木质纤维素燃料乙醇蒸馏废水的高温厌氧处理及菌群结构分析

【目的】为开发高效的高浓度木质纤维素燃料乙醇蒸馏废水厌氧处理及资源化利用工艺,以活性炭为载体,在实验室规模上对高温厌氧流化床反应器处理木质纤维素燃料乙醇蒸馏废水进行研究。【方法】反应器经65 d梯度驯化后启动,对工艺参数进行一系列优化,并通过基于16S rRNA基因的分子生态学技术分析厌氧污泥中的优势菌群。【结果】实验获得了最优的反应条件和处理效果:厌氧流化床反应器(Anaerobic fluidized bed reactor,AFBR)在温度55±1°C、有机负荷率(OLR)13.8 g COD/(L·d)及水力停留时间(HRT)48 h操作时,COD去除率达到90%以上,同时甲烷产率达到290 mL/g COD;菌群鉴定分析结果显示高温厌氧活性污泥中Clostridia所占比例最大,产甲烷菌属以Methanoculleus和Methanosarcina为主,其它功能菌群主要为Alphaproteobacteria等。【结论】AFBR反应器可高效降解木质纤维素燃料乙醇蒸馏废水并产生生物能源甲烷,其反应体系内微生物种类丰富。     

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.     

Sustainable bioethanol production combining biorefinery principles using combined raw materials from wheat undersown with clover-grass

To obtain the best possible net energy balance of the bioethanol production the biomass raw materials used need to be produced with limited use of non-renewable fossil fuels. Intercropping strategies are known to maximize growth and productivity by including more than one species in the crop stand, very often with legumes as one of the components. In the present study clover-grass is undersown in a traditional wheat crop. Thereby, it is possible to increase input of symbiotic fixation of atmospheric nitrogen into the cropping systems and reduce the need for fertilizer applications. Furthermore, when using such wheat and clover-grass mixtures as raw material, addition of urea and other fermentation nutrients produced from fossil fuels can be reduced in the whole ethanol manufacturing chain. Using second generation ethanol technology mixtures of relative proportions of wheat straw and clover-grass (15:85, 50:50, and 85:15) were pretreated by wet oxidation. The results showed that supplementing wheat straw with clover-grass had a positive effect on the ethanol yield in simultaneous saccharification and fermentation experiments, and the effect was more pronounced in inhibitory substrates. The highest ethanol yield (80% of theoretical) was obtained in the experiment with high fraction (85%) of clover-grass. In order to improve the sugar recovery of clover-grass, it should be separated into a green juice (containing free sugars, fructan, amino acids, vitamins and soluble minerals) for direct fermentation and a fibre pulp for pretreatment together with wheat straw. Based on the obtained results a decentralized biorefinery concept for production of biofuel is suggested emphasizing sustainability, localness, and recycling principles. JIMB 2008: BioEnergy—Special issue.     

Development of a versatile shuttle vector for gene expression in Geobacillus spp

An improved, versatile shuttle vector has been created for the metabolic engineering of Geobacillus spp. As kanamycin is the most thermo-tolerant of commonly used antibiotics, the gene encoding a thermostable kanamycin nucleotidyltransferase, together with the origin of replication from the G. stearothermophilus plasmid pBST1 were cloned into the Escherichia coli cloning vector pUC18. The resulting vector, named pUCG18, replicated in both organisms and could be transformed with an efficiency of 1 x 10(4) transformants per microg of DNA in G. thermoglucosidasius and was stable up to 68 degrees C with antibiotic selection. It was used to demonstrate expression of the pyruvate decarboxylase (pdc) gene from Zymomonas palmae in G. thermoglucosidasius at 45 degrees C. Sequence analysis of the pBST1 derived origin of replication revealed homology with a family of theta replicons that have previously only been found in strains of Bacillus megaterium.     

Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds

Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g l^–1 glucose, 18 g l^–1 xylose, 6 g l^–1 acetate, less than 0.1 g l^–1 of total furans, and 12 g l^–1 of soluble phenolic compounds (SPCs). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: (1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and (2) overliming with Ca(OH)₂. These reduced the phenolic compounds by 40% and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the overlimed hydrolysate showed a 60% higher volumetric productivity (0.45 g_ETOH l^–1 hr^–1). The bioethanol/sugar yield was over 90% in both strategies.     

