乙醇消耗菌的分离鉴定及其对基因工程集胞藻乙醇产量的影响

为研究产乙醇基因工程集胞藻培养液中的乙醇消耗菌污染情况及其对乙醇产量的影响,从污染的培养液中分离出4株乙醇消耗菌,通过16S rDNA、26S rDNA序列分析对分离出的菌株进行鉴定,并研究其乙醇消耗能力及对基因工程集胞藻乙醇产量的影响。结果表明,分离出的4株菌分别为红酵母(Rhodotorula sp.)、季也蒙酵母(Meyerozyma guilliermondii)、短波单胞菌(Brevundimonas sp.)、微杆菌(Microbacterium sp.)。其中乙醇消耗能力最强的是红酵母,乙醇比消耗速率达到391 g/(1015 cfu?d);其次为季也蒙酵母,乙醇比消耗速率为80.1 g/(1015 cfu?d);短波单胞菌和微杆菌的乙醇比消耗速率远低于红酵母和季也蒙酵母。将分离出的菌株与产乙醇集胞藻共培养7d后,污染红酵母、季也蒙酵母、短波单胞菌、微杆菌的实验组乙醇产量分别下降了53.8%、23.6%、40.7%、27.3%。4株菌对基因工程集胞藻的生长无明显影响,均通过直接消耗乙醇而降低集胞藻的乙醇产量。     

Improving the remaining activity of lignocellulolytic enzymes by membrane entrapment

The production of bioethanol by the conversion of lignocellulosic waste has attracted much interest in recent years because of its low cost and great potential availability. However, the high cost of the enzyme required for this conversion is often considered to be the major bottleneck in the commercial lignocellulosic ethanol industry. In this work, the hydrolysis of rice straw by free and entrapped lignocellulolytic enzymes (cellulase, xylanase and laccase) was carried out at pH 5.5 and 37 °C. The hydrolysis of rice straw by enzymes entrapped in a membrane produced a higher monosaccharide content: 601.05 mg/g rice straw for entrapped enzymes vs. 465.46 mg/g rice straw for free enzymes. This study has shown that enzyme entrapment is an important technique for the efficient use and reuse of enzymes in industrial applications and also for the rapid separation of saccharide products from the reaction medium, thus improving the remaining enzymatic activities.     

Laccase detoxification of steam-exploded wheat straw for second generation bioethanol

In this work we compared the efficiency of a laccase treatment performed on steam-exploded wheat straw pretreated under soft conditions (water impregnation) or harsh conditions (impregnation with diluted acid). The effect of several enzymatic treatment parameters (pH, time of incubation, laccase origin and loading) was analysed. The results obtained indicated that severity conditions applied during steam explosion have an influence on the efficiency of detoxification. A reduction of the toxic effect of phenolic compounds by laccase polymerization of free phenols was demonstrated. Laccase treatment of steam-exploded wheat straw reduced sugar recovery after enzymatic hydrolysis, and it should be better performed after hydrolysis with cellulases. The fermentability of hydrolysates was greatly improved by the laccase treatment in all the samples. Our results demonstrate the action of phenolic compounds as fermentation inhibitors, and the advantages of a laccase treatment to increase the ethanol production from steam-exploded wheat straw.     

Biofuels in China: opportunities and challenges

With rapid economic development, energy consumption in China has tripled in the past 20 yr, exceeding 2.4 billion tons of standard coal in 2006. The search for new green energy as substitutes for the nonrenewable energy resources has become an urgent task. China has a variety of climates and is rich in potential biofuel plant species. Corn and cassava are used as the main raw materials for bioethanol production in China. At the end of 2005, bioethanol productivity had increased to 1.02 million tons produced by four companies, and bioethanol-blended petrol accounted for 20% of the total petrol consumption in China. According to the Mid- and Long-term Development Plan for Renewable Energy, the consumption of biodiesel in China will reach 0.2 million tons in 2010 and 2.0 million tons in 2020. This review is intended to provide an introduction to the distribution and development of biofuel crops and biofuel industry in China.     

In vivo biotinylation of recombinant beta-glucosidase enables simultaneous purification and immobilization on streptavidin coated magnetic particles

Beta-glucosidase from Bacillus licheniformis was in vivo biotinylated in Escherichia coli and subsequently immobilized directly from cell lysate on streptavidin coated magnetic particles. In vivo biotinylation was mediated by fusing the Biotin Acceptor Peptide to the C-terminal of beta-glucosidase and co-expressing the BirA biotin ligase. The approach enabled simultaneous purification and immobilization of the enzyme from crude cell lysate on magnetic particles because of the high affinity and strong interaction between biotin and streptavidin. After immobilization of the biotinylated beta-glucosidase the specific activity (using p-nitrophenyl-β-d-glucopyranoside as substrate) was increased 6.5 fold (compared to cell lysate). Immobilization of the enzyme resulted in improved thermal stability compared to free enzyme; after 2 h of incubation (at 50 °C) the residual enzyme activity of immobilized and free beta-glucosidase was 67 and 13%, respectively. The recyclability of immobilized beta-glucosidase was examined and it was observed that the enzyme could be recycled at least 9 times and retain 89% of its initial activity.     

Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate

A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.     

ADH7启动子精细调控表达MSN2酿酒酵母菌株对 糠醛耐受的研究

【目的】构建自我精细调控表达应激转录调控基因MSN2的酿酒酵母(Saccharomyces cerevisiae)基因工程菌株,提高其对糠醛的耐受能力。【方法】以酿酒酵母BY4742基因组DNA为模板,采用PCR技术扩增获得ADH7启动子、CYC1终止子以及MSN2编码框序列,以pUG6质粒为载体构建含ADH7p-MSN2-CYC1t表达盒的重组表达质粒pUG6-AM。通过醋酸锂法,将线性化后的质粒pUG6-AM转入酿酒酵母BY4742,筛选阳性转化子,初步分析其对糠醛的耐受能力,采用荧光定量PCR技术检测MSN2基因及其调控代表基因的转录变化。【结果】构建了在ADH7启动子控制下表达MSN2的酿酒酵母基因工程菌株AM01,该菌株对糠醛耐受能力明显增强,MSN2基因的转录得到了自我精细调控,并提高了其调控基因的转录水平。【结论】以糠醛诱导表达基因的启动子精细调控应激转录调控基因MSN2的转录表达,既可提高酿酒酵母工程菌株对糠醛的耐受能力,又能避免其持续高效表达带来的副作用。     

Genome sequence of Acremonium strictum AAJ6 strain isolated from the Cerrado biome in Brazil and CAZymes expression in thermotolerant industrial yeast for ethanol production

Increased demand for biofuels promotes the search for new biomass-degrading fungi. Acremonium strictum is an environmentally widespread filamentous fungi found on plant debris; that secretes lignocellulose-degrading enzymes. A recently isolated A. strictum strain, AAJ6; native to the Brazilian Cerrado biome was evaluated for its capacity to degrade lignocellulosic substrates. In this study, whole-genome sequencing of AAJ6 was performed and 775 CAZy domains were identified which correlated to those of A. strictum strain DS1bioAY4a and other lignocellulolytic fungi; suggesting AAJ6 is a high CAZyme producer. We expressed the glycoside hydrolase families GH74 and GH3 from plasmid or genome-integrated to evaluate the ethanol production from cellulosic substrates in Brazilian industrial Saccharomyces cerevisiae strains (PE-2 and SA-1) evolved for thermotolerance (AMY12 and AMY35). Those expressing the genome-integrated enzymes showed the highest β-glucosidase activity and growth in medium with cellobiose at 40°C. The strain AGY005 (integrated cassettes) showed 19, 23 and 46% higher ethanol production in SHF, pSSF (partial hydrolysis SSF) and SSF processes, respectively, using Avicel, and ∼50% more ethanol using pre-treated sugarcane bagasse, compared to the strain with a plasmid-based expression. These results indicate the improved performance of thermotolerant industrial strains with genome-integrated CAZymes in the SSF process for 2G ethanol.     

Renewable and sustainable bioenergies production from palm oil mill effluent (POME): win-win strategies toward better environmental protection

Palm oil industry is one of the leading agricultural industries in Malaysia with average crude palm oil production of more than 13 million tonne per year. However, production of such huge amount of crude palm oil has consequently resulted to even larger amount of palm oil mill effluent (POME). POME is a highly polluting wastewater with high chemical oxygen demand (COD) and biochemical oxygen demand (BOD) in which can caused severe pollution to the environment, typically pollution to water resources. On the other hand, POME was identified as a potential source to generate renewable bioenergies such as biomethane and biohydrogen through anaerobic digestion. In other words, a combination of wastewater treatment and renewable bioenergies production would be an added advantage to the palm oil industry. In line with the world's focus on sustainability concept, such strategy should be implemented immediately to ensure palm oil is produced in an environmental friendly and sustainable manner. This review aims to discuss various technologies to convert POME to biomethane and biohydrogen in a commercial scale. Furthermore, discussion on using POME to culture microalgae for biodiesel and bioethanol production was included in the present paper as a new remedy to utilize POME with a greater beneficial return.