Optimisation of the biological pretreatment of wheat straw with white-rot fungi for ethanol production

The biological pretreatment of lignocellulosic biomass for the production of bioethanol is an environmentally friendly alternative to the most frequently used process, steam explosion (SE). However, this pretreatment can still not be industrially implemented due to long incubation times. The main objective of this work was to test the viability of and optimise the biological pretreatment of lignocellulosic biomass, which uses ligninolytic fungi (Pleurotus eryngii and Irpex lacteus) in a solid-state fermentation of sterilised wheat straw complemented with a mild alkali treatment. In this study, the most important parameters of the mechanical and thermal substrate conditioning processes and the most important parameters of the fungal fermentation process were optimised to improve sugar recovery. The largest digestibilities were achieved with fermentation with I. lacteus under optimised conditions, under which cellulose and hemicellulose digestibility increased after 21 days of pretreatment from 16 to 100 % and 12 to 87 %, respectively. The maximum glucose yield (84 %) of cellulose available in raw material was obtained after only 14 days of pretreatment with an overall ethanol yield of 74 % of the theoretical value, which is similar to that reached with SE.     

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.     

Recent process improvements for the ammonia fiber expansion (AFEX) process and resulting reductions in minimum ethanol selling price

The ammonia fiber expansion (AFEX) process has been shown to be an effective pretreatment for lignocellulosic biomass. Technological advances in AFEX have been made since previous cost estimates were developed for this process. Recent research has enabled lower overall ammonia requirements, reduced ammonia concentrations, and reduced enzyme loadings while still maintaining high conversions of glucan and xylan to monomeric sugars. A new ammonia recovery approach has also been developed. Capital and operating costs for the AFEX process, as part of an overall biorefining system producing fuel ethanol from biomass have been developed based on these new research results. These new cost estimates are presented and compared to previous estimates. Two biological processing options within the overall biorefinery are also compared, namely consolidated bioprocessing (CBP) and enzymatic hydrolysis followed by fermentation. Using updated parameters and ammonia recovery configurations, the cost of ethanol production utilizing AFEX is calculated. These calculations indicate that the minimum ethanol selling price (MESP) has been reduced from $1.41/gal to $0.81/gal.     

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.     

Dissolution of cellulose from AFEX-pretreated Zoysia japonica in AMIMCl with ultrasonic vibration

In this study, 1-allyl-3-methylimidazolium chloride (AMIMCl), an ionic liquid, was synthesized and characterized by a series of test methods. Pretreatment of Zoysia japonica by ammonia fiber expansion (AFEX) was shown to reduce significantly the mass of hemicellulose and lignin in biomass, thereby breaking the lignocellulosic structure. Z. japonica samples pretreated with AFEX showed reasonable solubility in AMIMCl upon ultrasonic treatment. The rate of cellulose regeneration from Z. japonica samples pretreated with AFEX increased with increase in applied power of ultrasonication within a certain power range from 0 to 110 W. The regeneration rate of cellulose from AFEX-pretreated Z. japonica reached a maximum of 97% when the ultrasonic power was 110 W. Fourier transform infrared spectroscopy and nuclear magnetic resonance analyses indicated that the regenerated cellulose was similar to microcrystalline cellulose.     

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.     

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.     

生物质生物预处理研究进展与展望

木质纤维素生物质分布广、产量大、可再生,用于制备生物基能源、生物基材料和生物基化学品。木质纤维素生物质组成复杂,包含纤维素、半纤维素和木质素等,木质素与半纤维素通过共价键、氢键交联形成独特的“包裹结构”,纤维素含有复杂的分子内与分子间氢键,上述因素制约着其资源化利用。生物预处理以其独特优越性成为生物质研究的重要方面。系统阐述了生物预处理过程中木质素降解和基团修饰对纤维素酶解的影响,纤维素含量及结晶区变化,半纤维素五碳糖利用,微观物理结构的改变。进一步提出了以生物预处理为核心的组合预处理、基于不同功能的多酶协同催化体系、木质纤维素组分分级利用和新型高效细菌预处理工艺是生物预处理未来发展的重要趋势。     

