The effects of nonionic surfactants on the pretreatment and enzymatic hydrolysis of recycled newspaper

The effects of surfactants on the pretreatment and enzymatic hydrolysis stages of recycled newspaper processing were examined. Newspaper substrate was pretreated with surfactants at 40°C and 400 rpm for 1 h, and the enzymatic digestibilities of the pretreated substrate were compared. NP-20 was 10–20% more effective as a surfactant than Tween-20 and Tween-80. To investigate the effects of the surfactants on the subsequent enzymatic hydrolysis stage, the newspaper was pretreated with NP-20 and then hydrolyzed in the presence of TW-20 or TW-80. TW-80 showed an approximate 7% higher digestibility than TW-20. The surfactant effect on the hydrolysis of the untreated newspaper was significant, whereas the surfactant effect on the hydrolysis of the surfactant-pretreated newspaper was marginal. When the digestibilities of the pure cellulose substrates (α-cellulose and filter paper) were examined, markedly different surfactant effects were observed. In contrast to the newspaper substrate, the surfactant-pretreated pure cellulose substrates had a significant effect on digestibility when they were hydrolyzed in the presence of a surfactant, indicating that the surfactant effect on digestibility is highly dependent on substrate type.     

The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock

Despite recent improvement in cellulase enzymes properties, the high cost associated with the hydrolysis step remains a major impediment to the commercialization of full-scale lignocellulose-to-ethanol bioconversion process. As part of a research effort to develop a commercial process for bioconversion of softwood residues, we have examined the potential for recycling enzymes during the hydrolysis of mixed softwood substrate pretreated by organosolv process. We have used response surface methodology to determine the optimal temperature, pH, ionic strength, and surfactant (Tween 80) concentration for maximizing the recovery of bound protein and enzyme activity from the residual substrates after hydrolysis. Data analysis showed that the temperature, pH and surfactant concentration were the major factors governing enzyme desorption from residual substrate. The optimized conditions were temperature 44.4 °C, pH 5.3 and 0.5% Tween 80. The optimal conditions significantly increased the hydrolysis yield by 25% after three rounds of hydrolysis. This bound enzyme desorption combining with free enzyme re-adsorption is a potential method to recover cellulase enzymes and reduce the cost of enzymatic hydrolysis.     

Properties important for solid–liquid separations change during the enzymatic hydrolysis of pretreated wheat straw

Objectives

The biochemical conversion of lignocellulosic biomass into renewable fuels and chemicals provides new challenges for industrial scale processes. One such process, which has received little attention, but is of great importance for efficient product recovery, is solid–liquid separations, which may occur both after pretreatment and after the enzymatic hydrolysis steps. Due to the changing nature of the solid biomass during processing, the solid–liquid separation properties of the biomass can also change. The objective of this study was to show the effect of enzymatic hydrolysis of cellulose upon the water retention properties of pretreated biomass over the course of the hydrolysis reaction.

Results

Water retention value measurements, coupled with ¹H NMR T₂ relaxometry data, showed an increase in water retention and constraint of water by the biomass with increasing levels of cellulose hydrolysis. This correlated with an increase in the fines fraction and a decrease in particle size, suggesting that structural decomposition rather than changes in chemical composition was the most dominant characteristic.

Conclusions

With increased water retained by the insoluble fraction as cellulose hydrolysis proceeds, it may prove more difficult to efficiently separate hydrolysis residues from the liquid fraction with improved hydrolysis.

     

Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates) were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine). Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains per se, but rather the limited accessibility of the enzymes to the cellulose chains due to the physical structure of the cellulosic substrate.     

Biomass‐water interactions correlate to recalcitrance and are intensified by pretreatment: An investigation of water constraint and retention in pretreated spruce using low field NMR and water retention value techniques

The underlying mechanisms of the recalcitrance of biomass to enzymatic deconstruction are still not fully understood, and this hampers the development of biomass based fuels and chemicals. With water being necessary for most biological processes, it is suggested that interactions between water and biomass may be key to understanding and controlling biomass recalcitrance. This study investigates the correlation between biomass recalcitrance and the constraint and retention of water by the biomass, using SO₂ pretreated spruce, a common feedstock for lignocellulosic biofuel production, as a substrate to evaluate this relationship. The water retention value (WRV) of the pretreated materials was measured, and water constraint was assessed using time domain Low Field Nuclear Magnetic Resonance (LFNMR) relaxometry. WRV increased with pretreatment severity, correlating to reduced recalcitrance, as measured by hydrolysis of cellulose using commercial enzyme preparations. Water constraint increased with pretreatment severity, suggesting that a higher level of biomass‐water interaction is indicative of reduced recalcitrance in pretreated materials. Both WRV and water constraint increased significantly with reductions in particle size when pretreated materials were further milled, suggesting that particle size plays an important role in biomass water interactions. It is suggested that WRV may be a simple and effective method for measuring and comparing biomass recalcitrance. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:146–153, 2017     

