Effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pine

The effects of surfactants addition on enzymatic hydrolysis and subsequent fermentation of steam exploded lodgepole pine (SELP) and ethanol pretreated lodgepole pine (EPLP) were investigated in this study. Supplementing Tween 80 during cellulase hydrolysis of SELP resulted in a 32% increase in the cellulose‐to‐glucose yield. However, little improvement was obtained from hydrolyzing EPLP in the presence of the same amount of surfactant. The positive effect of surfactants on SELP hydrolysis led to an increase in final ethanol yield after the fermentation. It was found that the addition of surfactant led to a substantial increase in the amount of free enzymes in the 48 h hydrolysates derived from both substrates. The effect of surfactant addition on final ethanol yield of simultaneous saccharification and fermentation (SSF) was also investigated by using SELP in the presence of additional furfural and hydroxymethylfurfural (HMF). The results showed that the surfactants slightly increased the conversion rates of furfural and HMF during SSF process by Saccharomyces cerevisiae. The presence of furfural and HMF at the experimental concentrations did not affect the final ethanol concentration either. The strategy of applying surfactants in cellulase recycling to reduce enzyme cost is presented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effects of the surfactant tween 80 on enzymatic hydrolysis of newspaper

The effects of Tween 80 on the enzymatic hydrolysis of newspaper were tested. By monitoring sugar production it was found that the surfactant (0.1%) increased the rate and extent of cellulose saccharification. Consequently, the rate of enzyme usage in the hydrolysis reactor was improved by 33%. In addition, in the presence of surfactant the recovery of enzymes was higher. Analysis of the enzyme solution showed that with Tween 80 present larger fractions of enzyme remained in solution throughout hydrolysis. Thus, it appears that the surfactant hindered the immobilization of the enzymes on the substrates by reducing the strength of adsorption.     

Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper

Effects of five types of nonionic surfactant having a polyoxyethylene glycol (POG) group on enzymatic hydrolysis of used newspaper were studied. The surfactants examined in this work always enhanced the saccharification. The optimum surfactant concentration was 0.05% (wt/substrate wt) in the case of POG(21) sorbitane oleic ester. Among the surfactants, POG phenyl ether types showed the highest enhancement effect, for example, with two times higher conversion at 80 h than that without surfactant. Using POG nonylphenyl ether series, the effects of surfactant were considered from the point of the HLB (hydrophile-lypophile balance) value. The GFC (gel filtration chromatography) analysis of free enzyme quantity were also done to study the effect of surfactant on enzyme adsorption onto substrate. As the HLB value increased, the free enzyme quantity and the conversion both increased. It appears that surfactants help the enzyme to desorb from the binding site on the substrate surface after the completion of saccharification at that site.     

Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass

The switchgrass (SG) samples pretreated by cellulose solvent‐ and organic solvent‐based lignocellulose fractionation were characterized by enzymatic hydrolysis, substrate accessibility assay, scanning electron microscopy, X‐ray diffraction (XRD), cross polarization/magic angle spinning (CP/MAS) ¹³C nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). Glucan digestibility of the pretreated SG was 89% at hour 36 at one filter paper unit of cellulase per gram of glucan. Crystallinity index (CrI) of pure cellulosic materials and SG before and after cellulose solvent‐based pretreatment were determined by XRD and NMR. CrI values varied greatly depending on measurement techniques, calculation approaches, and sample drying conditions, suggesting that the effects of CrI data obtained from dried samples on enzymatic hydrolysis of hydrated cellulosic materials should be interpreted with caution. Fast hydrolysis rates and high glucan digestibilities for pretreated SG were mainly attributed to a 16.3‐fold increase in cellulose accessibility to cellulase from 0.49 to 8.0 m²/g biomass, because the highly ordered hydrogen‐bonding networks in cellulose fibers of biomass were broken through cellulose dissolution in a cellulose solvent, as evidenced by CP/MAS ¹³C‐NMR and FTIR. Biotechnol. Bioeng. 2011; 108:521–529. © 2010 Wiley Periodicals, Inc.     

Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques

Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodgepople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto cellulose surface of the set of pretreated substrates were also carried out using an in situ UV-Vis spectrophotometric technique. The cellulose accessibilities measured by the solute exclusion and a cellulose-binding module (CBM)-containing green fluorescent protein (TGC) adsorption methods correlate well for both sets of samples. The substrate enzymatic digestibilities (SEDs) of the hornified substrates are proportional to the measured cellulose accessibilities. Approximately over 90% of the SED was contributed by the accessible pore surfaces of the hornified substrates, suggesting that the substrate external surface plays a minor role contributing to cellulose accessibility and SED. The cellulose accessibilities of the pretreated substrates correlated well with the amounts of cellulase adsorbed. The SEDs of these substrates directly correlated with the amounts of adsorbed cellulase.     

Enzymatic hydrolysis of cellulose by the cellobiohydrolase domain of CelB from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus

The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-β-d-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80 °C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80 °C with half-lives of 23 h and 39 h, respectively, and retained >94% activity after 48 h at 70 °C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain.     