Enhancing bioethanol production from delactosed whey permeate by upstream desalination techniques

Industrial cheese whey processing comprises generally the isolation of proteins and lactose, but the economic use for the residual molasses, the so‐called delactosed whey permeate (DWP), is still to be improved. One possibility to maximize valorization and to minimize waste water treatment is the conversion of the remaining lactose in the DWP to ethanol by the yeast Kluyveromyces marxianus. This fermentation process depends strongly on the total ash content of the DWP, as high salt concentrations inhibit yeast metabolism. Here, three different approaches were tested to lower the DWP salt content: (i) simple dilution; (ii) nanofiltration; and (iii) electrodialysis. Lactose consumption, ethanol production and time‐dependent yields were compared between the three methods. A dilution of DWP to 60% v/v led to fermentation taking less than 80 h and yield above 7% AbV (alcohol by volume). After nanofiltration, 7.5% AbV was produced in about 80 h, and after electrodialysis, 11% AbV was produced in about 52 h. On the one hand the technical treatments (nanofiltration and electrodialysis) led to enhanced productivity in the fermentations, but, on the other hand, elaborate and extensive preprocessing is needed. Overall, ethanol production from DWP could be enhanced by prior partial desalination.     

一株富含碳水化合物微藻的筛选和分子鉴定

微藻生长快,单位体积碳水化合物产率高,是发酵生产生物乙醇的理想原料。本研究采用通气培养系统,对初筛得到的10株微藻进行分批培养,以单位体积碳水化合物产率为主要指标,筛选富含碳水化合物的优良藻种。研究结果显示：10株微藻的生物质干重、可溶性糖含量、碳水化合物含量和碳水化合物产率变化范围分别在0.922~1.965 g/L、4.42%~19.23%、26.8%~60.9% 和36.17~149.67 mg·L-1·d-1之间,其中藻株GZ-57的碳水化合物产率和可溶糖含量最高,分别为149.67 mg·L-1·d-1 和19.23%,表明藻株GZ-57是一株具有培养潜力的高产碳水化合物微藻。进一步对其进行形态特征及基于18S rDNA、ITS序列的分子系统学分析,发现藻株GZ-57与栅藻科（Scenedesmaceae）链带藻属（Desmodesmus）的极大链带藻（Desmodesmus maximus）亲缘关系较近,因此将其鉴定为极大链带藻（Desmodesmus maximus）。     

Temporal quantitative proteomics of Saccharomyces cerevisiae in response to a nonlethal concentration of furfural

Furfural, one of the main inhibitory compounds in lignocellulosic hydrolytes, inhibits the growth and ethanol production rate of yeast. To get a global view of the dynamic expression pattern of proteins in Saccharomyces cerevisiae during the fermentation with the introduction of 8 g/L furfural, the protein samples were taken before the addition of furfural, during the initial phase of furfural conversion and immediately after the conversion of furfural for comparative proteomic analysis with iTRAQ on a LC‐ESI‐MS/MS instrument. A comparison of the temporal expression pattern of 107 proteins related to protein synthesis between the reference cultures and the furfural‐treated cultures showed that a temporal downregulation of these proteins was retarded after the addition of furfural. The expression levels of 20 enzymes in glucose fermentation and 5 enzymes in the tricarboxylic acid cycle were reduced by furfural, with notably delayed temporal downregulations of Glk1p, Tdh1p, Eno1p and Aco1p, which is correlated to the reduced ethanol formation rate and glucose consumption rate by 66.7 and 60.4%, respectively. In the presence of furfural, proteins catalyzing the upper part of the super pathway of sulfur amino acid biosynthesis were repressed at all time points, which is related to the inhibited growth of furfural‐treated yeast. The expressions of 18 proteins related to stress response showed increased trends, including several highly induced heat shock proteins and proteins related to cellular signaling pathways.     

Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism.     

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r = 0.8–0.9) and dilution (up to 0.48 h^?1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160 g/l at r = 0.9 and dilution rate of 0.2 h^?1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5 g/l h^?1 could be achieved with feeding concentrations of 80–200 g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48 g/l h^?1.