Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

Particle concentration and yield stress of biomass slurries during enzymatic hydrolysis at high‐solids loadings

Effective and efficient breakdown of lignocellulosic biomass remains a primary barrier for its use as a feedstock for renewable transportation fuels. A more detailed understanding of the material properties of biomass slurries during conversion is needed to design cost‐effective conversion processes. A series of enzymatic saccharification experiments were performed with dilute acid pretreated corn stover at initial insoluble solids loadings of 20% by mass, during which the concentration of particulate solids and the rheological property yield stress (τ_y) of the slurries were measured. The saccharified stover liquefies to the point of being pourable (τ_y ≤ 10 Pa) at a total biomass conversion of about 40%, after roughly 2 days of saccharification for a moderate loading of enzyme. Mass balance and semi‐empirical relationships are developed to connect the progress of enzymatic hydrolysis with particle concentration and yield stress. The experimental data show good agreement with the proposed relationships. The predictive models developed here are based on established physical principles and should be applicable to the saccharification of other biomass systems. The concepts presented, especially the ability to predict yield stress from extent of conversion, will be helpful in the design and optimization of enzymatic hydrolysis processes that operate at high‐solids loadings. Biotechnol. Bioeng. 2009; 104: 290–300 © 2009 Wiley Periodicals, Inc.     

青贮对柳枝稷制取燃料乙醇转化过程的影响

青贮是一种传统的生物质原料保存方法,广泛应用于纤维素乙醇炼制领域尚需要考察其对原料品质和下游乙醇转化过程的影响。文中以秋季(初、中和末)收割的柳枝稷为原料,通过青贮、高温水热(LHW)预处理、纤维素酶水解和同步糖化与发酵(SSF)实验对上述问题予以回答。结果显示,秋季初收割的柳枝稷以不同湿度青贮后pH均小于4.0,干重损失小于2%,各主要成分与青贮前相比无明显变化;LHW预处理中青贮样品半纤维素水解率普遍高于未贮存样品,但青贮同样使原料获得了更高的发酵抑制物产生水平;青贮柳枝稷葡萄糖、木糖和半乳糖产量(预处理+酶水解)高于未贮存柳枝稷;经过168 h的SSF,青贮样品乙醇浓度为12.1 g/L,未贮存的秋季初、秋季中和秋季末柳枝稷为底物的浓度分别为10.3 g/L、9.7 g/L和10.6 g/L。综上,青贮有助于提高柳枝稷LHW预处理效率、酶水解率和乙醇产量。     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Optimization of Dilute Acid Pretreatment and Enzymatic Hydrolysis of <Emphasis Type="Italic">Phalaris aquatica</Emphasis> L. Lignocellulosic Biomass in Batch and Fed-Batch Processes

Phalaris aquatica L., a rich in holocellulose (69.80 %) and deficient in lignin (6.70 %) herbaceous, perennial grass species, was utilized in a two-step (biomass pretreatment-enzymatic hydrolysis) saccharification process for sugars recovery. The Taguchi methodology was employed to determine the dilute acid pretreatment and enzymatic hydrolysis conditions that optimized hemicellulose conversion (75.04 %), minimized the production of inhibitory compounds (1.41 g/L), and maximized the cellulose to glucose yield (69.69 %) of mixed particulate biomass (particles <1000 μm) under batch conditions. The effect of biomass particle size on saccharification process efficiency was also investigated. It was found that small-size biomass particles (53–106 μm) resulted in maximum hemicellulose conversion (81.12 %) and cellulose to glucose yield (93.24 %). The determined optimal conditions were then applied to a combined batch pretreatment process followed by a fed-batch enzymatic hydrolysis process that maximized glucose concentration (62.24 g/L) and yield (92.48 %). The overall efficiency of the saccharification process was 88.13 %.     

