Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp

The modular endoglucanase Cel9B from Paenibacillus barcinonensis is a highly efficient biocatalyst, which expedites pulp refining and reduces the associated energy costs as a result. In this work, we set out to identify the specific structural domain or domains responsible for the action of this enzyme on cellulose fibre surfaces with a view to facilitating the development of new cellulases for optimum biorefining. Using the recombinant enzymes GH9–CBD3c, Fn3–CBD3b, and CBD3b, which are truncated forms of Cel9B, allowed us to assess the individual effects of the catalytic, cellulose binding, and fibronectin‐like domains of the enzyme on the refining of TCF kraft pulp from Eucalyptus globulus. Based on the physico‐mechanical properties obtained, the truncated form containing the catalytic domain (GH9–CBD3c) has a strong effect on fibre morphology. Comparing its effect with that of the whole cellulase (Cel9B) revealed that the truncated enzyme contributes to increasing paper strength through improved tensile strength and burst strength and also that the truncated form is more effective than the whole enzyme in improving tear resistance. Therefore, the catalytic domain of Cel9B has biorefining action on pulp. Although cellulose binding domains (CBDs) are less efficient toward pulp refining, evidence obtained in this work suggests that CBD3b alters fibre surfaces and influences paper properties as a result. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Carboxymethyl cellulase production optimization from Glutamicibacter arilaitensis strain ALA4 and its application in lignocellulosic waste biomass saccharification

Abstract

In this context, carboxymethyl cellulase (CMCase) production from Glutamicibacter arilaitensis strain ALA4 was initially optimized by one factor at a time (OFAT) method using goat dung as proficient feedstock. Two-level full factorial design (2⁵ factorial matrix) using first-order polynomial model revealed the significant (p?<?0.05) influence of pH, moisture, and peptone on CMCase activity. Central composite design at N?=?20 was further taken into account using a second-order polynomial equation, and thereby liberated maximum CMCase activity of 4925.56?±?31.61?U/g in the goat dung medium of pH 8.0 and 100% moisture containing 1% (w/w) peptone, which was approximately two fold increment with respect to OFAT method. Furthermore, the partially purified CMCase exhibited stability not only at high pH and temperature but also in the presence of varied metal ions, organic solvents, surfactants, and inhibitors with pronounced residual activities. The enzymatic hydrolysis using partially purified CMCase depicted the maximum liberation of fermentable sugars from alkali pretreated lignocellulosic wastes biomass in the order of paddy straw (13.8?±?0.15?mg/g)?>?pomegranate peel (9.1?±?0.18?mg/g)?>?sweet lime peel (8.37?±?0.16?mg/g), with saccharification efficiency of 62.1?±?0.8, 40.95?±?0.4, and 37.66?±?0.4%, respectively after 72?hr of treatment.     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.     

Environmental implications of recombinant DNA technology

Applications of recombinant DNA technology are discussed as a backdrop for evaluation of the environmental impacts of this technology. Some of applications include using traditional biological techniques for specific purposes, including nitrogen fixation, microbial pesticides, and waste treatment. In these instances the final product lies along a continuum, beginning with an organism marginally performing its function, and ending with one that is highly specialised and very efficient in what it does. One may move along this continuum toward the ‘perfect’ microorganism by using traditional methodologies of mutagenesis and selection, recombinant DNA technology, or a combination of the two.     

Use of laccase in pulp and paper industry

Laccase, through its versatile mode of action, has the potential to revolutionize the pulping and paper making industry. It not only plays a role in the delignification and brightening of the pulp but has also been described for the removal of the lipophilic extractives responsible for pitch deposition from both wood and nonwood paper pulps. Laccases are capable of improving physical, chemical, as well as mechanical properties of pulp either by forming reactive radicals with lignin or by functionalizing lignocellulosic fibers. Laccases can also target the colored and toxic compounds released as effluents from various industries and render them nontoxic through its polymerization and depolymerization reactions. This article reviews the use of both fungal and bacterial laccases in improving pulp properties and bioremediation of pulp and paper mill effluents.     

木质纤维生物质资源高效利用分子技术的开发

木质纤维生物质是地球上最丰富的可再生生物质资源,可为造纸、化工、纺织和生物能源等工业提供重要的原材料。木质纤维生物质主要包括木质素、纤维素和半纤维素三种生物多聚物成分。如何利用分子手段改造这些生物聚合物,提高它们的工业利用率是目前高度关注的问题。综述了近年来木质纤维多聚物在生物合成与改造方面的研究进展,展望了利用分子技术改造植物木质纤维生物质实现其高效利用的前景。     

生物酶在造纸工业绿色制造中的应用

近年来随着生物技术的发展,生物酶制剂的生产水平不断提高,促进了酶制剂在生物制浆、生物漂白、废纸生物脱墨、酶法纸浆改善性能及树脂生物控制等方面的应用,体现了酶技术在减轻制浆造纸工业环境污染、改善纸浆抄造性能等方面的潜力。文中重点介绍在不同制浆造纸原料及工艺中酶的选用、复配和应用技术及原理,以及酶制剂的应用效率及其对制浆造纸中节能减排和绿色环保的意义。     

Reaction of estuarine ecosystems to effluent from pulp and paper industry

A major problem associated with the discharge of pulp or paper-mill effluent into shallow estuarine ecosystems, such as the upper L'Etang in Canada or the Don in Scotland, is the development of anoxic conditions in water column and sediment. The effluent modifies the balance, within the ecosystem, between the supply of, and demand for, oxygen. The lignosulphonate component of pulpmill effluent blocks the transmission of light energy to photosynthetic green plants. The oxygen demand is increased by the microbial degration of the additional organic matter in water and sediment. The addition of cellulosic pulp fibre to the sediment also results in an increase in the concentration of their metabolic by-product, sulphide. The oxidation of sulphide exerts an additional oxygen demand on the system, which is an important factor in the development and maintenance of anoxic conditions. It is suggested that temperature is an important factor in controlling the release of sulphide from the sediment into the overlying water and in modifying the toxicity of sulphide towards the bacterial populations and, therefore, the capacity of the ecosystem to cope with the effluent.     