Application of high throughput pretreatment and co‐hydrolysis system to thermochemical pretreatment. Part 1: Dilute acid

Because conventional approaches for evaluating sugar release from the coupled operations of pretreatment and enzymatic hydrolysis are extremely time and material intensive, high throughput (HT) pretreatment and enzymatic hydrolysis systems have become vital for screening large numbers of lignocellulosic biomass samples to identify feedstocks and/or processing conditions that significantly improve performance and lower costs. Because dilute acid pretreatment offers many important advantages in rendering biomass highly susceptible to subsequent enzymatic hydrolysis, a high throughput pretreatment and co‐hydrolysis (HTPH) approach was extended to employ dilute acid as a tool to screen for enhanced performance. First, a single‐step neutralization and buffering method was developed to allow effective enzymatic hydrolysis of the whole pretreated slurry. Switchgrass and poplar were then pretreated with 0.5% and 1% acid loadings at a 5% solids concentration, the resulting slurry conditioned with the buffering approach, and the entire mixture enzymatically hydrolyzed. The resulting sugar yields demonstrated that single‐step neutralizing and buffering was capable of adjusting the pH as needed for enzymatic saccharification, as well as overcoming enzyme inhibition by compounds released in pretreatment. In addition, the effects of pretreatment conditions and biomass types on susceptibility of pretreated substrates to enzymatic conversion were clearly discernible, demonstrating the method to be a useful extension of HTPH systems. Biotechnol. Bioeng. 2013; 110: 754–762. © 2012 Wiley Periodicals, Inc.     

Production and characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carbon source

Cellulases and hemicellulases are key enzymes in the production of alternative fuels and chemicals from lignocellulosic biomass-an abundant renewable resource. Carbon source selection is an important factor in the production of cellulases and hemicellulases. Rice straw--a potential ethanol source--has recently gained considerable interest in Asian countries. Here, we investigated the production of cellulases by using rice straw subjected to various pretreatments as substrates in order to produce cellulases at low costs; we also identified the enzymes' characteristics. Rice straw cutter milled to <3mm was pretreated by wet disk milling, dry ball milling, or hot-compressed water treatment (HCWT). Pretreated rice straw and commercial cellulose, Solka Floc (SF), were used as carbon sources for cellulase production by the fungus Acremonium cellulolyticus. Filter paper cellulase, β-xylanase, and β-xylosidase production from ball- and disk-milled samples were higher than those from SF. Enzymatic activity was absent in cultures where HCWT rice straw was used as carbon source. Wet disk-milled rice straw cultures were more suitable for enzymatic hydrolysis of pretreated rice straw than SF cultures. Thus, wet disk milling may be a suitable pretreatment for producing substrates for enzymatic hydrolysis and generating inexpensive carbon sources for cellulase production.     

Influence of solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive tree biomass

Olive tree pruning biomass, pretreated by either liquid hot water or steam explosion under selected conditions, was used as a substrate for enzymatic hydrolysis. The pretreated material was further submitted to alkaline delignification, the objective being to improve hydrolysis yields as well as increasing cellulose content in the pretreated feedstock. The enzymatic hydrolysis of pretreated residues was performed using a commercial cellulase mixture supplemented with β-glucosidase, using a solid loading range from 2 to 30% (w/v). The influence of substrate concentration on the enzymatic hydrolysis yield and on glucose concentration was studied. Comparative results with and without a delignification step are presented. Enzymatic hydrolysis at high substrate concentration (≥20%) is possible, yielding a concentrated glucose solution (>50 g/L). Nevertheless, a cellulose fraction of the pretreated residue remains unaltered.     

