Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.     

过程工程在木质纤维素发酵抑制物解除中的应用

发酵抑制物对宿主细胞产生毒害作用,是木质纤维素生物炼制的主要瓶颈之一。减少抑制物含量、解除抑制作用是提高发酵效率的重要环节。本文讨论了木质纤维素发酵抑制物的来源、组成、特点以及相应的解除方法,提出了"源头降低抑制物—纤维素木质素分级转化"炼制模式和"发酵促进剂设计技术",为木质纤维素发酵抑制物的解除及木质纤维素开发利用提供了全新的技术路线。     

Yeast strains for ethanol production from lignocellulosic hydrolysates during in situ detoxification

Yeast strains Y1, Y4 and Y7 demonstrated high conversion efficiencies for sugars and high abilities to tolerate or metabolize inhibitors in dilute-acid lignocellulosic hydrolysates. Strains Y1 and Y4 completely consumed the glucose within 24 h in dilute-acid lignocellulosic hydrolysate during in situ detoxification, and the maximum ethanol yields reached 0.49 g and 0.45 g ethanol/g glucose, equivalent to maximum theoretical values of 96% and 88.2%, respectively. Strain Y1 could metabolize xylose to xylitol with a yield of 0.64 g/g xylose, whereas Y4 was unable to utilize xylose as a substrate. Strain Y7 was able to consume sugars (glucose and xylose) within 72 h during hydrolysate in situ detoxification, producing a high ethanol yield (equivalent to 93.6% of the maximum theoretical value). Y1 and Y7 are the most efficient yeast strains yet reported for producing ethanol from non-detoxified dilute-acid lignocellulosic hydrolysates. These findings offer huge potential for improving the economics of bio-ethanol production from lignocellulosic hydrolysates.     

Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors

This work describes a novel approach to detoxify lignocellulosic hydrolysates and facilitate the analysis of inhibitory compounds, namely supercritical fluid extraction (SFE). The efficiency of the fermentation of lignocellulosic dilute-acid hydrolysates depends upon the composition of the hydrolysate and the organism used. Furthermore, it has been shown that inhibitors in the hydrolysate reduce the fermentation yield. This knowledge has given rise to the need to identify and remove the inhibiting compounds. Sample clean-up or work-up steps, to provide a clean and concentrated sample for the analytical system, facilitate the characterization of inhibitors, or indeed any compound in the hydrolysates. Removal of inhibitors was performed with countercurrent flow supercritical fluid extraction of liquid hydrolysates. Three different groups of inhibitors (furan derivatives, phenolic compounds, and aliphatic acids) and sugars were subsequently analyzed in the hydrolysate, extracted hydrolysate, and extract. The effect of the SFE treatment was examined with respect to fermentability with Saccharomyces cerevisiae. Not only did the extraction provide a clean and concentrated sample (extract) for analysis, but also a hydrolysate with increased fermentability as well as lower concentrations of inhibitors such as phenolics and furan derivatives.     

Sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust enzymatic saccharification of hardwoods

This study demonstrates sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust bioconversion of hardwoods. With only about 4% sodium bisulfite charge on aspen and 30‐min pretreatment at temperature 180°C, SPORL can achieve near‐complete cellulose conversion to glucose in a wide range of pretreatment liquor of pH 2.0–4.5 in only about 10 h enzymatic hydrolysis. The enzyme loading was about 20 FPU cellulase plus 30 CBU β‐glucosidase per gram of cellulose. The production of fermentation inhibitor furfural was less than 20 mg/g of aspen wood at pH 4.5. With pH 4.5, SPORL avoided reactor corrosion problem and eliminated the need for substrate neutralization prior to enzymatic hydrolysis. Similar results were obtained from maple and eucalyptus. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Potential of termite-based biomass pre-treatment strategies for use in bioethanol production

Abstract When considering the current state of the biorefinery industry, it is readily apparent that industrial cellulose and hemicellulose digestion processes are relatively advanced, whereas enzymatic pre-treatment strategies for biomass delignification and cellulose solubilization are not well developed. The need for efficient biomass pre-treatment strategies presents a significant opportunity for researchers studying lignocellulose digestion in termites and other insects. With an emphasis on industrial biomass pre-treatment, this review provides an overview of: (i) industrial biorefining operations (feedstocks, processing, and economics); (ii) recent findings from termite research that have revealed candidate enzymes; and (iii) research needs and opportunities for consideration by entomologists working in this area. With respect to research findings, recently identified candidate lignases (laccases, catalases, peroxidases, esterases), other potentially important detoxification enzymes (cytochrome P450, superoxide dismutase), and phenolic acid esterases (carboxylesterases) that may assist in hemicellulose solubilization are overviewed. Regarding research needs and opportunities, several approaches for identification of candidate pre-treatment enzymes from upstream, symbiont-free gut regions are also described.     

