Strategies for reducing supplemental medium cost in bioethanol production from waste house wood hydrolysate by ethanologenic Escherichia coli: inoculum size increase and coculture with Saccharomyces cerevisiae

In this paper, we report a simultaneous realization of both efficient ethanol production and saving medium nutrient (corn steep liquor [CSL]) during bioethanol fermentation of overliming-treated hydrolysate of waste house wood (WHW) using ethanologenic Escherichia coli KO11. In cultivation using WHW hydrolysate supplemented with 4% (v/v) CSL and 0.2 g-dry cell weight (DCW)/l E. coli KO11 cells, the overall ethanol yield reached 84% of the theoretical value at 61 h. When we conducted the cultivation with 1% CSL to reduce the supplemental medium cost, the overall ethanol yield remained in the range of 66-72% even at 90 h. We proposed two alternative methods for increasing the overall yield even with 1% CSL. The first method involved increasing the inoculum size of E. coli KO11 up to 0.8 g-DCW/l, where 83% of the overall yield was attained at 60 h of cultivation. The second method involved the coculture of 0.2 g-DCW/l E. coli KO11 together with 0.02 g-DCW/l of Saccharomyces cerevisiae TJ1, and the overall yield reached 81% at 47 h of cultivation.     

Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production

Hydrolysates of lignocelluloses hydrolyzed by diluted sulfuric acid contain toxic compounds that inhibit ethanol production by Saccharomyces cerevisiae and the ethanologenic recombinant Escherichia coli KO11. We investigated the biological detoxification of a hydrolysate of waste house wood (WHW) by a thermophilic bacterium, Ureibacillus thermosphaericus. When the hydrolysate was treated with this bacterium at 50 degrees C for 24 h, the ethanol production rate by S. cerevisiae increased markedly and was comparable to that for the hydrolysate treated with an excess amount of calcium hydroxide (overliming). Chromatographic analysis of synthetic hydrolysates containing furfural or 5-hydroxymethyl furfural that are considered to be major toxic compounds in hydrolysates revealed that U. thermosphaericus degrades these compounds. In the WHW hydrolysates, however, the concentrations of these compounds were not decreased markedly by the bacterium. These results suggest that the bacterium degrades minor but more toxic compounds or phenolic compounds in the WHW hydrolysates. The combination of bacterial and overliming treatments of hydrolysates minimized significantly the decrease in ethanol production rate by E. coli KO11 as fermentation proceeded. Because the bacterium grows rapidly and does not consume sugars, our biological detoxification should be useful for bioethanol production from acid hydrolysates of lignocelluloses.     

重组大肠杆菌KO11利用单糖发酵乙醇条件优化

对重组大肠杆菌KO11利用单糖转化乙醇的能力进行了研究与优化,结果表明,KO11对己糖中的半乳糖、甘露糖、葡萄糖和戊糖中的岩藻糖、木糖均有较高的乙醇转化率。通过对KO11利用葡萄糖与木糖发酵的考察发现,以4%(v/v)接种量接种于含葡萄糖20 g/L的LB培养基中,KO11在pH 5.5、37℃、150 r/min振荡培养4 h后再静置发酵20 h能够达到乙醇转化率94.81%;将其接种于含同样浓度木糖的LB培养基在相同的pH、发酵温度与振荡速度下振荡12 h后再静置发酵15 h达到乙醇转化率94.15%。另外,KO11具有较高糖耐受性,发酵葡萄糖与木糖混合糖时优先利用葡萄糖,并且在43℃时仍表现出较高的乙醇转化率,为利用该菌种进行纤维质水解液发酵提供了参考依据。     

Ethanol production from non-pretreated napiergrass through a simultaneous saccharification and fermentation process followed by a pentose fermentation with Escherichia coli KO11

Efficient ethanol production from lignocellulosic napiergrass (Pennisetum purpureum Schumach) was examined by the combination of the simultaneous saccharification and fermentation (SSF) with commercial cellulase and Saccharomyces cerevisiae NBRC 2044 and subsequent pentose fermentation (PF) by Escherichia coli KO11. Under the optimized conditions, the combination of the SSF and PF processes resulted in the production of 144 mg g(-1) of ethanol from the non-pretreated napiergrass powder. The ethanol yield was 44.2% of the theoretical yield based on the hexose (37.5 g) and pentose (26.5 g) derived from 100 g of dry powdered napiergrass.     

Disruption of ptsG gene and manXYZ operon of ethanol-producing Escherichia coli KO11: Effects on glucose and xylose utilization and ethanol production

Ethanol-producing Escherichia coli strain KO11 consumed 99% of the glucose and only 13% of the xylose in a mixture of glucose (60g/L) and xylose (40g/L) during the 72-h fermentation at 30°C. The deletion mutants ΔptsG, ΔmanXYZ, and ΔptsG/manXYZ utilized 42%, 78%, and 35% of the glucose and 50%, 32%, and 32% of the xylose, respectively.     