蒸汽爆破处理沙柳原料酶解条件优化

研究蒸汽爆破预处理对沙柳原料酶解效果的影响,通过响应曲面实验设计法优化蒸汽爆破处理沙柳原料的酶解工艺。结果表明,蒸汽爆破预处理沙柳原料的最佳蒸汽爆破处理条件：压力3.5 MPa、维压时间300 s; 蒸汽爆破最佳酶解条件：pH 4.8、温度53.5 ℃、 每克底物酶加量29.8 FPU。在最优条件下,蒸汽爆破处理沙柳原料的酶解率可以达到最大值87.92%,并验证了数学模型的有效性,试验结果表明蒸汽爆破预处理可以有效提高沙柳原料的水解率。     

Enriched arabinoxylan in corn fiber for value-added products

A two-step process is evaluated to separate the hexose component in wet milling corn fibers from the pentose component for production of value-added products. Corn fibers were first pretreated with hot water at 121°C for 1 h followed by glucoamylase hydrolysis to remove starch. The remaining solid was then treated with hot water at 140–170°C followed by an enzymatic hydrolysis to further separate the hexose and pentose components. After the second pretreatment, the enzymatic digestibility of cellulose was much better than that of arabinoxylan. As a result, up to 90% arabinoxylan in corn fibers was retained in a solid form after the enzyme hydrolysis, while most of the hexose components were removed.     

Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses

This study investigated the effect of fiber hornification during drying on lignocellulosic substrate enzymatic saccharification. Two chemically pretreated wood substrates and one commercial bleached kraft hardwood pulp were used. Heat drying at 105 and 150°C and air drying at 50% RH and 23.8°C for different durations were applied to produce substrate with various degrees of hornification. It was found that substrate enzymatic digestibilities (SEDs) of hornified substrates made from the same never-dried sample correlate very well to an easily measurable parameter, water retention value (WRV), and can be fitted by a Boltzmann function. The hornification-produced SED reduction at a given degree of hornification as the percentage of the total SED reduction when the substrate is completely hornified depends on two parameters. The first is WRVˉ, which is primarily a function of the effective enzyme molecule size, and Δ, which is related to the substrate pore size distribution shape. The low values of SED(CH), SED of a completely hornified substrate, obtained from curve fittings for the three sets of samples studied, suggest that enzyme accessibility to cellulose is mainly through the pores in the cell wall rather than substrate external surface. The SEDs of hornified substrates were found to correlate to Simons' staining measurements well. A new parameter was proposed to better correlate enzyme accessibility to cellulose using the two-color Simons' staining technique.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.     

斑茅酶解转化可发酵单糖的液氨预处理及参数优化

斑茅(Saccharum arundinaceum Retz.)的生物产量高,对土壤条件要求低,可作为纤维素乙醇生产的原料作物在我国南方地区广泛种植.实验以斑茅为原料,采用液氨预处理法克服其水解顽抗性,并添加纤维素酶进行酶解,运用高效液相色谱(HPLC)测定了酶解液中的单糖含量.实验结果表明在纤维素酶添加量为15FPU/(g当量葡聚糖)、预处理原料含水率为80％、预处理温度为130℃、预处理驻留时间为10 min、液氨与生物质的质量比例为2∶1时,葡聚糖和木聚糖的总转化率分别为69.34％和82.60％,相比于未作预处理的原料分别提高了573％和1 056％,单糖产量提高8倍.实验结果表明液氨预处理对斑茅是一种有效的预处理方式,并优于稀酸或湿爆法预处理,与酸预处理和氨爆法(AFEX)处理效果接近.     

The effect of bovine serum albumin on batch and continuous enzymatic cellulose hydrolysis mixed by stirring or shaking

Bovine serum albumin (BSA) was applied as a model non-catalytic protein to enzymatic hydrolysis of Avicel and dilute acid pretreated corn stover at different reaction conditions to improve the understanding of its ability to enhance cellulose hydrolysis. Addition of BSA improved the 72 h hydrolysis yields in shake flasks by up to 26% for both substrates by reducing de-activation of the exoglucanases and by facilitating reductions in particle size and crystallinity during a magnetically stirred pre-incubation step. The enzyme stabilizing effect of BSA addition was most striking for batch hydrolysis in a stirred tank reactor, with glucose yields increasing by 76% after 72 h for Avicel and by 40% after 145 h for corn stover. Application of BSA to continuous hydrolysis for a mean residence time of 24h gave 33% and 40% higher glucose yields for corn stover and Avicel compared to the controls.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

