Hydrolysis of lignocellulosic materials for ethanol production: a review

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.     

Ethanol production from lignocellulosic biomass of energy cane

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum based liquid fuels. The release of many new sugarcane varieties by the United States Department of Agriculture to be used as energy crops is a promising feedstock alternative. Energy cane produces large amounts of biomass that can be easily transported, and production does not compete with food supply and prices because energy cane can be grown on marginal land instead of land for food crops. The purpose of this study was to evaluate energy cane for lignocellulosic ethanol production. Energy cane variety L 79-1002 was pretreated with weak sulfuric acid to remove lignin. In this study, 1.4 M sulfuric acid pretreated type II energy cane had a higher ethanol yield after fermentation by Klebsiella oxytoca without enzymatic saccharification than 0.8 M and 1.6 M sulfuric acid pretreated type II energy cane. Pretreated biomass was inoculated with K. oxytoca for cellulose fermentation and Pichia stipitis for hemicellulose fermentation under simultaneous saccahrification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) conditions. For enzymatic saccharification of cellulose, the cellulase and ??-glucanase cocktail significantly increased ethanol production compared to the ethanol production of fermented acid pretreated energy cane without enzymatic saccharification. The results revealed that energy cane variety L 79-1002 produced maximum cellulosic ethanol under SHF (6995 mg/L) and produced 3624 mg/L ethanol from fermentation of hemicellulosic sugars.     

Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products

Pulps with residual lignin ranging from 6.4-27.4% (w/w) were prepared from mixed softwoods using a proprietary biorefining technology (the Lignol process) based on aqueous ethanol organosolv extraction. The pulps were evaluated for bioconversion using enzymatic hydrolysis of the cellulose fraction to glucose and subsequent fermentation to ethanol. All pulps were readily hydrolyzed without further delignification. More than 90% of the cellulose in low lignin pulps (< or =18.4% residual lignin) was hydrolyzed to glucose in 48 h using an enzyme loading of 20 filter paper units/g cellulose. Cellulose in a high lignin pulp (27.4% residual lignin) was hydrolyzed to >90% conversion within 48 h using 40 filter paper units/g. The pulps performed well in both sequential and simultaneous saccharification and fermentation trials indicating an absence of metabolic inhibitors. Chemical and physical analyses showed that lignin extracted during organosolv pulping of softwood is a suitable feedstock for production of lignin-based adhesives and other products due to its high purity, low molecular weight, and abundance of reactive groups. Additional co-products may be derived from the hemicellulose sugars and furfural recovered from the water-soluble stream.     

Optimization of alkaline pretreatment of coffee pulp for production of bioethanol

The use of lignocellulosic raw materials in bioethanol production has been intensively investigated in recent years. However, for efficient conversion to ethanol, many pretreatment steps are required prior to hydrolysis and fermentation. Coffee stands out as the most important agricultural product in Brazil and wastes such as pulp and coffee husk are generated during the wet and dry processing to obtain green grains, respectively. This work focused on the optimization of alkaline pretreatment of coffee pulp with the aim of making its use in the alcoholic fermentation. A central composite rotatable design was used with three independent variables: sodium hydroxide and calcium hydroxide concentrations and alkaline pretreatment time, totaling 17 experiments. After alkaline pretreatment the concentration of cellulose, hemicellulose, and lignin remaining in the material, the subsequent hydrolysis of the cellulose component and its fermentation of substrate were evaluated. The results indicated that pretreatment using 4% (w/v) sodium hydroxide solution, with no calcium hydroxide, and 25 min treatment time gave the best results (69.18% cellulose remaining, 44.15% hemicelluloses remaining, 25.19% lignin remaining, 38.13 g/L of reducing sugars, and 27.02 g/L of glucose) and produced 13.66 g/L of ethanol with a yield of 0.4 g ethanol/g glucose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:451–462, 2014     

Metabolic engineering applications to renewable resource utilization

Lignocellulosic materials containing cellulose, hemicellulose, and lignin are the most abundant renewable organic resource on earth. The utilization of renewable resources for energy and chemicals is expected to increase in the near future. The conversion of both cellulose (glucose) and hemicellulose (hexose and pentose) for the production of fuel ethanol is being studied intensively, with a view to developing a technically and economically viable bioprocess. Whereas the fermentation of glucose can be carried out efficiently, the bioconversion of the pentose fraction (xylose and arabinose, the main pentose sugars obtained on hydrolysis of hemicellulose), presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize both glucose and pentoses, and convert them to useful compounds, such as ethanol. Metabolic strategies seek to generate efficient biocatalysts (bacteria and yeast) for the bioconversion of most hemicellulosic sugars to products that can be derived from the primary metabolism, such as ethanol. The metabolic engineering objectives so far have focused on higher yields, productivities and expanding the substrate and product spectra.     