Energy requirements and process design considerations in compression-milling pretreatment of cellulosic wastes for enzymatic hydrolysis

The effectiveness of compression-milling pretreatment of lignocellulosics for enzymatic hydrolysis has been demonstrated for a wide variety of substrate sources. Reductions in the degree of crystallinity and the degree of polymerization of cellulose and partial destruction of the structural integrity of lignocellulosics brought about by compression milling significantly increase the susceptibility of cellulose to enzymatic hydrolysis. The enzymatic hydrolysis yield was found to be directly related to the specific energy input to the cellulosic substrate (kWh/1b substrate) by compression milling, and the energy input can be controlled by the milling time. The enzymatic hydrolysis yeilds from cellulosic materials pretreated by compression milling also vary significantly depending on the source and kind, the composition milling also vary significantly depending on the source and kind, the composition (contents of lignin and other components), and the structure. The power requirements for compression milling which renders equivalent hydrolysis yields also depend on the source and kind of lignocellulosics to be pretreated. For newspaper, the specific energy input required for 55% sugar yield is estimated as 0.3 kWh/lb substrate including 15% power loss. The additional sugar yield gained from the enzymatic hydrolysis of compression-milled newspaper (over and above the sugar yield of untreated substrate) is determined as 453 g sugar/kWh energy input.     

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5 FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively. However, when higher enzyme loadings were employed, the greater availability of the enzyme could overcome the limitations imposed by both the lignin’s restrictions on cellulose accessibility and direct binding of the enzymes, resulting in a near complete hydrolysis of the cellulose.     

乙醇预处理麦秆的酶解性能研究简

采用H2 SO4催化和自催化乙醇法对麦秆进行预处理,比较预处理后麦秆的主要化学组成、纤维素酶解性能和半同步糖化发酵生产乙醇特性,并进行物料衡算。结果表明：H2 SO4催化和自催化乙醇预处理过程中纤维素固体回收率大于90％。添加非离子表面活性剂吐温20和吐温80没有显著提高H2 SO4催化乙醇预处理后纤维素的酶解葡萄糖得率及半同步糖化发酵过程中乙醇的产量,而对自催化乙醇处理后麦秆的酶解和半同步糖化发酵过程有一定程度的促进作用,相应的酶解葡聚糖转化率由72.7％提高到85.0％,而半同步糖化发酵过程中乙醇质量浓度提高了11.4％。物料衡算结果表明：酸催化和自催化乙醇预处理后葡聚糖回收率分别为91.0％和95.4％;半同步糖化发酵生产乙醇的得率分别为10.4和11.6 g（按100 g原料计）。     

Effect of different non-ionic surfactants on the biodegradation of PAHs by diverse aerobic bacteria

The aim of this work was to evaluate the effect of several non-ionic surfactants (Tween-80, Triton X-100 and Tergitol NP-10) on the ability of different bacteria (Enterobacter sp., Pseudomonas sp. and Stenotrophomonas sp.) to degrade polycyclic aromatic hydrocarbons (PAHs). Bacterial cultures were performed at 25 °C in an orbital shaker under dark conditions in BHB medium containing 1% of surfactant and 500 mg l⁻¹ of each PAH. Experiments performed with Tween-80 showed the highest cell density values and maximum specific growth rate because this surfactant was used as a carbon source by all bacteria. High degree of PAHs degradation (>90%) was reached in 15 days in all experiments. Toxicity increased at early times using Tween-80 but decreased to low levels in a short time after the firsts 24 h. On the other hand, Triton X-100 and Tergitol NP-10 were not biodegraded and toxicity kept constant along time. However, PAHs-degradation rate was higher, especially by the action of Enterobacter sp. with Tween-80 or Triton X-100. Control experiments performed without surfactant showed a significant decrease in biomass growth rate with a subsequent loss of biodegradation activity likely due to a reduced solubility and bioavailability of PAHs in absence of surfactant.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.     

Cell-type-dependent enzymatic hydrolysis of palm residues: chemical and surface characterization of fibers and parenchyma cells

Chemical and surface characteristics of sulfite-pretreated royal palm sheath (RPS) fibers and parenchyma cells were investigated in order to study cell-type-dependent biomass hydrolysis by cellulase. Size, chemical composition, cellulose crystallinity and the exposure of cellulose microfibrils in pretreated RPS biomass affected the enzymatic accessibility and digestibility of different cell-type substrates.     

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.     

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.     

A minimal set of bacterial cellulases for consolidated bioprocessing of lignocellulose

Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step.     

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.     

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.     

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

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

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.     

Comparative studies on hydrothermal pretreatment and enzymatic saccharification of leaves and internodes of alamo switchgrass

Hydrothermal pretreatment was performed on the leaves and internodes portions of Alamo switchgrass, Panicum virgatum L., to enhance the digestibility of cellulose towards cellulase. It was observed that extractives free leaves portion provided 18.1% lower pretreatment gravimetrical yield and 33.8% greater cellulose-to-glucose yield than internodes portion. The degree of polymerization (DP) and ultrastructure of cellulose were determined by gel-permeation chromatography and solid-state cross polarization/magic angle spinning ¹³C NMR experiments. The results suggested that hydrothermal pretreatment hydrolyzed amorphous cellulose and yielded a product enriched in paracrystalline cellulose. Furthermore, the DP of cellulose was reduced to one third of the origin value after hydrothermal pretreatment. The resulting biomass after pretreatment for leaves and internodes has similar cellulose ultrastructure and chemical profiles. The results of the enzymatic hydrolysis studies of cellulose suggest that the reduced DP of cellulose of pretreated switchgrass was an important factor influencing the enhanced digestibility of pretreated switchgrass.