Simultaneous saccharification and fermentation of amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without supplemental cellulase

A derivative of Klebsiella oxytoca M5A1 containing chromosomally integrated genes for ethanol production from Zymomonas mobilis (pdc, adhB) and endoglucanase genes from Erwinia chrysanthemi (celY, celZ) produced over 20 000 U endoglucanase l^–1 activity during fermentation. In combination with the native ability to metabolize cellobiose and cellotriose, this strain was able to ferment amorphous cellulose to ethanol (58–76% of theoretical yield) without the addition of cellulase enzymes from other organisms.     

Manipulating corn germplasm to increase recombinant protein accumulation

Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.     

Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048. © 2010 Wiley Periodicals, Inc.     

Microbial and enzymatic control of pitch in the pulp and paper industry

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.     

Mechanisms prevalent during bioremediation of wastewaters from the pulp and paper industry

Bioremediation of wastewaters represents an important treatment methodology, especially when examined against the backdrop of ever-stricter legislation that is evolving in order to regulate effluent release into the environment. It has been reported that bioremediation specifically holds promise in solving environmental problems. Crucial questions surrounding the treatment of effluents include: efficiency of the process, economic feasibility, legal requirements, and the mechanisms involved in the remediation process. Of all these issues mentioned, the last requires special attention. This paper investigates these matters and focuses on techniques that are currently employed to determine the efficiency of bioremediation and mechanisms involved therein. The physiological significance of biosorption is also examined, as this subject has not been fully addressed in previous publications.     

Environmental assessment of enzyme assisted processing in pulp and paper industry

Background Aims and Scope  The pulp and paper (P&P) industry is traditionally known to be a large contributor to environmental pollution due its large consumptions of energy and chemicals. Enzymatic processing, however, offers potential opportunities for changing the industry towards more environmentally friendly and efficient operations compared to the conventional methods. The aims of the present study has been to investigate whether the enzyme technology is a more environmentally sound alternative than the conventional ways of producing paper. The study addresses five enzyme applications by quantitative means and discusses the environmental potential of a range of other enzyme applications by qualitative means. Methods  LCA is used as analytical tool and modelling is facilitated in SimaPro software. Foreground LCA data are production/company specific and collected from P&P technology service providers, specific P&P companies and P&P researchers. The background data on energy systems, auxiliary chemicals, etc. are primarily taken from the ecoinvent database. Results  The study shows that fossil energy consumption and potential environmental impacts (global warming, acidification, nutrient enrichment, photochemical smog formation) induced by enzyme production are low compared with the impacts that they save when applied in bleach boosting, refining, pitch control, deinking, and stickies control. Discussion  The general explanation is that small amounts of enzyme provide the same function as large amounts of chemicals and that enzymatic processes generally require less fossil energy inputs than conventional processes. Data quality assessments and sensitivity analyses indicate that this observation is robust for all processes except deinking, although the results are subject to uncertainty and much variation. Conclusions and Recommendations  The environmental improvements that can be achieved by application of enzymatic solutions in the P&P industry are promising. To get a greater penetration of enzymatic solutions in the market and to harvest the environmental advantages of biotechnological inventions, it is recommended that enzymatic solutions should be given more attention in, for instance, ‘Best Available Technology’ notes within the framework of the European Directive on Integrated Pollution Prevention and Control (IPPC). ESS-Submission Editor: Roland Hischier (roland.hischier@empa.ch)     

武汉市造纸行业资源代谢分析

资源代谢问题是造成产业系统生态环境影响的主要原因.在分析武汉市造纸行业资源代谢的基础上从资源输入-资源使用-环境胁迫-系统循环共生4个方面构建武汉市造纸行业资源代谢分析指标体系.运用模糊综合评判法和问题树模型对2007年武汉市造纸业的原材料、水、能源等资源的代谢与全国造纸行业平均水平进行了比较分析.结果表明:(1)武汉市造纸业资源代谢水平总体上评价等级为优,但某些指标与全国平均水平比还略有差距;(2)从资源输入角度分析,原材料输入生态效率及水资源输入生态效率等级为优,能源输入生态效率等级为良;(3)从资源使用角度分析,单位产品的原材料消耗及单位产品的水资源消耗等级为优,单位产品的能源消耗等级为良;(4)从环境胁迫角度分析,单位污染物工业总产值及单位产品污染物排放等级均为优,污染物达标排放率等级为良;(5)从系统循环共生分析,企业清洁生产达标率等级为良,资源持续利用及污染物治理等级为优,而中水回用率等级为差.通过代谢分析可得,武汉市造纸行业能源输入生态效率、单位产品能源消耗与全国平均水平比还有一定距离,整个行业中企业清洁生产比例还具有提升的空间;中水回用方面较弱,需要特别加强.