A sustainable woody biomass biorefinery

Woody biomass is renewable only if sustainable production is imposed. An optimum and sustainable biomass stand production rate is found to be one with the incremental growth rate at harvest equal to the average overall growth rate. Utilization of woody biomass leads to a sustainable economy. Woody biomass is comprised of at least four components: extractives, hemicellulose, lignin and cellulose. While extractives and hemicellulose are least resistant to chemical and thermal degradation, cellulose is most resistant to chemical, thermal, and biological attack. The difference or heterogeneity in reactivity leads to the recalcitrance of woody biomass at conversion. A selection of processes is presented together as a biorefinery based on incremental sequential deconstruction, fractionation/conversion of woody biomass to achieve efficient separation of major components. A preference is given to a biorefinery absent of pretreatment and detoxification process that produce waste byproducts. While numerous biorefinery approaches are known, a focused review on the integrated studies of water-based biorefinery processes is presented. Hot-water extraction is the first process step to extract value from woody biomass while improving the quality of the remaining solid material. This first step removes extractives and hemicellulose fractions from woody biomass. While extractives and hemicellulose are largely removed in the extraction liquor, cellulose and lignin largely remain in the residual woody structure. Xylo-oligomers, aromatics and acetic acid in the hardwood extract are the major components having the greatest potential value for development. Higher temperature and longer residence time lead to higher mass removal. While high temperature (>200°C) can lead to nearly total dissolution, the amount of sugars present in the extraction liquor decreases rapidly with temperature. Dilute acid hydrolysis of concentrated wood extracts renders the wood extract with monomeric sugars. At higher acid concentration and higher temperature the hydrolysis produced more xylose monomers in a comparatively shorter period of reaction time. Xylose is the most abundant monomeric sugar in the hydrolysate. The other comparatively small amounts of monomeric sugars include arabinose, glucose, rhamnose, mannose and galactose. Acetic acid, formic acid, furfural, HMF and other byproducts are inevitably generated during the acid hydrolysis process. Short reaction time is preferred for the hydrolysis of hot-water wood extracts. Acid hydrolysis presents a perfect opportunity for the removal or separation of aromatic materials from the wood extract/hydrolysate. The hot-water wood extract hydrolysate, after solid-removal, can be purified by Nano-membrane filtration to yield a fermentable sugar stream. Fermentation products such as ethanol can be produced from the sugar stream without a detoxification step.     

Biomass pretreatment: fundamentals toward application

Development of sustainable energy systems based on renewable biomass feedstocks is now a global effort. Lignocellulosic biomass contains polymers of cellulose, hemicellulose, and lignin, bound together in a complex structure. Liquid biofuels, such as ethanol, can be made from biomass via fermentation of sugars derived from the cellulose and hemicellulose within lignocellulosic materials, but the biomass must be subjected to pretreatment processes to liberate the sugars needed for fermentation. Production of value-added co-products along-side biofuels through integrated biorefinery processes creates the need for selectivity during pretreatment. This paper presents a survey of biomass pretreatment technologies with emphasis on concepts, mechanism of action and practicability. The advantages and disadvantages, and the potential for industrial applications of different pretreatment technologies are the highlights of this paper.     

Bioethanol production from non-starch carbohydrate residues in process streams from a dry-mill ethanol plant

Slurries obtained from process streams in a starch-to-ethanol plant, Agroetanol AB in Norrk?ping, Sweden, were used to assess the potential increase in bioethanol yield if heat treatment followed by enzymatic hydrolysis were applied to the residual starch-free cellulose and hemicellulose fractions. The effects of different pretreatment conditions on flour (the raw material), the stream after saccharification of starch, before fermentation, and after fermentation were studied. The conditions resulting in the highest concentration of glucose and xylose in all streams were heat treatment at 130 degrees C for 40 min with 1% H(2)SO(4). Mass-balance calculations over the fermentation showed that approximately 64%, 54%, 75% and 67% of the glucan, xylan, galactan and arabinan, respectively, in the flour remained water insoluble in the process stream after fermentation without any additional treatment. Utilizing only the starch in the flour would theoretically yield 425 L ethanol per ton flour. By applying heat pretreatment to the water-insoluble material prior to enzymatic hydrolysis, the ethanol yield could be increased by 59 L per ton flour, i.e. a 14% increase compared with starch-only utilization, assuming fermentation of the additional pentose and hexose sugars liberated.     

Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations?

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.     

Pretreatments to enhance the digestibility of lignocellulosic biomass

Lignocellulosic biomass represents a rather unused source for biogas and ethanol production. Many factors, like lignin content, crystallinity of cellulose, and particle size, limit the digestibility of the hemicellulose and cellulose present in the lignocellulosic biomass. Pretreatments have as a goal to improve the digestibility of the lignocellulosic biomass. Each pretreatment has its own effect(s) on the cellulose, hemicellulose and lignin; the three main components of lignocellulosic biomass. This paper reviews the different effect(s) of several pretreatments on the three main parts of the lignocellulosic biomass to improve its digestibility. Steam pretreatment, lime pretreatment, liquid hot water pretreatments and ammonia based pretreatments are concluded to be pretreatments with high potentials. The main effects are dissolving hemicellulose and alteration of lignin structure, providing an improved accessibility of the cellulose for hydrolytic enzymes.     

玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化

木质纤维素高浓度还原糖水解液的获得是纤维乙醇产业化发展的方向。在发酵工业领域,分批补料法是实现这一目标的重要研究途径。本研究采用分批补料法对获得高浓度玉米秸秆酶解还原糖的条件进行了优化。以稀硫酸预处理的玉米秸秆为原料,考察了液固比、补加量与补加时间对分批补料糖化的影响。结果表明,秸秆高浓度酶解液条件的初始物料为20％ (重量/体积),木聚糖酶220 U/g (底物),纤维素酶6 FPU/g (底物),果胶酶50 U/g (底物),在24 h、48 h后分批补加8％预处理后的物料,同时添加与补料量相应的木聚糖酶20 U/g (底物),纤维素酶2 FPU/g (底物),72 h后,最终糖化结果与非补料法相比,还原糖浓度从48.5 g/L提高到138.5 g/L,原料的酶解率最终达到理论值的62.5％。试验结果表明补料法可以显著提高秸秆水解液还原糖浓度。     

Fusarium oxysporum: status in bioethanol production

Fermentation of lignocellulosic materials to ethanol and other solvents provides an alternative way of treating wastes and producing chemical feedstocks and fuel additives. Considerable efforts have been made in past 10 years to improve the process based on lignocellulosic biomass and hydrolysate that contains a complex mixture of sugars, decomposition products of sugars, and sometimes the inhibitory levels of soluble lignin. Despite the relative abundance of D-xylose in crop and forest residues it has not been found efficiently fermentable by most of the microorganisms. Recent research has revealed that D-xylose may be fermented to ethanol and organic acids. Recently, several strains of Fusarium oxysporum have been found to have potential for converting not only D-xylose, but also cellulose to ethanol in a one-step process. Distinguishing features of F. oxysporum for ethanol production in comparison to other organisms are identified. These include the advantage of in situ cellulase production and cellulose fermentation, pentose fermentation, and the tolerance of sugars and ethanol. The main disadvantage is the slow conversion rate when compared with yeast.     

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。     

Characterization of sugars from model and enzyme-mediated pulp hydrolyzates using high-performance liquid chromatography coupled to evaporative light scattering detection

High-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector was used to quantitatively determine glucose and cellobiose in hydrolyzates from the production of cellulose nanofillers from modified lignocellulosic materials. Prevail Carbohydrate ES 5 μ column proved more suitable for achieving the chromatographic separation of the model pulp hydrolyzate into its constituent sugars than the YMC-Pack Polyamine column. Linear calibration curves for the various sugars in the mixtures were developed. Glucose and cellobiose were clearly detectable in pulp hydrolyzates obtained from enzyme-mediated hydrolysis of recycled pulp, pine and hardwood dissolving pulps. Finally, the amount of glucose in the pulp hydrolyzates was generally higher than cellobiose.     

Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification

The production of fermentable sugars from olive tree biomass was studied by dilute acid pretreatment and further saccharification of the pretreated solid residues. Pretreatment was performed at 0.2%, 0.6%, 1.0% and 1.4% (w/w) sulphuric acid concentrations while temperature was in the range 170-210 degrees C. Attention is paid to sugar recovery both in the liquid fraction issued from pretreatment (prehydrolysate) and that in the water-insoluble solid (WIS). As a maximum, 83% of hemicellulosic sugars in the raw material were recovered in the prehydrolysate obtained at 170 degrees C, 1% sulphuric acid concentration, but the enzyme accessibility of the corresponding pretreated solid was not very high. In turn, the maximum enzymatic hydrolysis yield (76.5%) was attained from a pretreated solid (at 210 degrees C, 1.4% acid concentration) in which cellulose solubilization was detected; moreover, sugar recovery in the prehydrolysate was the poorest one among all the experiments performed. To take account of fermentable sugars generated by pretreatment and the glucose released by enzymatic hydrolysis, an overall sugar yield was calculated. The maximum value (36.3 g sugar/100 g raw material) was obtained when pretreating olive tree biomass at 180 degrees C and 1% sulphuric acid concentration, representing 75% of all sugars in the raw material. Dilute acid pretreatment improves results compared to water pretreatment.     

Bioethanol from fermentation of cassava pulp in a fibrous-bed bioreactor using immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense

There is an increasing worldwide interest in bioethanol production from agricultural, industrial, and urban residues for both ecological and economic reasons. The acid hydrolysis of cassava pulp to reducing sugars and their fermentation to ethanol were evaluated in a fibrousbed bioreactor with immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense. A maximum yield of total reducing sugars of 53.5% was obtained after 8 h of hydrolysis at 85oC in 0.4 mol/L hydrochloric acid with a solid-to-liquid ratio of 1:20, which was optimized by using an orthogonal design based on preliminary experiments. In the FBB, the fed-batch fermentation, using glucose as the sole carbon source, gave a maximum ethanol production of 38.3 g/L with a yield of 0.364 g/g in 100 h; whereas the fed-batch fermentation, using xylose as the sole carbon source, gave 34.1 g/L ethanol with a yield of 0.342 g/g in 135 h. When cassava pulp hydrolysate was used as a carbon source, 39.1 g/L ethanol with a yield of 0.123 g/g cassava pulp in185 h was observed, using the fed-batch fermentation model. In addition, for repeated batch fermentation of cassava pulp hydrolysate carried out in the fibrous-bed bioreactor, long-term operation with high ethanol yield and volumetric productivity were achieved. The above results show that the acid hydrolysate of cassava pulp can be used for ethanol production in a fibrous-bed bioreactor, although some inhibition phenomena were observed during the process of fermentation.     

Two-stage pretreatment of rice straw using aqueous ammonia and dilute acid

Liberation of fermentable sugars from recalcitrant lignocellulosic biomass is one of the key challenges in production of cellulosic ethanol. Here we developed a two-stage pretreatment process using aqueous ammonia and dilute sulfuric acid in a percolation mode to improve production of fermentable sugars from rice straw. Aqueous NH? was used in the first stage which removed lignin selectively but left most of cellulose (97%) and hemicellulose (77%). Dilute acid was applied in the second stage which removed most of hemicellulose, partially disrupted the crystalline structure of cellulose, and thus enhanced enzymatic digestibility of cellulose in the solids remaining. Under the optimal pretreatment conditions, the enzymatic hydrolysis yields of the two-stage treated samples were 96.9% and 90.8% with enzyme loadings of 60 and 15FPU/g of glucan, respectively. The overall sugar conversions of cellulose and hemicellulose into glucose and xylose by enzymatic and acid hydrolysis reached 89.0% and 71.7%, respectively.     

Isolation and characterization of a Trichodermastrain capable of fermenting cellulose to ethanol

The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1). This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol. Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol, >5.0 g l(-1), were obtained by the fermentation of glucose. Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobically) a wide range of carbon sources. In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.     

Bioethanol production from steam-exploded rice husk by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Rice husk is one of the most abundant types of lignocellulosic biomass. Because of its significant amount of sugars, such as cellulose and hemicellulose, it can be used for the production of biofuels such as bioethanol. However, the complex structure of lignocellulosic biomass, consisting of cellulose, hemicellulose and lignin, is resistant to degradation, which limits biomass utilization for ethanol production. The protection of cellulose by lignin contributes to the recalcitrance of lignocelluloses to hydrolysis. Therefore, we conducted steam-explosion treatment as pretreatment of rice husk. However, recombinant Escherichia coli KO11 did not ferment the reducing sugar solution obtained by enzymatic saccharification of steam-exploded rice husk. When the steam-exploded rice husk was washed with hot water to remove inhibitory substances and M9 medium (without glucose) was used as a fermentation medium, E. coli KO11 completely fermented the reducing sugar solution obtained by enzymatic saccharification of hot water washing-treated steam-exploded rice husk to ethanol. We report here the efficient production of bioethanol using steam-exploded rice husk.     

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose‐metabolizing pathways. In this study, a novel two‐stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non‐Saccharomyces microorganism and coupled to a glucose‐utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two‐stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae‐based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014