The effect of high intensity mixing on the enzymatic hydrolysis of concentrated cellulose fiber suspensions

Enzymatic hydrolysis of lignocellulosic biomass in a high shear environment was examined. The conversion of cellulose to glucose in samples mixed in a torque rheometer producing shear flows similar to those found in twin screw extruders was greater than that of unmixed samples. In addition, there is a synergistic effect of mixing and enzymatic hydrolysis; mixing increases the rate of cellulose conversion while the increased conversion facilitates mixing. The synergy appears to result in part from particle size reduction, which is more significant when hydrolysis occurs during intense mixing.     

Application of an integrated statistical design to optimize the cold enzyme hydrolysis conditions for ethanol production

Cold enzyme hydrolysis was investigated on the ethanol production by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing. An integrated statistical design, which incorporated single factor design, response surface methodology (RSM) and weighting coefficient method, was used to determine the optimum hydrolysis conditions leading to maximum biomass, ethanol concentration and starch utilization ratio. After the studied ranges of α-amylase, glucoamylase and liquefaction time were identified by single factor design, RSM was used to further optimize the hydrolysis conditions for each objective. The results showed that, under hydrolysis condition optimized with RSM, biomass, ethanol concentration and starch utilization ratio reached 4.401 ± 0.042 × 10⁸ cells/ml, 14.81 ± 0.23% (wt.%) and 94.52 ± 0.53%, respectively. Finally, multi-objective optimization (MOO) was applied to obtain a compromised result of three desirable responses by weighting coefficient methodology. Biomass of 4.331 ± 0.038 × 10⁸ cells/ml, ethanol concentration of 14.12 ± 0.21% (wt.%) and starch utilization ratio of 92.88 ± 0.21% were simultaneous obtained when hydrolysis at pH 5.9 for 114 min with 233 IU/g_starch α-amylase and 778 IU/g_starch glucoamylase. The optimized conditions were shown to be feasible and reliable through verification tests.     

Evaluation of cellulase recycling strategies for the hydrolysis of lignocellulosic substrates

Recycling of cellulases should lower the overall cost of lignocellulosiic bioconversion processes. In this study, three recycling strategies were evaluated to determine their efficiencies over five successive rounds of hydrolysis. The effect of lignin on recycling was examined by comparing water-washed, steam-exploded birch (WB; 32% lignin) and WB which had been further extracted with alkali and peroxide (PB; 4% lignin). When the cellulases were recovered from the residual substrates after partial hydrolysis of both substrates, the recovered cellulase activity toward the mixture of fresh and residual substrates decreased after each recycling step. When the cellulases in the supernatants were also recycled, up to 20% more activity could be recovered. In both of these cases, the recovered activities did not correspond to the activities expected from the amount of cellulase protein recovered during recycling. The best recovery was obtained when the cellulases were recovered from both the residue and the supernatant after complete hydrolysis of the PB substrate. In this case, all of the originally added cellulase activity could be recovered for four consecutive hydrolysis rounds. However, when the same recycling strategy was carried out using the WB substrate, the recovered cellulase activity declined quickly with each recycling round. In all three of the recycling strategies, lower cellulase activities were recovered from the substrates with higher lignin contents. (c) 1995 John Wiley & Sons, Inc.     

