Optimization of Bioethanol Production from Enzymatic Treatment of Argan Pulp Feedstock

Argan pulp is an abundant byproduct from the argan oil process. It was investigated to study the feasibility of second-generation bioethanol production using, for the first time, enzymatic hydrolysis pretreatment. Argan pulp was subjected to an industrial grinding process before enzymatic hydrolysis using Viscozyme L and Celluclast 1.5 L, followed by fermentation of the resulting sugar solution by Saccharomyces cerevisiae. The argan pulp, as a biomass rich on carbohydrates, presented high saccharification yields (up to 91% and 88%) and an optimal ethanol bioconversion of 44.82% and 47.16% using 30 FBGU/g and 30 U/g of Viscozyme L and Celluclast 1.5 L, respectively, at 10%_w/v of argan biomass.     

Rotation of Cellulose Ribbons During Degradation with Fungal Cellulase

Degradation of bacterial cellulose with a commercial cellulase, Celluclast 1.5L (Novo Nordisk), from the fungus Trichoderma reesei, causes a rotational movement of the cellulose microfibrils. Purified cellulases (CBH I, CBH II, and EG II) do not induce rotation of bacterial cellulose, however, ratios of CBH I and EG II do cause rotation of bacterial cellulose. Equimolar amounts of CBH I or CBH II and EG II do not result in motion during degradation. Based on these observations, we provide further evidence supporting, at least on theoretical grounds, the hypothesis that cellulose chains have intrinsic chirality. As the cellulase enzymes interact with and degrade the cellulose fibrils, the crystalline structure of the cellulose is altered, allowing the linear cellulose polymers to relax into a lower energy state, thus relieving the strain induced by crystallization of the nascent -glucan chains during the biogenesis of the microfibril. This conversion of crystalline bacterial ribbons into more relaxed conformations produces the rotation observed during the treatment of bacterial cellulose with cellulase.     

Comparison of Different Pretreatment Strategies for Enzymatic Hydrolysis of Wheat and Barley Straw

In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L from Trichoderma reesei, and a beta-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, approximately 48% (g g-1), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of approximately 39% (g g-1). Addition of extra enzyme (Celluclast 1.5 L+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32-39 g L-1, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.     

Effects of Substrate Loading on Enzymatic Hydrolysis and Viscosity of Pretreated Barley Straw

In this study, the applicability of a “fed-batch” strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a β-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.     

Effect of cationic surfactant cetyltrimethylammonium bromide on the enzymatic hydrolysis of cellulose

Effect of cationic surfactants alkyltrimethylammonium bromide (C_nTAB) with varied alkyl chain lengths on the enzymatic hydrolysis of Avicel and the surface charge of cellulase was investigated. Enzymatic hydrolysis of Avicel increased linearly from 42.1 to 61.4 % with the increase of the concentration of cetyltrimethylammonium bromide (C₁₆TAB) logarithmically from 0.0001 to 0.01 mM, and reached a maximum value at the concentration of 0.01–0.03 mM. When the concentration was increased further, the cellulase solution became positively charged and the enzymatic hydrolysis of Avicel decreased rapidly. With the increasing alkyl chain length, C_nTAB provided more proton and neutralized the negative charge of cellulase more obviously. Therefore, the required concentration of C_nTAB could be less to enhance the enzymatic hydrolysis of Avicel. In addition, C₁₆TAB could enhance enzymatic hydrolysis efficiency of corncob at high solid content from 35.0 to 56.3 %; C₁₆TAB could reduce about 60 % of the cellulase loading in the enzymatic hydrolysis of corncob to obtain the same glucose yield. Effect of C₁₆TAB on the enzymatic hydrolysis of typical pretreated softwood and hardwood was also investigated. This study laid the foundation for using C_nTAB to recover cellulase, and provided the design direction for cellulase with higher activity and better stability by adjusting its hydrophilicity and chargeability.     

The structural changes in crystalline cellulose and effects on enzymatic digestibility

The enzymatic hydrolysis of cellulose I achieves almost complete digestion when sufficient enzyme loading as much as 20 mg/g-substrate is applied. However, the yield of digestion reaches the limit when the enzyme dosage is decreased to 2 mg/g-substrate. Therefore, we have performed three pretreatments such as mercerization, dissolution into phosphoric acid and EDA treatment. Transformation into cellulose II hydrate by mercerization and dissolution into phosphoric acid were not sufficient because substrate changed to highly crystalline structure during saccharification. On the other hand, in the case of crystalline conversion of cellulose I to III_I by EDA, almost perfect digestion was achieved even in enzyme loading as small as 0.5 mg/g-substrate, furthermore, hydrolyzed residue was typical cellulose I. The structural analysis of substrate after saccharification provides an insight into relationships between cellulose crystalline property and cellulase toward better enzymatic digestion.     

