Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.     

Strain improvement of thermotolerant Saccharomyces cerevisiae VS strain for better utilization of lignocellulosic substrates

AIM: Pentose-utilizing yeast development by protoplast fusion and sequential mutations and ethanol fermentation using lignocellulosic substrate. METHODS AND RESULTS: Protoplasts of thermotolerant Saccharomyces cerevisiae and mesophilic, xylose-utilizing Candida shehatae were fused by electrofusion. The fusants were selected based on their growth at 42 degrees C and ability to utilize xylose. The selected best fusant was mutated sequentially and 3 mutant fusants obtained were tested for their stability. The mutant fusant CP11 was found to be stable and used for lignocellulosic fermentation. The Prosopis juliflora wood material was hydrolysed with 1% sulphuric acid initially for 18 h at room temperature and then for 20 min at 121 degrees C. The acid hydrolysate was separated and used for detoxification by ethyl acetate and overliming. The hard cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 50 degrees C. The substrate (15% w/v) yielded 84 g l(-1) sugars, representing 80% (w/w) hydrolysis of carbohydrate content present in the lignocellulosic material. The acid and enzyme hydrolysates were then equally mixed and used for fermentation with the developed fusant yeast (CP11). The fusant yeast gave an ethanol yield of 0.459 +/- 0.012 g g(-1), productivity of 0.67 +/- 0.015 g l(-1) h(-1) and fermentation efficiency of 90%. CONCLUSIONS: Protoplast fusion followed by sequential mutations method gave a stable and good performing fusant with maximum utilization of reducing sugars in the media. SIGNIFICANCE AND IMPACT OF THE STUDY: This new method could be applied to develop fusants for better biotechnological applications.     

Characterization and potential industrial applications of five novel,thermotolerant, fermentative,yeast strains

All five novel, thermotolerant, alcohol-producing Kluyveromyces marxianus yeast strains, capable of growth at 52°C, grew aerobically on lactose (growth rates of 0.43 to 0.5 h^-1), whey permeate (0.38 to 0.43 h^-1), cellobiose (0.18 to 0.3 h^-1) and xylose (0.18 to 0.26 h^-1). They also grew anaerobically at 40 (0.18 to 0.19 h^-1) and 45°C (0.09 to 0.14 h^-1). All fermented glucose to ethanol and tolerated ethanol at 95 g l^-1 at 45°C. Their potential for industrial alcohol production, possibly in combination with the dairy and wood-related industries, is discussed.I.M. Banat was and R. Marchant is with the School of Applied Biology and Chemical Sciences, University of Ulster. Coleraine BT52 1SA, UK. I.M. Banat is now with the Department of Biology, UAE University, P.O. Box 17551. Al Ain. Abu-Dhabi, United Arab Emirates     

Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain

A Bacillus licheniformis strain, 189, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 degrees C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 degrees C and 100 degrees C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.     

Isolation by genetic and physiological characteristics of a fuel-ethanol fermentative Saccharomyces cerevisiae strain with potential for genetic manipulation

Fuel ethanol fermentation process is a complex environment with an intensive succession of yeast strains. The population stability depends on the use of a well-adapted strain that can fit to a particular industrial plant. This stability helps to keep high level of ethanol yield and it is absolutely required when intending to use recombinant strains. Yeast strains have been previously isolated from different distilleries in Northeast Brazil and clustered in genetic strains by PCR-fingerprinting. In this report we present the isolation and selection of a novel Saccharomyces cerevisiae strain by its high dominance in the yeast population. The new strain, JP1 strain, presented practically the same fermentative capacity and stress tolerance like the most used commercial strains, with advantages of being highly adapted to different industrial units in Northeast Brazil that used sugar cane juice as substrate. Moreover, it presented higher transformation efficiency that pointed out its potential for genetic manipulations. The importance of this strain selection programme for ethanol production is discussed.     

Genetic improvement of thermo-tolerance in wine Saccharomyces cerevisiae strains by a backcross approach

During red wine fermentation, high temperatures may cause stuck fermentation by affecting the physiology of fermenting yeast. This deleterious effect is the result of the complex interaction of temperature with other physicochemical parameters of grape juice, such as sugar and lipid content. The genetic background of fermenting yeast also interacts with this complex matrix and some strains are more resistant to high temperatures than others. Here, the temperature tolerance of nine commercial starters was evaluated, demonstrating that, at high sugar concentrations, half of them are sensitive to temperature. Using a classical backcross approach, one thermo-sensitive commercial starter was genetically improved by introducing quantitative trait loci conferring resistance to temperature. With this breeding program it is possible to obtain a thermo-resistant strain sharing most of its genome with the initial commercial starter. The parental and improved strains were compared for population growth and fermentation ability in various conditions. Despite their common genetic background, these two strains showed slight physiological differences in response to environmental changes that enable identification of the key physiological parameters influencing stuck fermentation.     