Mushroom spent straw: a potential substrate for an ethanol-based biorefinery

Rice straw (RS) is an important lignocellulosic biomass with nearly 800 million dry tons produced annually worldwide. RS has immense potential as a lignocellulosic feedstock for making renewable fuels and chemicals in a biorefinery. However, because of its natural recalcitrance, RS needs thermochemical treatment prior to further biological processing. Ammonia fiber expansion (AFEX) is a leading biomass pretreatment process utilizing concentrated/liquefied ammonia to pretreat lignocellulosic biomass at moderate temperatures (70–140°C). Previous research has shown improved cellulose and hemicellulose conversions upon AFEX treatment of RS at 2:1 ammonia to biomass (w/w) loading, 40% moisture (dwb) and 90°C. However, there is still scope for further improvement. Fungal pretreatment of lignocellulosics is an important biological pretreatment method that has not received much attention in the past. A few reasons for ignoring fungal-based pretreatments are substantial loss in cellulose and hemicellulose content and longer pretreatment times that reduce overall productivity. However, the sugar loss can be minimized through use of white-rot fungi (e.g. Pleutorus ostreatus) over a much shorter duration of pretreatment time. It was found that mushroom spent RS prior to AFEX allowed reduction in thermochemical treatment severity, while resulting in 15% higher glucan conversions than RS pretreated with AFEX alone. In this work, we report the effect of fungal conditioning of RS followed by AFEX pretreatment and enzymatic hydrolysis. The recovery of other byproducts from the fungal conditioning process such as fungal enzymes and mushrooms are also discussed. JIMB-2008: BioEnergy—Special issue.     

Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood

The impact of oxidative modification and partial removal of lignin by laccase-mediator treatments on the enzymatic hydrolysis of steam-pretreated softwood (SPS) was evaluated. Two mediators, N-hydroxy-N-phenylacetamide (NHA) and its acetylated precursor, were oxidized by the laccase from Trametes hirsuta, and their effects on the activity of cellulolytic enzymes and on the hydrolysis yield of SPS were examined. Both simultaneous and sequential combinations of laccase-mediator treatments with commercial cellulases increased the sugar yield in the enzymatic hydrolysis of SPS. The maximal increase was 21% when a sequential treatment was applied. Laccase treatment alone was also shown to improve hydrolysis. NHA oxidized by laccase inhibited significantly the cellulases of Trichoderma reesei, but the presence of the solid substrate protected the activities against oxidative inactivation. Surface analysis of the lignocellulosic substrate before and after the laccase and cellulase treatments revealed an enrichment of lignin and an increase of carboxylic groups on the surface of the hydrolysis residue.     

Valorization of olive stones for xylitol and ethanol production from dilute acid pretreatment via enzymatic hydrolysis and fermentation by Pachysolen tannophilus

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195 °C for 5 min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40 FPU g⁻¹, while in the fermentation carried out with a cell concentration of 2 g L⁻¹ a yield of 0.44 g xylitol/g d-xylose and a global volumetric productivity of 0.11 g L⁻¹ h⁻¹ were achieved.     

Effect of Fenton's pretreatment on cotton cellulosic substrates to enhance its enzymatic hydrolysis response

Fenton’s reagent that generates reactive hydroxyl radical species was evaluated for its effectiveness as a pretreatment agent on cotton cellulosic substrates to increase its susceptibility to cellulase enzyme. Response surface methodology was used to optimize four different process variables viz., time of reaction; substrate size and concentrations of Fe²⁺ and H₂O₂. Overall, the cellulose substrates treated at 0.5 mM concentration of Fe²⁺, 2% concentration of H₂O₂ for a reaction period of 48 h gave the highest enzyme activity as determined using the response surface methodology. Cellulose substrates with high aspect ratio recorded better enzyme response than that with low aspect ratio which is supported by copper number estimation. The cellulosic substrate prepared using a combination of optimized Fenton’s pretreatment conditions and/or enzyme hydrolysis were studied and characterized by atomic force microscopy and scanning electron microscopy. Additionally, degree of polymerization analysis gives further insight into the degradation during Fenton’s reaction.     

家蚕肠道纤维素酶菌株的筛选及产酶条件的优化研究

从5龄家蚕肠道分离筛选得到一株产纤维素酶菌株BMC-2,以羧甲基纤维素酶(CMCase)比活力为响应值,通过Plackett-Burman试验设计、最陡爬坡试验和Box-Behnken试验设计对菌株BMC-2产纤维素酶发酵条件进行优化,结果表明,发酵时间、发酵温度、培养基初始pH和转速对CMCase比活力具有显著影响,其影响程度由大到小依次为发酵时间、培养基初始pH、发酵温度、转速.确定菌株BMC-2产纤维素酶最优发酵条件为:发酵时间94.35 h,发酵温度30.3℃,培养基初始pH 7.01,转速179 r/min.在此条件下, CMCase比活力理论值为25.801 U/mg,验证值为25.526 U/mg,较产酶条件优化前提高了1倍,预测模型可靠性高,可应用于菌株BMC-2产纤维素酶条件的优化.     