Enhanced enzymatic hydrolysis of lignocellulosic biomass pretreated by low-pressure steam autohydrolysis

Summary The use of low-pressure steam autohydrolysis in the pretreatment of corn stover and hybrid poplar has been assessed. In terms of yield of prehydrolyzed solids, minimal by-product formation and extent of subsequent enzymatic saccharification, the results of low-pressure steam pretreatment were found to be as good as or better than those reported for more severe pretreatment processes. Almost complete saccharification of the cellulose in the prehydrolyzed biomass solids was obtained within 24h with a commercial cellulase preparation — Celluclast. The presence of grinding elements (glass beads) during the enzymatic hydrolysis was found to increase the extent of saccharification by 40% to 50% over controls without any grinding elements.     

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.     

Improving the fermentation performance of saccharomyces cerevisiae by laccase during ethanol production from steam‐exploded wheat straw at high‐substrate loadings

Operating the saccharification and fermentation processes at high‐substrate loadings is a key factor for making ethanol production from lignocellulosic biomass economically viable. However, increasing the substrate loading presents some disadvantages, including a higher concentration of inhibitors (furan derivatives, weak acids, and phenolic compounds) in the media, which negatively affect the fermentation performance. One strategy to eliminate soluble inhibitors is filtering and washing the pretreated material. In this study, it was observed that even if the material was previously washed, inhibitory compounds were released during the enzymatic hydrolysis step. Laccase enzymatic treatment was evaluated as a method to reduce these inhibitory effects. The laccase efficiency was analyzed in a presaccharification and simultaneous saccharification and fermentation process at high‐substrate loadings. Water‐insoluble solids fraction from steam‐exploded wheat straw was used as substrate and Saccharomyces cerevisiae as fermenting microorganism. Laccase supplementation reduced strongly the phenolic content in the media, without affecting weak acids and furan derivatives. This strategy resulted in an improved yeast performance during simultaneous saccharification and fermentation process, increasing significantly ethanol productivity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Variation in Cell Wall Composition and Accessibility in Relation to Biofuel Potential of Short Rotation Coppice Willows

Short rotation coppice (SRC) willow is currently emerging as an important dedicated lignocellulosic energy crop in the UK. However, investigation into the variation between species and genotypes in their suitability for liquid transport biofuel processing has been limited. To address this, four traits relevant to biofuel processing (composition, enzymatic saccharification, response to pretreatment and projected ethanol yields) were studied in 35 genotypes of willow including Europe’s leading SRC willow cultivars. Large, genotype-specific variation was observed for all four traits. Significant positive correlations were identified between the accessibility of glucan to enzymatic saccharification before and after pretreatment as well as glucose release and xylose release via acid hydrolysis during pretreatment. Of particular interest is that the lignin content of the biomass did not correlate with accessibility of glucan to enzymatic saccharification. The genotype-specific variations identified have implications for SRC willow breeding and for potential reductions in both the net energy expenditure and environmental impact of the lignocellulosic biofuel process chain. The large range of projected ethanol yields demonstrate the importance of feedstock selection based on an ideotype encompassing the performance of both field biomass growth and ease of conversion.     

A Weibull statistics‐based lignocellulose saccharification model and a built‐in parameter accurately predict lignocellulose hydrolysis performance

Renewable energy from lignocellulosic biomass has been deemed an alternative to depleting fossil fuels. In order to improve this technology, we aim to develop robust mathematical models for the enzymatic lignocellulose degradation process. By analyzing 96 groups of previously published and newly obtained lignocellulose saccharification results and fitting them to Weibull distribution, we discovered Weibull statistics can accurately predict lignocellulose saccharification data, regardless of the type of substrates, enzymes and saccharification conditions. A mathematical model for enzymatic lignocellulose degradation was subsequently constructed based on Weibull statistics. Further analysis of the mathematical structure of the model and experimental saccharification data showed the significance of the two parameters in this model. In particular, the λ value, defined the characteristic time, represents the overall performance of the saccharification system. This suggestion was further supported by statistical analysis of experimental saccharification data and analysis of the glucose production levels when λ and n values change. In conclusion, the constructed Weibull statistics‐based model can accurately predict lignocellulose hydrolysis behavior and we can use the λ parameter to assess the overall performance of enzymatic lignocellulose degradation. Advantages and potential applications of the model and the λ value in saccharification performance assessment were discussed.     

