纤维素乙醇基因工程研究进展

天然微生物代谢木质纤维素生成乙醇的途径和能力各不相同,通过基因工程的手段,将不同菌种的优良基因加以重组和改造,提高乙醇产率及降低成本,是当前纤维素乙醇研究的重要课题。综述了已发现的能够代谢纤维素产乙醇的天然微生物的种类、特性、代谢机理及构建重组菌株的方法和研究进展,并对基因工程开发纤维素乙醇工程菌的前景和存在的问题进行分析。     

世界纤维素乙醇生产发展趋势

第一代乙醇装置通常使用淀粉基（如谷物、小麦或甜高粱）原料或糖基（如甘蔗或甜菜）原料。利用淀粉基和糖基原料来生产乙醇已开拓了新的市场,但它们与人争粮。纤维素乙醇,也称之为“第二代”乙醇,它以非粮作物为原料。     

纤维素乙醇的生物炼制及研究进展

纤维素乙醇是以农业废弃生物质中的纤维素为主要原料、通过微生物发酵转化而成的生物燃料产品。作为一种绿色可再生替代能源,纤维素乙醇具有显著的能量收益和碳减排效益,对保障我国可持续发展、能源安全以及环境友好意义重大。然而,纤维素乙醇的生物炼制过程面临着难点和挑战。本文围绕纤维素原料及其预处理、纤维素酶水解和纤维素乙醇发酵工艺3个方面,介绍纤维素乙醇生物炼制的工艺流程及特征,剖析纤维素乙醇生产的主要技术瓶颈,并基于菌株抑制物胁迫耐性、碳源利用以及乙醇合成强化3个方面,总结了近年来纤维素乙醇生物炼制的研究进展,最后对纤维素乙醇未来的研究重点和发展前景进行了展望。     

纤维素乙醇制备中纤维素酶相关技术专利分析

生物乙醇制备中,纤维素酶相关技术是关键技术之一。本文概述了纤维素乙醇工艺以及纤维素酶,并利用专利分析的方法对纤维素乙醇制备中纤维素酶相关技术发展态势进行了研究,揭示了这一技术领域的专利申请的时空分布、主要技术领域及研究热点、重要专利权人及其竞争领域。     

万吨级乙醇秸秆汽爆炼制产业化技术与示范

大力推进纤维素燃料乙醇产业化,是改善能源结构、实现经济可持续发展的重要途径。文章综述了纤维素乙醇产业化技术发展状况,剖析了纤维素乙醇产业化关键过程存在的问题,提出相应解决对策并构建了具有自主知识产权的纤维素乙醇产业化技术体系。基于此技术体系,建立了万吨级乙醇秸秆炼制产业化技术与示范,实现了秸秆乙醇规模化连续生产和经济运行,具有重要的示范和推广价值。     

嗜热菌在纤维素乙醇中的研究进展及应用前景

利用纤维素生产乙醇的研究中,传统的中温微生物在基质范围、酶活性及热稳定性等方面存在不足,难以满足工业需求.嗜热菌具有独特的降解纤维素、半纤维素和生产乙醇的耐高温酶系和代谢途径,不但热稳定性高、而且底物范围广泛,在生物质能学领域有重要的研究价值和应用潜力.综述了在纤维素乙醇研究中具有潜力的嗜热菌的种类、特性、代谢机理和研究进展,并对嗜热菌的应用前景进行了分析与展望.     

木薯纤维素乙醇发酵的纤维素酶成本评价

木薯中的纤维素成分约占木薯干重的10％(W/W).文中以木薯燃料乙醇生产的木薯纤维素酒渣为原料,从纤维素酶成本角度评估了三种利用木薯纤维素组分发酵生产乙醇的方法,包括木薯纤维素酒渣的直接糖化和乙醇发酵、木薯纤维素酒渣预处理后的糖化与乙醇发酵、木薯乙醇发酵中同步淀粉与纤维素糖化以及乙醇发酵.结果表明,前两种方法的纤维素利用效率不高,酶成本分别达到13602、11659元/吨乙醇.第三种方法,即在木薯乙醇发酵过程同时加入糖化酶和纤维素酶,进行同步淀粉与纤维素糖化,进而进行乙醇发酵,木薯纤维素乙醇的收益最高.发酵结束时的乙醇浓度从101.5g/L提高到107.0g/L,纤维素酶成本为3 589元/吨乙醇.此方法利用木薯纤维素与木薯淀粉同时进行,不会带来额外的设备及操作投入,酶成本低于产品乙醇价格,可实现盈利,因此第三种方法为木薯纤维用于乙醇发酵的最适方法,本研究结果将为木薯乙醇产业深度利用木薯纤维提供依据.     