响应面法优化灰色链霉菌产纤维素酶的工艺研究

以灰色链霉菌为原料,在单因素试验的基础上,采用响应面法试验,优化灰色链霉菌产纤维素酶活性的发酵条件。结果表明,单因素试验灰色链霉菌产纤维素酶活性的最适发酵条件:碳源为CMC-Na,氮源为明胶,温度为28℃,pH为7.0,转速为130 r/min。响应面法试验优化灰色链霉菌产纤维素酶活性最佳发酵条件为:温度27.7℃,pH值6.9,转数130.3 r/min,在此优化条件下,灰色链霉菌产纤维素平均酶活性为6.103 U/mL(n=3),与模型的预测值(6.217 U/mL)比较接近,误差为1.83%,证明了该响应面模型具有可靠性。     

The potential bioavailability of organic C,N, and P through enzyme hydrolysis in soils of the Mojave Desert

Increases in the growth rate of plants and microbes in the Mojave Desert in response to predicted increases in precipitation and CO₂ due to global climate change may induce nutrient limitations. This study was designed to measure the pool of potentially bioavailable nutrients in soils of the Mojave Desert. Soils were collected from shrub and interspace microsites and then subjected to amendment with buffered solutions of an excess of various enzymes. The products of each enzyme reaction were then measured and the maximum quantity of hydrolyzable substrates was calculated. In interspace and shrub microsite soils, respectively, 14.5 and 9.7% of the organic C in the form cellulose, 60.0–97.8% and 61.2–100.0% of the organic N in the form protein, and 44.0 and 57.5% of the organic P was hydrolyzable. There were significant differences between microsites for hydrolyzable substrate using all enzyme amendments, except protease. We propose that accumulations of hydrolyzable organic C, N, and P in the Mojave Desert could be a result of the persistently dry soil conditions often found in desert ecosystems and the immobilization of enzymes, which may result in low diffusivity of soil substrates and enzymes and, accordingly, little degradation of organic C, N, and P. Alternatively, rapid nutrient cycling and immobilization by soil microorganisms could account for accumulations of organic C, N, and P. Further refinement of the methods used in this study could lead to a valuable tool for the assessment of potential bioavailability of nutrients in a variety of soils.     

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes.The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan.When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.     

Zero-waste biorefinery of oleaginous microalgae as promising sources of biofuels and biochemicals through direct transesterification and acid hydrolysis

Oleaginous microalgae are considered as promising sources of biofuels and biochemicals due to their high lipid content and other high-value components such as pigments, carbohydrate and protein. This study aimed to develop an efficient biorefinery process for utilizing all of the components in oleaginous microalgae. Acetone extraction was used to recover microalgal pigments prior to processes for the other products. Microalgal lipids were converted into biodiesel (fatty acid methyl ester, FAME) through a conventional two-step process of lipid extraction followed by transesterification, and alternatively a one-step direct transesterification. The comparable FAME yields from both methods indicate the effectiveness of direct transesterification. The operating parameters for direct transesterification were optimized through response surface methodology (RSM). The maximum FAME yield of 256 g/kg-biomass was achieved when using chloroform:methanol as co-solvents for extracting and reacting reagents at 1.35:1 volumetric ratio, 70 °C reaction temperature, and 120 min reaction time. The carbohydrate content in lipid-free microalgal biomass residues (LMBRs) was subsequently acid hydrolyzed into sugars under optimized conditions from RSM. The maximum sugar yield obtained was 44.8 g/kg-LMBRs and the protein residues were recovered after hydrolysis. This biorefinery process may contribute greatly to zero-waste industrialization of microalgae based biofuels and biochemicals.     