家具生产中木质废料的来源、控制与处理

分析了家具制造企业原料输入和废料产生情况,针对家具生产过程木质废料的产生情况和废料形式进行了剖析,结合生产实际提出了家具生产木质废料的控制与处理措施。     

利用重组大肠杆菌发酵生产L-茶氨酸

L-茶氨酸是茶叶中一种独特的非蛋白质氨基酸。γ-谷氨基甲酰胺合成酶可以催化L-谷氨酸、乙胺和ATP合成L-茶氨酸,但反应成本较高。为了提高L-茶氨酸的生产效率,该研究开发了直接发酵生产L-茶氨酸的新方法。首先,在大肠杆菌基因组上双拷贝Methylovorus mays来源的γ-谷氨酰甲胺合成酶基因gmas,获得了一株遗传稳定的用于L-茶氨酸生产的重组菌株。其次,在5 L罐中采用流加乙胺的方式发酵20 h,L-茶氨酸产量达到30. 45 g/L,糖酸转化率达到20.17%。最后,根据L-茶氨酸的物化性质拟定出合适的分离提取路线,从发酵液中获得了98.51%纯度的L-茶氨酸成品,总提取收率可达到70.34%。该方法操作简单,原料成本较低,具有较好的工业应用前景。     

蒸汽爆破尾叶桉木材纤维素酶水解液发酵单细胞蛋白

用高效液相色谱对蒸汽爆破尾叶桉木材的纤维素酶水解液进行了还原糖和乙酸含量分析,通过酿酒酵母和假丝酵母混合接种,对水解液进行了摇瓶发酵单细胞蛋白的工艺研究,确定了发酵的最佳工艺条件。结果表明,水解液中主要含有葡萄糖和木糖,用双菌种混合发酵单细胞蛋白的最佳发酵条件为:酿酒酵母(Sacchromyces cerevisive):假丝酵母(Candida utilis2.587)为1:3,发酵温度31℃,初始pH4.5,初始还原糖浓度17～18g/L,发酵时间27h。     

Quality assessment of pellets and briquettes made from glued wood waste

European Journal of Wood and Wood Products - In accordance with sustainable economic and social development, Europe supports the use of energy from renewable sources to decrease the use of fossil...     

木浆中段废水的脱色处理

为解决木浆中段废水色度难于被生化法所降低的问题，分别采用混凝法，Fenton试剂法，电解法和活性炭吸附法进行了试验研究。     

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.     

Release of CFC-11 from disposal of polyurethane foam waste

The halocarbon CFC-11 has extensively been used as a blowing agent for polyurethane (PUR) insulation foams in home appliances and for residential and industrial construction. Release of CFCs is an important factor in the depletion of the ozone layer. For CFC-11 the future atmospheric concentrations will mainly depend on the continued release from PUR foams. Little is known about rates and time frames of the CFC release from foams especially after treatment and disposal of foam containing waste products. The CFC release is mainly controlled by slow diffusion out through the PUR. From the literature and by reevaluation of an old reported experiment, diffusion coefficients in the range of 0.05-1.7 x 10(-14) m2 s-1 were found reflecting differences in foam properties and experimental designs. Laboratory experiments studying the distribution of CFC in the foam and the short-term releases after shredding showed that about 40% of the CFC is solubilized in the PUR phase, and that up to 10% of the total content will be released within a few weeks if the foam is shredded down to 2-cm sized pieces. For smaller pieces the quick release will be larger. Fifty percent of residual CFC content will be released within 9-300 years from 2-cm pieces based on the range in diffusion coefficients reported. For larger pieces the initial release is insignificant, and the release time frames are much longer than for the shredded foam.     

重组大肠杆菌全细胞合成苯基乳酸的研究

在E.coli BL21(DE3)中过量表达D-乳酸脱氢酶基因(D-ldh),并优化该重组菌全细胞转化苯丙酮酸钠合成苯基乳酸的条件。通过单因素实验和正交实验优化诱导表达条件,并在此基础上对全细胞转化苯丙酮酸钠合成苯基乳酸进行了优化。结果表明,菌体OD600为1.2时添加IPTG至终浓度0.2mmol/L,25℃诱导4h后收集菌体具有最佳转化活性;最优分批转化条件:p H7.0,8.0g/L苯丙酮酸钠,20g/L葡萄糖,1%(v/v)吐温-80,菌体浓度20g/L(干重),37℃,转速200r/min转化0.5h,苯基乳酸产量和转化率分别达到4.91g/L,56%。在上述优化条件上通过流加苯丙酮酸钠和葡萄糖,经6h转化,苯基乳酸最终产量达到17.23g/L,转化率为54%。研究结果表明该重组大肠杆菌成功转化苯丙酮酸合成苯基乳酸,具有较好的应用前景,为系统化代谢工程改造大肠杆菌生物合成苯基乳酸的进一步研究和应用提供了有用的技术参数。     