纤维素乙醇工程化探讨

出于对能源安全、大气污染的担忧以及促进农村经济发展的考虑,世界许多国家使用乙醇作为含氧添加剂或交通运输燃料来替代汽油。纤维素乙醇生产原料丰富,且具有明显的低碳排放特性而备受关注。随着全球范围内几套大型纤维素乙醇示范装置的相继试车,工程化问题将得到解决,并有望在2015-2016年完成装置的经济性考核,逐步进入商业化阶段。为避免原料"与人争粮,与粮争地",1代燃料乙醇将逐步向2代纤维素乙醇过渡。本文在综述近期国内外纤维素乙醇产业化概况的基础上,从化学工程和生物工程的角度对预处理、酶制剂及酶解工艺、戊糖/己糖共发酵菌株及工艺、装备等几个方面的技术进展进行剖析,讨论了工程化遇到的主要问题,探讨了我国纤维素乙醇技术的发展方向。     

甜菜渣中多糖的分析

近年来,发现动植物蛋白、酶、核酸、多糖组成的生物大分子具有多种多样的生理活性,特别是在免疫、抗肿瘤、抗病毒和心血管疾病的防治方面引起了国内外学者的关注。因此,对植物的有效成分的研究开始活跃起来。本文以甜菜渣中多糖成分含量进行了分析,结果如下: 采自制糖后的甜菜渣为原料,提取甜菜渣     

纤维素酶在生物质转化中的应用研究进展

纤维素酶是木质纤维素转化的重要酶系,主要是由内切β-1,4-葡聚糖酶、外切β-1,4-葡聚糖酶和β-葡萄糖苷酶组成,水解不同位置的糖苷键从而形成葡萄糖。本文总结了纤维素酶近几年来的研究开发与应用研究进展,包含产酶菌和菌种选育、辅助蛋白,介绍了纤维素酶来源和组成、纤维素酶工业化生产的最新研究进展以及纤维素酶在生物质转化方面的应用,纤维素酶在1.5代、2代纤维素燃料乙醇及生物质制气中都有重要的应用。     

Technology and economics of ethanol production from fodder beets via solid-phase fermentation

Summary Operating conditions for our semi-continuous, solid-phase fermentation system were optimized for conversion of fodder beets to fuel ethanol and distiller's wet feed (DWF). This information was then used to estimate operating parameters achievable in a commercial plant, and likely baseline production costs of such a plant. Initial acidification of pulp to pH 2.9–3.2 was effective in controlling bacterial contamination. The maximum operating capacity of the fermentor was approximately 92%, with 75% used for commercial application. A fermentation time of 24 h was sufficient to completely ferment the beet pulp to 8–9% (v/v) ethanol. Based on these parameters, a fodder beet cost of $19.25/metric ton ($17.50/ton), other operating and capital costs, and a PF credit of $0.14/L ($0.53/gal), ethanol production costs were estimated to be $0.49/L ($1.87/gal).     

Direct prediction of bioethanol yield in sugar beet pulp using near infrared spectroscopy

Sugar beets are a raw material for the production of sugar and ethanol. The decision on which end product to pursue could be facilitated by fast and reliable means of predicting the potential ethanol yield from the beets. A Near Infrared (NIR) Spectroscopy-based approach was tested for the direct prediction of the potential bioethanol production from sugar beets. A modified partial least squares (MPLS) regression model was applied to 125 samples, ranging from 21.9 to 31.0 gL(-1) of bioethanol in sugar beet brei. The samples were analyzed in reflectance mode in a Direct Contact Food Analyser (DCFA) FOSS-NIRSystems 6500 monochromator, with standard error of cross validation (SECV), standard error of prediction (SEP), coefficient of determination (r(2)) and coefficient of variation (CV) of 0.51, 0.49, 0.91 and 1.9 gL(-1), respectively. The NIR technique allowed direct prediction of the ethanol yield from sugar beet brei (i.e. the product obtained after sawing beets with a proper machine) in less than 3 min.     