Thermal decomposition kinetics of wheat straw treated by Phanerochaete chrysosporium

Combining biological pretreatment with thermal processing may offer an alternative strategy for efficient conversion of lignocellulosic biomass into fuels and chemicals. The thermal decomposition kinetics of biologically pretreated wheat straw by Phanerochaete chrysosporium was investigated in this study using thermogravimetry (TG) - deconvoluted thermogravimetry (DTG) techniques and the Friedman method. This study revealed that biological pretreatment reduced the thermal degradation temperature of the biomass significantly. Relying on the thermal behavior of the biologically pretreated wheat straw, we proposed two biomass degradation phases during the biological degradation of wheat straw. The first phase of biodegradation (within 10 days of biological pretreatment) improved the efficiency of pyrolysis by reducing the temperature demand. In the second phase (after 10 days), although the efficiency of pyrolysis displayed the similar trend as the first phase, it showed a significant increase in activation energy demand. This process is greatly influenced by the residual lignin and cellulose ratios in the biomass. These experimental results will be useful in developing a biological pretreatment based thermochemical conversion process for lignocellulosic biomass.     

Evaluation of pretreatment methods for lignocellulosic ethanol production from energy cane variety L 79-1002

Approximately half of the 80 billion tons of crop produced annually around the world remains as residue that could serve as a renewable resource to produce valuable products such as ethanol and butanol. Ethanol produced from lignocellulosic biomass is a promising renewable alternative to diminishing oil and gas liquid fuels. Sugarcane is an important industry in Louisiana. The recently released variety of “energy cane” has great potential to sustain a competitive sugarcane industry. It has been demonstrated that fuel-grade ethanol can be produced from post harvest sugarcane residue in the past, but optimized ethanol production was not achieved. Optimization of the fermentation process requires efficient pretreatment to release cellulose and hemicellulose from lignocellulosic complex of plant fiber. Determining optimal pretreatment techniques for fermentation is essential for the success of lignocellulosic ethanol production process. The purpose of this study was to evaluate three pretreatment methods for the energy cane variety L 79-1002 for maximum lignocellulosic ethanol production. The pretreatments include alkaline pretreatment, dilute acid hydrolysis, and solid-state fungal pretreatment process using brown rot and white rot fungi. Pretreated biomass was enzymatically saccharified and subjected to fermentation using a recombinant Escherichia coli FBR5. The results revealed that all pretreatment processes produced ethanol. However, the best result was observed in dilute acid hydrolysis followed by alkaline pretreatment and solid-state fungal pretreatment.     

Rapid quantification of major reaction products formed during thermochemical pretreatment of lignocellulosic biomass using GC-MS

Accurate quantification of reaction products formed during thermochemical pretreatment of lignocellulosic biomass would lead to a better understanding of plant cell wall deconstruction for production of cellulosic biofuels and biochemicals. However, quantification of some process byproducts, most notably acetamide, acetic acid and furfural, present several analytical challenges using conventional liquid chromatography methods. Therefore, we have developed a high-throughput gas chromatography based mass spectrometric (GC-MS) method in order to quantify relevant compounds without requiring time-consuming sample derivatization prior to analysis. Solvent extracts of untreated, ammonia fiber expansion (AFEX) treated and dilute-acid treated corn stover were analyzed by this method. Biomass samples were extracted with acetone using an automated solvent extractor, serially diluted and directly analyzed using the proposed GC-MS method. Acetone was the only solvent amongst water, methanol and acetonitrile that did not contain detectable background levels of the target compounds or facilitate a buildup of plant-derived residues in the GC injector, which decreased analytical reproducibility. Quantitative results were based on the method of standard addition and external standard calibration curves.     

Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover

Ionic liquid (IL) and ammonia fiber expansion (AFEX) pretreatments were studied to develop the first direct side-by-side comparative assessment on their respective impacts on biomass structure, composition, process mass balance, and enzymatic saccharification efficiency. AFEX pretreatment completely preserves plant carbohydrates, whereas IL pretreatment extracts 76% of hemicellulose. In contrast to AFEX, the native crystal structure of the recovered corn stover from IL pretreatment was significantly disrupted. For both techniques, more than 70% of the theoretical sugar yield was attained after 48 h of hydrolysis using commercial enzyme cocktails. IL pretreatment requires less enzyme loading and a shorter hydrolysis time to reach 90% yields. Hemicellulase addition led to significant improvements in the yields of glucose and xylose for AFEX pretreated corn stover, but not for IL pretreated stover. These results provide new insights into the mechanisms of IL and AFEX pretreatment, as well as the advantages and disadvantages of each.     

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.     

Lignin Structural Alterations in Thermochemical Pretreatments with Limited Delignification

Lignocellulosic biomass has a complex and rigid cell wall structure that makes biomass recalcitrant to biological and chemical degradation. Among the three major structural biopolymers (i.e., cellulose, hemicellulose, and lignin) in plant cell walls, lignin is considered the most recalcitrant component and generally plays a negative role in the biochemical conversion of biomass to biofuels. The conversion of biomass to biofuels through a biochemical platform usually requires a pretreatment stage to reduce the recalcitrance. Pretreatment renders compositional and structural changes of biomass with these changes ultimately governing the efficiency of the subsequent enzymatic hydrolysis. Dilute acid, hot water, steam explosion, and ammonia fiber expansion pretreatments are among the leading thermochemical pretreatments with a limited delignification that can reduce biomass recalcitrance. Practical applications of these pretreatment are rapidly developing as illustrated by recent commercial scale cellulosic ethanol plants. While these thermochemical pretreatments generally lead to only a limited delignification and no significant change of lignin content in the pretreated biomass, the lignin transformations that occur during these pretreatments and the roles they play in recalcitrance reduction are important research aspects. This review highlights recent advances in our understanding of lignin alterations during these limited delignification thermochemical pretreatments, with emphasis on lignin chemical structures, molecular weights, and redistributions in the pretreated biomass.     

Pretreatments for Enhanced Enzymatic Hydrolysis of Pinewood: a Review

Pinewood is an abundant source of lignocellulosic biomass that has potential to be used as renewable feedstock in biorefineries for conversion into advanced biofuels and other value-added chemicals. However, its structural recalcitrance, due to the compact packing of its major components, viz. cellulose, hemicellulose and lignin, high lignin content, and high cellulose crystallinity, is a major bottleneck in its widespread use as a biorefinery feedstock. Typical chemical, thermal, and biological pretreatment technologies are aimed at removing lignin and hemicellulose fractions for improving enzyme accessibility and digestibility of cellulose. This review highlights common pine pretreatment procedures, associated key parameters and resulting enzymatic hydrolysis yields. The challenges and limitations are also discussed as well as potential strategies to overcome them, providing an essential source of information to realize pine as a compelling biorefinery biomass source.     

AFEX Pretreatment and Enzymatic Conversion of Black Locust (Robinia pseudoacacia L.) to Soluble Sugars

Black locust (Robinia pseudoacacia L.), like willow and poplar, is a hardwood species which can be grown in coppice for bioenergy production, and because of its nitrogen-fixing ability, it can be cultivated with higher yields on less productive land. For these experiments, we examined the feasibility of using ammonia fiber expansion (AFEX) pretreatment to increase the saccharification yields from black locust grown for bioethanol production, as well as examine the impact of posttreatments (hot-water washing and additional size reduction) on sugar yields. The optimal AFEX conditions for black locust were 180°C, 1.0?g NH₃/g dry biomass, 2.5?g H₂O/g dry biomass, for 30?min residence time, and of the parameters tested, temperature had the greatest impact on yields. Yields from the sample without posttreatment and hydrolyzed at the standard enzyme loading were very low: <30% glucose and ??50% hemicellulose. Both hot-water washing and size reduction improved yields; however, size reduction had a more significant effect indicating that increasing enzyme accessibility is more important for digestibility as opposed to the removal of soluble inhibitors. The effect of size reduction was comparable to that obtained by quadrupling the enzyme loading, increasing glucose yields by ??20?C30% and hemicellulose yields by ??20%. Untreated black locust is known to contain compounds which are inhibitory to both enzymes and microorganisms and AFEX pretreatment neutralizes this inhibitory effect to some extent.     

Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies

In order to investigate changes in substrate chemical and physical features after pretreatment, several characterizations were performed on untreated (UT) corn stover and poplar and their solids resulting pretreatments by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough, lime, and SO₂ technologies. In addition to measuring the chemical compositions including acetyl content, physical attributes determined were biomass crystallinity, cellulose degree of polymerization, cellulase adsorption capacity of pretreated solids and enzymatically extracted lignin, copper number, FT-IR responses, scanning electron microscopy (SEM) visualizations, and surface atomic composition by electron spectroscopy of chemical analysis (ESCA). Lime pretreatment removed the most acetyl groups from both corn stover and poplar, while AFEX removed the least. Low pH pretreatments depolymerized cellulose and enhanced biomass crystallinity much more than higher pH approaches. Lime pretreated corn stover solids and flowthrough pretreated poplar solids had the highest cellulase adsorption capacity, while dilute acid pretreated corn stover solids and controlled pH pretreated poplar solids had the least. Furthermore, enzymatically extracted AFEX lignin preparations for both corn stover and poplar had the lowest cellulase adsorption capacity. ESCA results showed that SO₂ pretreated solids had the highest surface O/C ratio for poplar, but for corn stover, the highest value was observed for dilute acid pretreatment with a Parr reactor. Although dependent on pretreatment and substrate, FT-IR data showed that along with changes in cross linking and chemical changes, pretreatments may also decrystallize cellulose and change the ratio of crystalline cellulose polymorphs (Iα/Iβ).     

Interactions between fungal growth, substrate utilization and enzyme production during shallow stationary cultivation of Phanerochaete chrysosporium on cotton stalks

Microbial pretreatment of lignocellulosic feedstocks is an environment friendly alternative to physio-chemical pretreatment methods. A better understanding of the interactive fungal mechanisms in biological systems is essential for enhancing performance and facilitating scale-up and commercialization of this pretreatment technique. In this study, mathematical models were developed for describing cellulose and hemicellulose consumption, lignin degradation, cellulase and ligninolytic enzyme production and oxygen uptake associated with the growth of Phanerochaete chrysosporium during a 14-day shallow stationary submerged fungal pretreatment process on cotton stalks. Model parameters were estimated and validated by Statistics Toolbox in MatLab 7.1. Models yielded sufficiently accurate predictions for cellulose and hemicellulose consumption (R2=0.9772 and 0.9837), lignin degradation (R2=0.9879 and 0.8682) and ligninolytic enzyme production (R2=0. 8135 and 0.9693) under both 1-day and 3-day oxygen flushing conditions, respectively. The predictabilities for fungal growth (R2=0.6397 and 0.5750) and cellulase production (R2=0.0307 and 0.3046) for 1-day and 3-day oxygen flushing, respectively, and oxygen uptake (R2=0.5435) for 3-day oxygen flushing were limited.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

The Influence of Hemicellulose and Lignin Removal on the Enzymatic Digestibility from Sugarcane Bagasse

Sugarcane bagasse (SCB) was pretreated with liquid hot water (LHW) and aqueous ammonia (AA), with the objective of investigating the influence of hemicellulose and lignin removal on the enzymatic digestibility and sugar recovery. The experimental results show that LHW and aqueous ammonia have a good performance in terms of hemicellulose dissolution and lignin removal respectively. The biggest xylan recovery of 74.3 % was obtained for LHW pretreatment at 160 °C, 5 %?w/v for 20 min with the xylan dissolution of 83.1 %. And the biggest lignin removal of 84.0 % was obtained for aqueous ammonia pretreatment at 160 °C, 10 %?w/v for 60 min. Moreover, the aperture and surface area of the sample were enlarged by the liquid hot water, which improves the accessibility of the substrate to the enzyme. The lignin removal caused by aqueous ammonia pretreatment can reduce the absorption of enzyme. In addition, the correlation between the compositional change and the enzymatic digestibility indicates that the removal of hemicellulose was more effective than lignin for destruction of the hemicellulose–lignin–cellulose structure.