Behavior of Escherichia coli cells and Bacillus cereus spores on poplar wood crates by impedance measurements

Dry poplar wood crates and glass surfaces (bottoms of Pyrex flasks) were contaminated with aqueous suspensions of Escherichia coli Collection de l'Institut Pasteur (CIP) 54.8 cells or Bacillus cereus CIP 78.3 spores. After different periods of storage at 25 degrees C, the number of cells still present on both surfaces was determined by impedance microbiology. The physical and chemical properties of the wood greatly and rapidly decreased the number of cells. After 4 h of contact time, B. cereus residual metabolic activity corresponded to less than 10% of the initial inoculum level. With a low inoculum level of 2.4 x 10(2) E. coli cells, sterility of the wood was obtained after a contact time of 24 h. With a higher inoculum level of 10(6) E. coli cells, only a few viable bacteria, corresponding to the metabolic activity of four cells, could be recovered after a prolonged contact time of 145 h. Conversely, under the same conditions of storage, when bacteria were deposited on inert and nonporous materials such as glass surfaces, growth occurred. After 24 h of contact time at 25 degrees C, bacterial populations were about 10(9) cells for E. coli and 10(7) cells for B. cereus. Under our experimental conditions after a prolonged contact time, wood exhibited growth-inhibiting properties and cells were no longer metabolically active. These results indicate that the potential for cross-contamination of foods stored directly in contact with previously contaminated poplar wood crates is low under our experimental conditions.     

Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant

Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.     

Characterization of Shiga toxigenic Escherichia coli isolated from foods

The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.     

酶解南美白对虾下脚料制备抗氧化酶解液的研究

以南美白对虾加工下脚料(虾头虾壳)为原料,采用内源酶、中性蛋白酶,通过控制pH、水料比、酶解温度、酶解时间和酶用量等条件进行酶解,从而制取抗氧化酶解液,以还原能力、清除羟基自由基能力为指标选择最优的酶解条件。结果表明,内源酶最佳酶解条件为:pH为6,水料比为2∶1,酶解温度为40℃、酶解时间为6h,此时还原力为0.668,·OH清除率为67.24%;中性蛋白酶的最佳酶解条件为:酶用量为60U/g原料,pH为6,水料比为2∶1,酶解温度为60℃、酶解时间为5h,此时还原力为0.672,·OH清除率为68.43%。在最佳酶解条件下,两种酶解液的还原力与·OH清除率大小排列为:中性蛋白酶>内源酶,但两者相差不大,从经济能源的综合因素考虑,选用内源酶较合适。     

全细胞催化合成L-苯基乳酸重组大肠杆菌的构建

苯基乳酸是一种新型天然广谱抑菌物质,L-乳酸脱氢酶是微生物转化苯丙酮酸合成L-苯基乳酸的关键酶,通过构建过量表达L-乳酸脱氢酶的重组大肠杆菌,提高大肠杆菌合成L-苯基乳酸(苯基乳酸,phenyllactic acid,PLA)的能力。以Bacillus megaterium Z2013513基因组为模板,经PCR扩增得到L-乳酸脱氢酶基因,连接表达载体p ET-28a(+)并导入到大肠杆菌BL21(DE3),获得重组大肠杆菌BL21(DE3)/p ET-28a-ldh L。SDS-PAGE电泳和酶活分析表明,约在40 ku处出现显著特异性条带,粗酶液酶活力达3.4 U/mg。在37℃、200 r/min条件下,25 g/L(干重)重组大肠杆菌经60 min将70.32 mmol/L苯丙酮酸全细胞转化合成50.59 mmol/L L-苯基乳酸。由于具有较高的产物光学纯度(96.98%e.e.)和底物摩尔转化率(71.94%),表明一步生物转化法能高效地合成L-苯基乳酸。     

木材造纸废弃物基质对番茄生长发育的影响

利用木材造纸废弃物（造纸除尘木屑和造纸污泥）制作蔬菜栽培基质。以造纸除尘木屑∶造纸污泥（体积比）分别为3∶1、5∶1、7∶1等3种类型的栽培基质进行番茄无土栽培的比较研究。结果表明：不同配比木材造纸废弃物栽培基质对番茄生长发育、产量的影响均有明显差别。番茄在除尘木屑和污泥基质3∶1的情况下,每667m2番茄产量达8224.6kg,较对照土壤栽培增产13.3%,是较适宜的栽培基质配方。本实验证明,木材造纸废弃物可为大棚作物无土栽培提供一种新的基质资源。