A continuous, farm-scale, solid-phase fermentation process for fuel ethanol and protein feed production from fodder beets

Fuel ethanol (95%) was produced from fodder beets in two farm-scale processes. In the first process, involving conventional submerged fermentation of the fodder beets in a mash, ethanol and a feed (PF) rich in protein, fat, and fiber were produced. Ethanol yields of 70 L/metric ton (7 gal/ton) were obtained; however, resulting beers had low ethanol concentrations [3-5% (v/v)]. The high viscosity of medium and low sugar, beet mashes caused mixing problems which prevented any further increase of beet sugar in the mash. The severely limited the maximum attainable ethanol concentration during fermentation, thereby making the beer costly to distill into fuel ethanol and the process energy inefficient. In order to achieve distillably worthwhile ethanol concentrations of 8-10% (v/v), we developed and tested a solid-phase fermentation process (continuous). In preliminary trials, this system produced fermented pulp with over 8% (v/v) ethanol corresponding to an ethanol yield of 87 L/metric ton (21 gal/ton). Production costs with this novel process are $0.47/L ($1.77/gal) and the energy balance is 2.11. These preliminary cost estimates indicate that fodder beets are potentially competitive with corn as an ethanol feedstock. Additional research, however, is warranted to more precisely refine individual costs, energy balances and the actual value of the PF.     

Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets ( Beta vulgaris ssp. maritima ) has to be considered. In this study the transfer of BNYVV- (beet necrotic yellow vein virus) resistance and herbicide-tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.     

Solid phase fermentation of foder beets for ethanol production: Effect of grind size

Solid phase fermentation of pulped fodder beets was studied to see what effect beet particle size had on various fermentation parameters. All trials were run in 4-l stainless stell containers and hammermilled pulp was initially adjusted to pH 3.0 to control bacterial contaminants. The maximum yeast population that built up in the pulp was independent of the hammermill screen size (0.476–1.905 cm) and averaged 2.0−2.3 × 10⁸ cells/ml. Pulp from finer screens (0.476–0.953 cm) took 19–22 h to reach a peak yeast population while pulp from coarser screens (1.270–1.905 cm) took a slightly longer 24–28 h. The time to reach maxium ethanol concentration was not affected by screeen size and averaged 28–30 h. Ethanol yields dropped slightly form 85–87% of theoretical with the finest screens to 83–84% with the coarset screens. The maximum ethanol concentration observed was 7.96% (v/v) and the average of all runs was 7.63% (v/v). Fermentation efficiency averaged 98–99% thoughout. The lack of a response to grinding fodder beets with different screens was due to their wet fibrous nature which hindered free flow of pulp though the screens. Pulp was, instead, extruded though the screens, forming particles of generally similar size. Our results indicate that the primary consideration for grind size is energy consumption for grinding. Therefore, if a hammermill is used, a large screen (1.270–1.905 cm) which requires less energy should be employed so as to minimize energy consumption. This strategy does not result in longer fermentation times or reduced ethanol yields.     

Azetidine-2-carboxylic acid in the food chain

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world’s supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.     

The origin and evolution of weed beets: consequences for the breeding and release of herbicide-resistant transgenic sugar beets

Populations of weed beets have expanded into European sugar beet production areas since the 1970s, thereby forming a serious new weed problem for this crop. We sampled seeds in different French populations and studied mitochondrial DNA, chloroplast DNA and life-cycle variability. Given the maternal inheritance of the mitochondrial and chloroplastic genomes and the nuclear determinism of the annual habit, we were able to determine the maternal origin and evolution of these weed beet populations. Our study shows that they carry the dominant allele B for annual habit at high frequency. The main cytoplasmic DNA type found in northern weed beet populations is the cytoplasmic male-sterile type characteristic of sugar beets. We were able to determine that these populations arise from seeds originating from the accidental pollinations of cultivated beets by adventitious beets in the seed production area, which have been transported to the regions where sugar beets are cultivated. These seeds are supposedly the origin of the weed forms and a frequently disturbed cultivated environment has selected for annual habit and early flowering genotypes. We discuss the consequences of the weed beet populations for the breeding, seed production and release of herbicide-resistant transgenic sugar beets.     