Enzyme adsorption on SO2 catalyzed steam-pretreated wheat and spruce material

Lignocellulose is widely recognized as a sustainable substrate for biofuels production, and the enzymatic hydrolysis is regarded as a critical step for the development of an effective process for the conversion of cellulose into ethanol. One key factor affecting the overall conversion rate is the adsorption capacity of the cellulase enzymes to the surface of the insoluble substrate. Pretreatment has a strong impact on hydrolysis, which could be related to both chemical changes and morphological changes of the material. In the current work, the accessibility of four differently pretreated wheat straw substrates, two differently pretreated spruce materials, and Avicel cellulose was investigated. Adsorption isotherms (at 4 °C and 30 °C) for a cellulase preparation were obtained, and the rates of hydrolysis were determined for the different materials. Furthermore, the surface area and pore size distribution of the various materials were measured and compared to adsorption and hydrolysis properties, and the structures of the pretreated materials were examined using scanning electron microscopy (SEM).The results demonstrated a positive correlation between enzyme adsorption and the substrate specific surface area within each feedstock. Overall, the amount of enzyme adsorbed was higher for pretreated spruce than for the pretreated wheat straw, but this was not accompanied by a higher initial rate of hydrolysis for spruce. Also, the difference in the measured endoglucanase adsorption and overall FPU adsorption suggests that a larger fraction of the enzyme adsorbed on spruce was unproductive binding. The SEM analysis of the material illustrated the structural effects of pretreatment harshness on the materials, and suggested that increased porosity explains the higher rate of hydrolysis of more severely pretreated biomass.     

Partial acid hydrolysis of cellulosic materials as a pretreatment for enzymatic hydrolysis

Partial acid hydrolysis was studied as a per treatment to enhance enzymatic hydrolysis, such a pretreatment was carried out in a continuous flow reactor on oak corn Stover, newsprint, and Solka Floc at temperatures ranging from 160 to 220°C, acid concentration ranging from 0 to 1.2%, and a fixed treatment time of 0.22 min. The resulting slurries and solids were than hydrolyzed with Trichoderma ressei QM 9414 cellulase at 50°C for 48 hr. For all substrates except Solka Floc, increased glucose yields were achieved during enzymatic hydrolysis of the pretreated materials as compared to hydrolysis of the original substrate. In several cases, after pretreatment, 100° of the potential glucose content of the substrate was converted to glucose after 24hr of enzymatic hydrolysis. It is felt that the increased glucose yields achieved after this pretreatment are due to acid's removal of hemicellulose, reduced degree of polymerization, and possibly due to a change in the crystal structure of the cellulose.     

The effect of isolated lignins,obtained from a range of pretreated lignocellulosic substrates,on enzymatic hydrolysis

The influence of the residual lignin remaining in the cellulosic rich component of pretreated lignocellulosic substrates on subsequent enzymatic hydrolysis was assessed. Twelve lignin preparations were isolated by two isolation methods (protease treated lignin (PTL) and cellulolytic enzymatic lignin (CEL)) from three types of biomass (corn stover, poplar, and lodgepole pine) that had been pretreated by two processes (steam and organosolv pretreatments). Comparative analysis of the isolated lignin showed that the CEL contained lower amounts of carbohydrates and protein than did the PTL and that the isolated lignin from corn stover contained more carbohydrates than did the lignin derived from the poplar and lodgepole pine. The lower yields of acid insoluble lignin (AIL) obtained from the corn stover when using the PTL method indicated that the lignin from the corn stover had a higher hydrophilicity than did the lignin from the poplar and lodgepole pine. The isolated lignin preparations were added to the reaction mixture containing crystalline cellulose (Avicel) and their possible effects on enzymatic hydrolysis were assessed. It was apparent that the lignin isolated from lodgepole pine and steam pretreated poplar decreased the hydrolysis yields of Avicel, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly. The hydrolysis yields of the pretreated lignocellulose and those of Avicel containing the PTL showed good correlation, indicating that the nature of the residual lignin obtained after pretreatment significantly influenced hydrolysis. Biotechnol. Bioeng. 2010;105: 871–879. © 2009 Wiley Periodicals, Inc.     

Enzymatic hydrolysis of cellulose and various pretreated wood fractions

Three strains of Trichoderma-T. reesei C30, T. reesei QM9414, and Trichoderma species E-58-were used to study the enzymatic hydrolysis of pretreated wood substrates. ach of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which had been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis.