fermentation and ethanol production.     

Xylanase Production by Bacillus circulans D1 Using Maltose as Carbon Source

Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbon sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that d-maltose is the best inducer of the enzyme synthesis (7.05 U/mg dry biomass at 48 h), while d-glucose and d-arabinose lead to the production of basal levels of xylanase. The crude enzyme solution is free of cellulases, even when the microorganism was cultivated in a medium with d-cellobiose. When oat spelt xylan was supplemented with d-glucose, the repressive effect of this sugar on xylanase production was observed at 24 h, only when used at 5.0 g/L, leading to a reduction of 60% on the enzyme production. On the other hand, when the xylan medium was supplemented with d-xylose (3.0 or 5.0 g/L), this effect was more evident (80 and 90% of reduction on the enzyme production, respectively). Unlike that observed in the xylan medium, glucose repressed xylanase production in the maltose medium, leading to a reduction of 55% on the enzyme production at 24 h of cultivation. Xylose, at 1.0 g/L, induced xylanase production on the maltose medium. On this medium, the repressive effect of xylose, at 3.0 or 5.0 g/L, was less expressive when compared to its effect on the xylan medium.     

Use of Spectroscopic and Imaging Techniques to Evaluate Pretreated Sugarcane Bagasse as a Substrate for Cellulase Production Under Solid-State Fermentation

The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7 %). However, the high crystallinity (74 %) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g^?1, respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access.     

Production of Cyclodextrins by CGTase from Bacillus clausii Using Different Starches as Substrates

Cyclodextrins (CDs) are cyclic oligasaccharides composed by d-glucose monomers joined by α-1,4-d glicosidic linkages. The main types of CDs are α-, β- and γ-CDs consisting of cycles of six, seven, and eight glucose monomers, respectively. Their ability to form inclusion complexes is the most important characteristic, allowing their wide industrial application. The physical property of the CD-complexed compound can be altered to improve stability, volatility, solubility, or bio-availability. The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme capable of converting starch into CD molecules. In this work, the CGTase produced by Bacillus clausii strain E16 was used to produce CD from maltodextrin and different starches (commercial soluble starch, corn, cassava, sweet potato, and waxy corn starches) as substrates. It was observed that the substrate sources influence the kind of CD obtained and that this CGTase displays a β-CGTase action, presenting a better conversion of soluble starch at 1.0%, of which 80% was converted in CDs. The ratio of total CD produced was 0:0.89:0.11 for α/β/γ. It was also observed that root and tuber starches were more accessible to CGTase action than seed starch under the studied conditions.     

Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production

The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82?±?2 mg/g CAB-H and 730?±?20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44?±?2 mg/g CAB-H, and 450?±?50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0?±?0.2 g L^?1 and 3.33 g L^?1 h^?1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L^?1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L^?1), ethanol concentration and productivity were 8.2?±?0.1 g L^?1 and 2.7 g L^?1 h^?1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.     

Rapid Determination of Cellulose and Hemicellulose Contents in Corn Stover Using Near-Infrared Spectroscopy Combined with Wavelength Selection

The contents of cellulose and hemicellulose (C and H) in corn stover (CS) have an important influence on its biochemical transformation and utilization. To rapidly detect the C and H contents in CS by near-infrared spectroscopy (NIRS), the characteristic wavelength selection algorithms of backward partial least squares (BIPLS), competitive adaptive reweighted sampling (CARS), BIPLS combined with CARS, BIPLS combined with a genetic simulated annealing algorithm (GSA), and CARS combined with a GSA were used to select the wavelength variables (WVs) for C and H, and the corresponding regression correction models were established. The results showed that five wavelength selection algorithms could effectively eliminate irrelevant redundant WVs, and their modeling performance was significantly superior to that of the full spectrum. Through comparison and analysis, it was found that CARS combined with GSA had the best comprehensive performance; the predictive root mean squared errors of the C and H regression model were 0.786% and 0.893%, and the residual predictive deviations were 3.815 and 12.435, respectively. The wavelength selection algorithm could effectively improve the accuracy of the quantitative analysis of C and H contents in CS by NIRS, providing theoretical support for the research and development of related online detection equipment.     

Production of Crude Cellulase and Xylanase From Trichoderma harzianum PPDDN10 NFCCI-2925 and Its Application in Photocopier Waste Paper Recycling

This paper implies production of cellulase and xylanase enzyme using a potent strain of Trichoderma harzianum for the efficient deinking of photocopier waste papers. Different nutritional and environmental factors were optimized for higher production of cellulase along with xylanase. After fermentation, maximum enzyme extraction was achieved from fermented matter using a three-step extraction process with increased efficiency by 26.6–29.3 % over single-step extraction. Static solid state was found as the best fermentation type using wheat bran (WB) as carbon source and ammonium ferrous sulfate (0.02 M) as nitrogen source. Subsequently, inoculum size (8?×?10⁶ CFU/gds), incubation days (4 days), temperature (34 °C), initial pH (6.0), and moisture ratio (1:3) significantly affected the enzyme production. Cellulase and xylanase activities were found to be maximum at pH 5.5 and temperature 55–60 °C with good stability (even up to 6 h). Furthermore, this crude enzyme was evaluated for the deinking of photocopier waste papers without affecting the strength properties with improved drainage as an additional advantage. The crude enzyme-deinked pulp showed 23.6 % higher deinking efficiency and 3.2 % higher brightness than chemically deinked pulp. Strength properties like tensile, burst indices, and folding endurance were also observed to improve by 6.7, 13.4, and 10.3 %, respectively, for enzyme-deinked pulp. However, the tear index was decreased by 10.5 %. The freeness of the pulp was also increased by 21.6 % with reduced drainage time by 13.9 %.     

Horticultural Waste as the Substrate for Cellulase and Hemicellulase Production by Trichoderma reesei Under Solid-State Fermentation

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), β-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and β-xylosidase (10.4 U/g SDM). The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than the commercial enzyme blend.