Long distance pollen-mediated gene flow at a landscape level: the weed beet as a case study

Gene flow is a crucial parameter that can affect the organization of genetic diversity in plant species. It has important implications in terms of conservation of genetic resources and of gene exchanges between crop to wild relatives and within crop species complex. In the Beta vulgaris complex, hybridization between crop and wild beets in seed production areas is well documented and the role of the ensuing hybrids, weed beets, as bridges towards wild forms in sugar beet production areas have been shown. Indeed, in contrast to cultivated beets that are bi-annual, weed beets can bolt, flower and reproduce in the same crop season. Nonetheless, the extent of pollen gene dispersal through weedy lineages remains unknown. In this study, the focus is directed towards weed-to-weed gene flow, and we report the results of a pollen-dispersal analysis within an agricultural landscape composed of five sugar beet fields with different levels of infestation by weed beets. Our results, based on paternity analysis of 3240 progenies from 135 maternal plants using 10 microsatellite loci, clearly demonstrate that even if weedy plants are mostly pollinated by individuals from the same field, some mating events occur between weed beets situated several kilometres apart (up to 9.6 km), with rates of interfield-detected paternities ranging from 11.3% to 17.5%. Moreover, we show that pollen flow appears to be more restricted when individuals are aggregated as most mating events occurred only for short-distance classes. The best-fit dispersal curves were fat-tailed geometric functions for populations exhibiting low densities of weed beets and thin-tailed Weibull function for fields with weed beet high densities. Thus, weed beet populations characterized by low density with geographically isolated individuals may be difficult to detect but are likely to act as pollen traps for pollen emitted by close and remote fields. Hence, it appears evident that interfield pollen-mediated gene flow between weed beets is almost unavoidable and could contribute to the diffusion of (trans)genes in the agricultural landscape.     

Genetic diversity and gene flow between wild, cultivated and weedy forms of Beta vulgaris L. (Chenopodiaceae), assessed by RFLP and microsatellite markers

 Beets belonging to the species Beta vulgaris L. can be found in crop, wild and weedy forms, all of which are interfertile. We studied the intra-specific genetic relationships of about 300 individuals from 54 populations of various French geographic origins using nuclear molecular markers (five single-copy RFLP loci and one microsatellite locus). The patterns of diversity were congruent for both types of markers. Genetic diversity in wild beets appeared to be high, both in term of allele number and observed heterozygosity, whereas the narrowness of the cultivated-beet gene pool was confirmed. Genetic distances between all forms showed that weed beets in northern France are intermediates between sugar beet and inland wild beets in south-western France. This analysis allowed us to infer the paternal origin of weed beets and furthermore, is in agreement with a previous study which focused on their maternal origin: weed beet infesting sugar-beet fields originated from accidental and recurrent hybridization between cultivated lines and ruderal inland wild beets during the production of commercial seeds in south-western France. Inland wild beets are genetically close to Mediterranean coastal wild beets, but differ from other coastal forms (from Biscay, Brittany and northern France). The study of gene flow in the beet complex contributes to the risk assessment of transgenic beets. Received: 8 June 1998 / Accepted: 8 October 1998     

Mitochondrial minisatellite polymorphisms in fodder and sugar beets reveal genetic bottlenecks associated with domestication

Historically, sugar beets were selected from fodder beets. We used mitochondrial minisatellite loci to analyze cytoplasmic genetic diversity in fodder beet and sugar beet. Among the 8 sugar beet accessions examined we identified 3 multi-locus haplotypes. These 3 haplotypes were a subset of 5 haplotypes identified among the 29 fodder beet accessions examined. All but one haplotype in fodder beet comprised, in turn, a subset of 12 haplotypes identified previously in leaf beets. Such apparent decreases in cytoplasmic genetic diversity must result from genetic bottlenecks associated with domestication and the ensuing breeding processes. We also detected the haplotype associated with the male-sterile Owen cytoplasm of sugar beet in the fodder beet gene pool. Furthermore, the presence of a 39 kDa protein associated with the Owen cytoplasm was confirmed in two fodder beet plants by Western blot analysis. These results lead us to speculate that the Owen cytoplasm may have originated in fodder beet, from which sugar beet was derived.