Cellulase production of Trichoderma reesei rut C 30 using steam-pretreated spruce

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hem icellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and logen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C30. However, the conversion of cellulose using these enzymes was higher than that obtained with logen or Celluclast cellulases using steam-pretreated spruce as substrate.     

Enzymatic hydrolysis of different allomorphic forms of microcrystalline cellulose

This paper investigates the enzymatic hydrolysis of three main allomorphic forms of microcrystalline cellulose using different cellulases, from Trichoderma reesei and from Aspergillus niger, respectively. It was demonstrated that both the morphological and crystalline structures are important parameters that have a great influence on the course of the hydrolysis process. The efficiency of the enzymatic hydrolysis of cellulosic substrates was estimated by the amounts of reducing sugar and by the yield of the reaction. Changes in the average particle sizes of the cellulose allomorphs were determined during enzymatic hydrolysis. The accumulation of soluble sugar within the supernatant was used as a measure of the biodegradation process’s efficiency, and was established by HPLC-SEC analysis. Any modifications in the supramolecular structure of the cellulosic residues resulting from the enzymatic hydrolysis were determined by X-ray diffraction. The action of each cellulase was demonstrated by a reduction in the crystalline index and the crystallite dimensions of the corresponding allomorphic forms. The crystalline structure of allomorphic forms I and II did not suffer significant modifications, while cellulose III recorded a partial return to the crystalline structure of cellulose I. The microstructures of cellulose allomorph residues were presented using optical microscopy and scanning electron microscopy.     

Electron beam combined with hydrothermal treatment for enhancing the enzymatic convertibility of sugarcane bagasse

The use of microbial cellulolytic enzymes is the most efficient process to liberate glucose from cellulose in biomass without the formation of fermentation inhibitors. A combination of pretreatment technologies is an alternative way to increase the access of enzymes to cellulose, and consequently, the conversion yield. In this way, the present study reports on the enzymatic hydrolysis of SCB submitted to three kinds of pretreatment: electron beam processing (EBP), and EBP followed by hydrothermal (TH) and diluted acid (AH) treatment. SCB samples were irradiated using a radiation dynamics electron beam accelerator, and then submitted to thermal and acid (0.1% sulfuric acid) hydrolysis for 40 and 60 min at 180 °C. These samples were submitted to enzymatic hydrolysis (EH) using commercial preparations, including Celluclast 1.5 L and beta-glycosidase. The addition of diluted acid improved TH treatment allowing for a shorter application time. EBP with 50 kGy increased the enzymatic hydrolysis yield of cellulose by 20% after TH and 30% after AH.     

Improving enzymatic hydrolysis of industrial hemp (Cannabis sativa L.) by electron beam irradiation

The electron beam irradiation was applied as a pretreatment of the enzymatic hydrolysis of hemp biomass with doses of 150, 300 and 450 kGy. The higher irradiation dose resulted in the more extraction with hot-water extraction or 1% sodium hydroxide solution extraction. The higher solubility of the treated sample was originated from the chains scission during irradiation, which was indirectly demonstrated by the increase of carbonyl groups as shown in diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) spectra. The changes in the micro-structure of hemp resulted in the better response to enzymatic hydrolysis with commercial cellulases (Celluclast 1.5L and Novozym 342). The improvement in enzymatic hydrolysis by the irradiation was more evident in the hydrolysis of the xylan than in that of the cellulose.     

Hydrolysis behavior of various crystalline celluloses treated by cellulase of Tricoderma viride

Cellobiose and glucose are valuable products that can be obtained from enzymatic hydrolysis of cellulose. This study discusses changes in the crystalline form of celluloses to enhance the production of sugars and examines the effect on structural properties during enzymatic hydrolysis. Various crystalline celluloses consisting of group I (cell I, cell III_I, cell IV_I) and group II (cell II, cell III_II, cell IV_II) of similar DPs were prepared as starting materials. The similar DP values allowed a more direct comparison of the hydrolysis yields. The outcomes were analyzed and evaluated based on the residues and supernatants obtained from the treatment. As a result: (1) action of the cellulase of Trichoderma viride decreased both DP and crystallinity, with greater changes in group II celluloses, (2) the polymorphic interconversion process that occurred for cell III_I, cell IV_I, cell III_II and cell IV_II during the treatment was independent of the enzymatic hydrolysis, thus, the hydrolysis behaviors depended on the starting material of the celluloses, and (3) higher sugar production was obtained from cell III_I and group II. Therefore, the hydrolysis behavior of the various crystalline celluloses depended on the particular polymorph of the starting material.     

Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.     

Real-time observation of the swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei

The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.     