表面活性剂对木质纤维素酶解产糖的影响研究进展

木质纤维素是生产生物燃料乙醇的主要原料,其含量丰富、绿色环保以及可再生性,因此有效地利用木质纤维素有望解决能源短缺问题。表面活性剂能够有效地促进木质纤维素的酶解反应,通过探讨不同表面活性剂对酶解反应的影响及机理,为实际的酶解过程找到合适表面活性剂提供一定的理论指导。     

低水用量约束条件下的高固体含量纤维乙醇生物加工技术策略

木质纤维素原料生物转化生产纤维乙醇需要使用大量的水和蒸汽,从而使过程能耗和废水排放显著增加,大幅度增加了加工成本。最大限度地降低水和蒸汽用量对过程节能和废水减排并对最终成本控制极为重要。对极限低水用量约束条件下木质纤维素生物转化关键路径进行了实验研究和计算分析,确定了极低水和蒸汽用量的新型预处理技术,实现高效率预处理过程的废水零排放;采用独特的生物脱毒技术,用从自然界筛选的煤油霉菌Amorphotheca resinae ZN1对预处理原料中的抑制物进行了快速生物脱毒;对极限高固体含量下高粘度多相流物系在复杂抑制物胁迫下的酶水解与发酵行为以及放大准则进行了研究;建立了基于Aspen plus平台上的生物质加工物性数据库和严格热力学意义上的全过程流程模拟数学模型,实现了对过程的局部和全局设计与调优。这一综合技术在生物炼制微型工厂中进行了测试,并在纤维素乙醇工业示范装置中得到了应用。该研究结果将为构建具有工业实用价值的节能和清洁化木质纤维素生物转化技术提供依据。     

微生物降解利用木质纤维素的协同作用

木质纤维素复杂的结构组成,是制约高效降解利用这一资源、发展生物炼制的瓶颈。微生物的多酶(菌)体系可有效降解木质纤维素。除好氧微生物的游离酶协同系统之外,主要存在于厌氧细菌中的纤维小体也是有序、高效的协同降解纤维素的复合体系。近年来,在天然纤维小体研究的基础上,研究者们成功设计、构建了人工纤维小体,加深了对这一复合体系的组成单元的理性认识。另外,菌群共培养技术利用各组成菌株代谢途径的协同作用实现了木质纤维素的高效降解。最后,引入异源纤维素酶,可改造现有工程菌株的代谢网络,提高工程菌发酵生产终产物的能力。这些技术有利于实现一步转化生产乙醇的联合生物工艺,有助于提高生物炼制的产率、降低生产成本。     

蓝细菌乙醇光合细胞工厂的发展与展望

生物乙醇是极具应用潜力和代表性的生物能源产品之一。以蓝细菌为光合平台,利用二氧化碳和太阳能直接进行乙醇合成可以同时起到降低二氧化碳排放和提供可再生能源的效果,具有重要的研究与应用价值。本文回顾了蓝细菌乙醇光合细胞工厂相关技术的发展历程和现状,从途径优化、底盘选择和代谢工程策略等层面对其最新进展和所遇到的问题进行了总结介绍,并对该技术未来发展方向进行了展望。     

相容溶质四氢嘧啶的微生物合成研究进展北大核心CSCD

四氢嘧啶(ectoine)作为一种氨基酸的衍生物,是嗜盐微生物胞内重要的天然次级代谢物,具有保护细胞和稳定生物大分子的功能,可广泛应用于药物制备佐剂、器官移植与保存、皮肤创伤修复与新型化妆品研发等生物医学领域。由于四氢嘧啶的医用价值和商业市场需求,文中从野生菌株的筛选与诱变育种、构建基因工程菌株与系统代谢整合工程菌株、四氢嘧啶的优化发酵与生产工艺以及抽提纯化工艺等方面,系统论述了四氢嘧啶高效积聚和过量化生产研究策略,并展望多组学、计算生物学及“细胞工厂”等技术在后续四氢嘧啶高效生产上的应用前景。     

微生物木糖发酵产乙醇的代谢工程

利用木质纤维素发酵生产乙醇具有广泛的应用前景。而自然界中缺少有效转化木糖为乙醇的微生物是充分利用纤维素水解产物、提高乙醇产率、降低生产成本的关键因素。多年来研究者利用分子生物学技术对微生物菌株进行了代谢工程改造,使其能更有效地利用木糖生产乙醇。以下主要对运动发酵单胞菌、大肠杆菌和酵母等候选产乙醇微生物的木糖代谢工程研究进展进行了概述。     

Biotechnological approaches to overcome hybridization barriers and use of micropropagation tool for further improvement in Heliconia: a review

Plant Cell, Tissue and Organ Culture (PCTOC) - Heliconia, also known as ‘False-Bird-of-Paradise’, is a genus of flowering plants in the monotypic family of Heliconiaceae.The genus...     