Statistical optimization of one-step immobilization process for recombinant endoglucanase from Clostridium thermocellum

Endoglucanase CelA from Clostridium thermocellum (CtCelA) is a thermophilic endo-β-1,4-glucanase and has a low solubility when expressed in Escherichia coli. To make industrial application of CtCeA more appealing, artificial oil bodies (AOBs) was implemented for one-step renaturation and immobilization of recombinant CtCelA. CtCelA was first fused with oleosin (Ole-CtCelA), a structural protein of plant seed oils. Ole-CtCelA was overexpressed in E. coli, and its insoluble form was recovered and mixed with plant oils to assemble AOBs. Moreover, the Box–Behnken design and the central composite design were employed to optimize the condition for assembly of AOBs and the enzymatic reaction condition, respectively. Consequently, the approach led to the resumption of active CtCelA on AOBs. CtCelA-bound AOBs exhibited an optimum activity at 69 °C and pH 6.3 while the immobilized protein remained stable for several hours at 70 °C and after 5 repeated uses. Overall, it indicates a promise of this novel approach for direct processing and immobilization of recombinant CtCelA.     

Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid

Enzymatic hydrolysis of pretreated lignocellulosic substrates has emerged as an interesting option to produce sugars that can be converted to liquid biofuels and other commodities using microbial biocatalysts. Lignocellulosic substrates are pretreated to make them more accessible to cellulolytic enzymes, but the pretreatment liquid partially inhibits subsequent enzymatic hydrolysis. The presence of pretreatment liquid from Norway spruce resulted in a 63% decrease in the enzymatic saccharification of Avicel compared to when the reaction was performed in a buffered aqueous solution. The addition of 15 mM of a reducing agent (hydrogen sulfite, dithionite, or dithiothreitol) to reaction mixtures with the pretreatment liquid resulted in up to 54% improvement of the saccharification efficiency. When the reducing agents were added to reaction mixtures without pretreatment liquid, there was a 13-39% decrease in saccharification efficiency. In the presence of pretreatment liquid, the addition of 15 mM dithionite to Avicel, α-cellulose or filter cake of pretreated spruce wood resulted in improvements between 25 and 33%. Positive effects (6-17%) of reducing agents were also observed in experiments with carboxymethyl cellulose and 2-hydroxyethyl cellulose. The approach to add reducing agents appears useful for facilitating the utilization of enzymes to convert cellulosic substrates in industrial processes.     

杂色云芝生物预处理对硬木和软木酶水解的影响

节能减排的生物预处理技术是促进木质纤维素酶水解转化乙醇的有效途径。本试验首次研究了白腐菌杂色云芝(Trametes vesicolor)生物预处理对柳木(Salix babylonica,硬木)和杉木(Cunninghamia lanceolata,软木)纤维素酶水解的影响及作用机制。结果显示生物预处理使硬木和软木的最终转化率分别增加4.78倍和4.02倍。通过研究酶与基质的相互作用发现,预处理后木材基质与酶亲和力的增强并不一定导致酶水解初始转化率的提高;但水解过程中转化速率的下降速度随着解吸附指数增加而降低,说明生物处理主要通过减少纤维素酶对基质的不可逆吸附,延缓水解过程中基质转化速率的急剧下降,从而提高水解效率。不可逆吸附的降低与预处理过程中木质素的部分降解与改性有一定关系。     

Enzymatic removal of plant residues from wool: Application of experimental design techniques for optimization parameters