Understanding the effects of lignosulfonate on enzymatic saccharification of pure cellulose

The effects of lignosulfonate (LS) on enzymatic saccharification of pure cellulose were studied. Four fractions of LS with different molecular weight (MW) prepared by ultrafiltration of a commercial LS were applied at different loadings to enzymatic hydrolysis of Whatman paper under different pH. Using LS fractions with low MW and high degree of sulfonation can enhance enzymatic cellulose saccharification despite LS can bind to cellulase nonproductively. The enhancing effect varies with LS properties, its loading, and hydrolysis pH. Inhibitive effect on cellulose saccharification was also observed using LS with large MW and low degree of sulfonation. The concept of “LS-cellulase aggregate stabilized and enhanced cellulase binding” was proposed to explain the observed enhancement of cellulose saccharification. The concept was demonstrated by the linear correlation between the measured amount of bound cellulase and saccharification efficiency with and without LS of different MW in a range of pH.     

Effects of hemicellulose and lignin on enzymatic hydrolysis of cellulose from dairy manure

This study focused on the effect of hemicellulose and lignin on enzymatic hydrolysis of dairy manure and hydrolysis process optimization to improve sugar yield. It was found that hemicellulose and lignin in dairy manure, similar to their role in other lignocellulosic material, were major resistive factors to enzymatic hydrolysis and that the removal of either of them, or for best performance, both of them, improved the enzymatic hydrolysis of manure cellulose. This result combined with scanning electron microscope (SEM) pictures further proved that the accessibility of cellulose to cellulase was the most important feature to the hydrolysis. Quantitatively, fed-batch enzymatic hydrolysis of fiber without lignin and hemicellulose had a high glucose yield of 52% with respect to the glucose concentration of 17 g/L at a total enzyme loading of 1300 FPU/L and reaction time of 160 h, which was better than corresponding batch enzymatic hydrolysis.     

Effect of sodium dodecyl sulfate and cetyltrimethylammonium bromide catanionic surfactant on the enzymatic hydrolysis of Avicel and corn stover

Nonionic surfactants could effectively improve the enzymatic hydrolysis efficiency of lignocellulose, while small molecule anionic and cationic surfactants usually inhibited the enzymatic hydrolysis. The results showed that the anionic surfactant sodium dodecyl sulfate (SDS) could improve the enzymatic hydrolysis efficiency of Avicel at the concentration range of 0.1–1 mM, but it did inhibit enzymatic hydrolysis at higher concentration. Cationic surfactant cetyltrimethylammonium bromide (CTAB) was used to regulate the surface charge of SDS; thereby catanionic surfactant SDS-CTAB was formed. The effect of SDS-CTAB catanionic surfactant with varied molar ratios on the enzymatic hydrolysis of pure cellulose and corn stover at various enzymatic hydrolysis environments was investigated. SDS-CTAB could increase the enzymatic hydrolysis of corn stover at high solid loading from 33.3 to 42.4%. Using SDS-CTAB could reduce about 58% of the cellulase dosage to achieve 80% of the enzymatic hydrolysis of corn stover. SDS-CTAB catanionic surfactant could regulate the surface charge of cellulase in the hydrolyzate and reduce the non-productive adsorption of cellulase on the lignin, thereby improving the enzymatic hydrolysis efficiency of lignocellulose.     

The effect of agitation speed,enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 °C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2³ full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.     

Onsite Enzyme Production During Bioethanol Production from Biomass: Screening for Suitable Fungal Strains

Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5L used for hydrolysis of pre-treated biomass.     

Do cellulose binding domains increase substrate accessibility?

This article provides an overview of various theories proposed during the past five decades to describe the enzymatic hydrolysis of cellulose highlighting the major shifts that these theories have undergone. It also describes the effect of the cellulose-binding domain (CBD) of an exoglucanase/xylanase from bacterium Cellulomonas fimi on the enzymatic hydrolysis of Avicel. Pretreatment of Avicel with CBD_Cex at 4 and 37°C as well as simultaneous addition of CBD_Cex to the hydrolytic enzyme (Celluclast, Novo, Nordisk) reduced the initial rate of hydrolysis owing to irreversible binding of CBD proteins to the substrate's binding sites. Nonetheless, near complete hydrolysis was achieved even in the presence of CBD_Cex. Protease treatment of both pure and CBD_Cex-treated Avicel reduced the substrates' hydrolyzability, perhapsowing to proteolysis of the hydrolyzing enzyme (Celluclast) by the residual Proteinase K remaining in the substrate. Better protocols for comptete removal of CBD proteins from the substrate need to be developed to investigate the effect of CBD adsorption on cellulose digestibility.     

Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g^?1 cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g^?1 cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.