燃料乙醇发酵技术研究进展

在分析木质纤维素类生物质制备燃料乙醇原理基础上,重点对燃料乙醇转化过程的发酵工艺进行了论述。目前乙醇发酵工艺主要包括直接发酵、分步糖化发酵、同步糖化发酵、同步糖化共发酵和联合生物加工技术等,对这几种技术的研究现状进行了分析并对其发展趋势进行了展望,通过基因工程构建高效发酵菌种的联合生物加工技术将是未来高效发酵工艺的发展趋势,旨在为有效提高发酵菌株的底物代谢能力,获得高的乙醇产量提供重要参考。     

On-line biomass monitoring by capacitance measurement.

An in-situ, steam-sterilizable capacitance probe was used to follow the biomass concentration on-line, in bioreactors from 20 to 2000 l total volume. Microbial cultures of Saccharomyces cerevisiae, Pichia pastoris and Streptomyces virginiae were grown in batch and fed-batch culture in both defined and complex media in order to demonstrate the wide dynamic operating range of the instrument. A linear correlation was found between the on-line capacitance measurement and the off-line measurements (optical density, OD620; packed mycelial volume, PMV; biomass concentration X, and colony forming units, CFU ml-1) for biomass concentrations (dry cell weight) up to 30 g l-1 (St. virginiae), 106 g l-1 (S. cerevisiae) and 89 g l-1 (P. pastoris). The on-line capacitance measurement was slightly influenced by variations in agitation speed and strong extraneous radio frequencies. A specific capacitance constant (Cs) was defined for all microbial cells which was dependent on cell viability and cell size. The Cs was easy to calculate using the on-line capacitance measurement and an off-line estimation of biomass concentration. The Biomass Monitor proved suitable for precise on-line monitoring of both homogeneous (uni-cellular) and heterogeneous (mycelial) cultures in bioreactors.     

Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process

The biochemical conversion of cellulosic biomass to ethanol, a promising alternative fuel, can be carried out efficiently and economically using the simultaneous saccharification and fermentation (SSF) process. The SSF integrates the enzymatic hydrolysis of cellulose to glucose, catalyzed by the synergistic action of cellulase and beta-glucosidase, with the fermentative synthesis of ethanol. Because the enzymatic step determines the ethanol. Because the enzymatic step determines the availability of glucose to the ethanologenic fermentation, the kinetic of cellulose hydrolysis by cellulase and beta-glucosidase and the susceptibility of the two enzymes to inhibition by hydrolysis and fermentation products are of significant importance to the SSF performance and were investigated under realistic SSF conditions. A previously developed SSF mathematical model was used to conceptualize the depolymerization of cellulose. The model was regressed to the collected data to determine the values of the enzyme parameters and was found to satisfactorily predict the kinetics of cellulose hydrolysis. Cellobiose and glucose were identified as the strongest inhibitors of cellulase and beta-glucosidase, respectively. Experimental and modeling results are presented in light of the impact of enzymatic hydrolysis on fuel ethanol production. (c) 1993 Wiley & Sons, Inc.     

Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation

Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass‐to‐ethanol pipeline. Here, the feasibility of scaling‐up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H₂O₂/g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose‐ and xylose‐utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. 2012; 109:922–931. © 2011 Wiley Periodicals, Inc.     

QTL Mapping of Enzymatic Saccharification in Short Rotation Coppice Willow and Its Independence from Biomass Yield

Short rotation coppice (SRC) willows (Salix spp.) are fast-growing woody plants which can achieve high biomass yields over short growth cycles with low agrochemical inputs. Biomass from SRC willow is already used for heat and power, but its potential as a source of lignocellulose for liquid transport biofuels has still to be assessed. In bioethanol production from lignocellulose, enzymatic saccharification is used as an approach to release glucose from cellulose in the plant cell walls. In this study, 138 genotypes of a willow mapping population were used to examine variation in enzymatic glucose release from stem biomass to study relationships between this trait and biomass yield traits and to identify quantitative trait loci (QTL) associated with enzymatic saccharification yield. Significant natural variation was found in glucose yields from willow stem biomass. This trait was independent of biomass yield traits. Four enzyme-derived glucose QTL were mapped onto chromosomes V, X, XI, and XVI, indicating that enzymatic saccharification yields are under significant genetic influence. Our results show that SRC willow has strong potential as a source of bioethanol and that there may be opportunities to improve the breeding programs for willows for increasing enzymatic saccharification yields and biofuel production.     

Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars

Miscanthus x giganteus is a tall perennial grass whose suitability as an energy crop is presently being appraised. There is very little information on the effect of pretreatment and enzymatic saccharification of Miscanthus to produce fermentable sugars. This paper reports sugar yields during enzymatic hydrolysis from ammonia fiber expansion (AFEX) pretreated Miscanthus. Pretreatment conditions including temperature, moisture, ammonia loading, residence time, and enzyme loadings are varied to maximize hydrolysis yields. In addition, further treatments such as soaking the biomass prior to AFEX as well as washing the pretreated material were also attempted to improve sugar yields. The optimal AFEX conditions determined were 160 degrees C, 2:1 (w/w) ammonia to biomass loading, 233% moisture (dry weight basis), and 5 min reaction time for water-soaked Miscanthus. Approximately 96% glucan and 81% xylan conversions were achieved after 168 h enzymatic hydrolysis at 1% glucan loading using 15 FPU/(g of glucan) of cellulase and 64 p-NPGU/(g of glucan) of beta-glucosidase along with xylanase and tween-80 supplementation. A mass balance for the AFEX pretreatment and enzymatic hydrolysis process is presented.     

Real time monitoring biomass concentration in Streptomyces clavuligerus cultivations with industrial media using a capacitance probe

On-line monitoring biomass concentration in mycelial fed-batch cultivations of Streptomyces clavuligerus grown with soluble and partially insoluble complex media, was investigated with an in-situ capacitance probe fitted to an industrial pilot-plant tank. Standard off-line and on-line biomass determinations, including cell dry weight, packed mycelial volume, viscosity, DNA concentration and total CO(2) evolution in the exhaust gases, were performed throughout the experiments and compared to on-line capacitance measurements. Linear relations between capacitance and all other measurements were developed for both media that hold only in defined process phases, depending on the biomass state and the amount of insoluble matter present. For the industrial complex culture media good linear relations were obtained in the fast growth phase between capacitance and DNA concentration and total CO(2) evolution, while in the subsequent transition and stationary phases only with apparent viscosity was a reasonable correlation found. The capacitance probe was shown to be a valuable tool for real-time monitoring biomass concentration in industrial-like cultivation of mycelial streptomycetes.     

Immobilization of co-culture of Saccharomyces cerevisiae and Scheffersomyces stipitis in sodium alginate for bioethanol production using hydrolysate of apple pomace under separate hydrolysis and fermentation

Apple pomace as a substrate for bioethanol production is interesting due to its abundance and sustainable availability in varied states like Himachal Pradesh (H.P.), Jammu and Kashmir, Uttarakhand and Arunachal Pradesh, India. In the current study, apple pomace which is the main fruit industrial waste of H.P. was evaluated as feedstock for bioethanol production by the process of enzymatic saccharification using multiple carbohydrases. Microwave pretreatment of the apple pomace resulted in the efficient removal of lignin and crystalline structure of cellulose fibre. The enzymatic saccharification of the pretreated biomass was done by optimizing parameters for maximal saccharification leads to production of 27.50?mg/g of reduce, ng sugar. An enhanced ethanol yield of 44.46?g/l and fermentation efficiency of 58% by immobilized co-culture of Saccharomyces cerevisiae MTCC 3089 and Scheffersomyces stipitis NCIM 3498 under SHF as compared to fermentation performed with free yeast cells, i.e. 34.46?g/l of ethanol and 45% of fermentation efficiency.