This study was undertaken to find the optimum conditions of a new enzymatic process to remove plant residues from wool. Commercial enzymatic preparations of Celluclast 1.5 L and Pectinex Ultra SP-L were selected in order to hydrolyze the polysaccharides in primary plant cell walls and middle lamella, resulting into more fragile residues easier to be removed. Since it was intended to define the optimal conditions for enzyme application, a four-factor central composite design was selected to study the effects of pH, temperature, enzyme concentration and wetting agent concentration, on the two selected responses, i.e., soluble reducing sugars (RS) and alkali solubility (AS) of wool to detect plant degradation and to evaluate wool quality, respectively. Results demonstrated that enzyme concentration was the most significant effect in plant residues degradation. A total enzyme concentration loading of 20 mL of both diluted enzymatic preparations in equal parts per 1 L of incubation solution (42.970 U/L of Celluclast preparation and PG 29.3 nkat/L + PME 2.537 nkat/L of Pectinex preparation), yielded an equivalent amount of 240.127 mg of glucose per 1.0 g of plant residue, at the optimal conditions: 40.56 °C, pH 4.0 and 1 mL Plurafac/L. SEM analysis has indicated an identical and important degradation of the plant residues, when compared to the conventional carbonization process, and wool quality has been preserved.     

Biochar: a potential route for recycling of phosphorus in agricultural residues

Phosphorus (P) is a finite and dwindling resource, while an enormous amount of P flows to agricultural residues with increasing agricultural production. Therefore, the recycling of P in agricultural residues is critical for P sustainability in agricultural systems, which is dominated by the route of direct land application. Biochar production from agricultural residues and its subsequent land application have been suggested as solutions for waste biomass disposal, carbon sequestration, soil amendment/remediation, and crop production promotion. However, little attention has been paid to the contrasting effects of the land application of biochar vs. agricultural residues on the recycling of P accumulated in agricultural residues. Phosphorus in agricultural residues can be retained and transformed into stable forms of P in the resulting biochar. Thus, compared to agricultural residues, biochar provides lower amounts of labile P and releases its P more slowly while providing a long‐lasting P source, and the loss potential of P from biochar is reduced by low mobility of its P, indicating that biochar‐based P recycling route could substantially promote P recycling by acting as sustainable P source and diminishing the loss of P applied to soil.     

Isolation and characterization of Aspergillus sp. for the production of extracellular polysaccharides by response surface methodology

In this study, Aspergillus sp. was isolated for the production of extracellular polysaccharide. The process parameters were initially optimized by traditional methods. The cheap substrate, wheat bran was used for the production of extracellular polysaccharide in solid state fermentation. Supplementation of (1%, w/w) maltose, gelatin enhanced EPS production (5.36?mg/g). The salts such as, Cu²⁺ (4.9?mg/g), Ca²⁺ (3.5?mg/g), Zn²⁺ (2.9?mg/g), Mn²⁺ (3.4?mg/g) and Mg²⁺ (1.8?mg/g) stimulated EPS production. In two level full factorial experimental designs, the EPS yield varied from 3.18 to 11.65?mg/g wheat bran substrate with various combinations of the components supplemented with wheat bran substrate. Among these selected factors in central composite design, maltose significantly influenced on extracellular polysaccharide production.     

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L^?1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L^?1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397–406, 2017     

Improving the stability of uricase from Aspergillus flavus by osmolytes: Use of response surface methodology for optimization of the enzyme stability

Urate oxidase (UOX) is used for the treatment of hyperuricemia and gout. The low stability of protein drugs such as UOX is a significant drawback in their liquid solutions. In this study, UOX from Aspergillus flavus was overexpressed and purified. Then, the effect of osmolytes (glycerol, sorbitol, and sucrose) on UOX thermal stability was studied. The enzyme stability was optimized by the aid of osmolytes using response surface methodology (RSM). The thermal stability results and Pareto analysis showed that sucrose has a stronger stabilization effect on UOX relative to the other osmolytes and increases the enzyme stability up to 2.4-fold, solely. RSM results indicated that the stability of UOX increased up to 4.9-fold in the presence of 12 % of glycerol and sucrose. The combinatorial effect results indicated that the osmolytes affect each other stabilization effects on UOX, probably through influencing the hydrogen bonds and mixing the osmolytes at different concentrations does not always lead to increased stability of UOX. The results revealed that the effect of sorbitol on UOX stability was negligible in the presence of glycerol and sucrose. Finally, this study results showed that RSM can be used to select a proper set of osmolytes for enzyme stabilization.