Potential inhibitors from wet oxidation of wheat straw and their effect on ethanol production of Saccharomyces cerevisiae: wet oxidation and fermentation by yeast

Alkaline wet oxidation (WO) (using water, 6.5 g/L sodium carbonate and 12 bar oxygen at 195 degrees C) was used as pretreatment method for wheat straw (60 g/L), resulting in a hydrolysate and a cellulosic solid fraction. The hydrolysate consisted of soluble hemicellulose (8 g/L), low-molecular-weight carboxylic acids (3.9 g/L), phenols (0.27 g/L = 1.7 mM) and 2-furoic acid (0.007 g/L). The wet oxidized wheat straw hydrolysate caused no inhibition of ethanol production by Saccharomyces cerevisiae ATCC 96581. Nine phenols and 2-furoic acid, identified to be present in the hydrolysate, were each tested in concentrations of 50-100 times the concentration found in the hydrolysate for their effect on fermentation by yeast. At these high concentrations (10 mM), 4-hydroxybenzaldehyde, vanillin, 4-hydroxyacetophenone and acetovanillone caused a 53-67% decrease in the volumetric ethanol productivity in S. cerevisiae compared to controls with an ethanol productivity of 3.8 g/L. The phenol acids (4-hydroxy, vanillic and syringic acid), 2-furoic acid, syringaldehyde and acetosyringone were less inhibitory, causing a 5-16% decrease in ethanol productivity. By adding the same aromatic compounds to hydrolysate (10 mM), it was shown that syringaldehyde and acetovanillone interacted negatively with hydrolysate components on the ethanol productivity. Fermentation in WO hydrolysate, that had been concentrated 6 times by freeze-drying, lasted 4 hours longer than in regular hydrolysate; however, the ethanol yield was the same. The longer fermentation time could not be explained by an inhibitory action of phenols alone, but was more likely caused by inhibitory interactions of phenols with carboxylic acids, such as acetic and formic acid.     

Cyanobacteria as an eco-friendly resource for biofuel production: A critical review

Cyanobacteria are photosynthetic microorganisms which can be found in various environmental habitats. These photosynthetic bacteria are considered as promising feedstock for the production of the third- and the fourth-generation biofuels. The main subject of this review is highlighting the significant aspects of the biofuel production from cyanobacteria. The most recent investigations about the extraction or separation of the bio-oil from cyanobacteria are also adduced in the present review. Moreover, the genetic engineering of cyanobacteria for improving biofuel production and the impact of bioinformatics studies on the designing better-engineered strains are mentioned. The large-scale biofuel production is challenging, so the economic considerations to provide inexpensive biofuels are also cited. It seems that the future of biofuels is strongly dependent to the following items; understanding the metabolic pathways of the cyanobacterial species, progression in the construction of the engineered cyanobacteria, and inexpensive large-scale cultivation of them.     

Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review

Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies.     

Lactic acid bacteria as a tool for biovanillin production: A review

Vanilla is the most commonly used natural flavoring agent in industries like food, flavoring, medicine, and fragrance. Vanillin can be obtained naturally, chemically, or through a biotechnological process. However, the yield from vanilla pods is low and does not meet market demand, and the use of vanillin produced by chemical synthesis is restricted in the food and pharmaceutical industries. As a result, the biotechnological process is the most efficient and cost-effective method for producing vanillin with consumer-demanding properties while also supporting industrial applications. Toxin-free biovanillin production, based on renewable sources such as industrial wastes or by-products, is a promising approach. In addition, only natural-labeled vanillin is approved for use in the food industry. Accordingly, this review focuses on biovanillin production from lactic acid bacteria (LAB), which is generally recognized as safe (GRAS), and the cost-cutting efforts that are utilized to improve the efficiency of biotransformation of inexpensive and readily available sources. LABs can utilize agro-wastes rich in ferulic acid to produce ferulic acid, which is then employed in vanillin production via fermentation, and various efforts have been applied to enhance the vanillin titer. However, different designs, such as response surface methods, using immobilized cells or pure enzymes for the spontaneous release of vanillin, are strongly advised.