酿酒酵母单倍体菌株分离筛选及其产腺苷甲硫氨酸的初步研究

获得产腺苷甲硫氨酸的二倍体酿酒酵母CGMCC 2842遗传育种单倍体亲本。采用不同产孢培养基考察了酿酒酵母的产孢率,并对酿酒酵母CGMCC 2842进行了生孢培养分离子囊孢子得到单倍体菌株,确定单倍体配型,测定不同单倍体菌株腺苷甲硫氨酸含量。从分离的七株单倍体菌株(6株a型和1株α型)中筛选出一株产腺苷甲硫氨酸较高的a配型的单倍体菌株,经250 m L摇瓶发酵48 h后产腺苷甲硫氨酸1.10 g/L。筛选得到了一株产腺苷甲硫氨酸较高a型的单倍体菌株,为菌株的进一步遗传育种改良和腺苷甲硫氨酸微生物发酵法规模化生产奠定了基础。     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae

To produce an industrial strain of Saccharomyces cerevisiae that metabolizes xylose, we constructed a rDNA integration vector and YIp integration vector, containing the xylose-utilizing genes, XYL1 and XYL2, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and XKS1, which encodes xylulokinase (XK) from S. cerevisiae, with the G418 resistance gene KanMX as a dominant selectable marker. The rDNA results in integration of multiple copies of the target genes. The industrial stain of S. cerevisiae NAN-27 was transformed with the two integration vectors to produce two recombinant strains, S. cerevisiae NAN-127 and NAN-123. Upon transformation, multiple copies of the xylose-utilizing genes were integrated into the genome rDNA locus of S. cerevisiae. Strain NAN-127 consumed twice as much xylose and produced 39% more ethanol than the parent strain, while NAN-123 consumed 10% more xylose and produced 10% more ethanol than the parent strain over 94 h.     

大白口蘑菌株"厦3"的分离驯化研究

采用菌柄组织分离,从野生大白口蘑获得菌株“厦3”,经转管驯化后,在适宜温度下,菌丝满管时间从32d左右缩短到15d左右。以棉籽壳基质的菌丝长速最快,沟叶结缕草基质次之,杂木屑基质最慢;经覆土袋栽,沟叶结缕草基质的生物效率（155.8%）高于木屑基质（80.1%）,增产94.5%。脱袋栽,在草料上的生物效率达123.9％。     

Characterization of technological features of dry yeast (strain I-7-43) preparation, product of electrofusion between Saccharomyces cerevisiae and Saccharomyces diastaticus, in industrial application

The aim of the study was to verify the technological usability and stability of biotechnological features of active dry distillery yeast preparation (strain I-7-43 with amylolytic abilities) applied to full-scale production of agricultural distillery. Various reduced doses of glucoamylase preparation (San-Extra L) were used for starch saccharification, from 90% to 70% in relation to the full standard dose of preparation. The dry distillery yeast I-7-43 were assessed positively in respect to fermentation activity and yield of ethanol production. Application of the dry yeast I-7-43 preparation in distillery practice lowers the costs of spirit production by saving the glucoamylase preparation (up to 30%) used in the process of mash saccharification. Concentrations of the volatile fermentation by-products in raw spirits obtained from fermentations with application of I-7-43 strain were on the levels guaranteeing good organoleptic properties of distillates.     

Fermentation of high concentrations of lactose to ethanol by engineered flocculent Saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l⁻¹ in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l⁻¹ h⁻¹, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.     

Diversity of Y region at HML locus in a Saccharomyces cerevisiae strain isolated from a Sardinian wine

Several mutations in genes involved in Saccharomyces mating type switching may affect the homothallic behaviour in wine yeasts. In this study the semi-homothallic (Hq) segregation of a flor wine yeast strain was analysed. We aimed to understand the molecular basis of this behaviour in a flor autochthonous strain, verifying the MAT locus status by a PCR-based HO gene disruption and sequencing of the Y region of the HML, HMR and MAT loci, after nested PCR. Presence of ORFs a1 and a2 in the Y region of the HML locus was found. At the ORF a2 at HML locus, a mutation in the stop codon was found, so the a2 ORF contains 33 more bases.     

Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor-controlled bioreactor

AIMS: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. METHODS AND RESULTS: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1.45-fold improved than its wild parent with respect to ethanol productivity (7.2 g l-1 h-1), product yield (0.44 g ethanol g-1 substrate utilized) and specific ethanol yield (19.0 g ethanol g-1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P相似文献   

Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme.

Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.     

Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae

Summary The PGI1 gene of Saccharomyces cerevisiae coding for the glycolytic enzyme phosphoglucose isomerase has been cloned by complementation of a mutant strain (pgi1) with a strongly reduced phosphoglucose isomerase activity. A genomic library constructed in the yeast multicopy vector YEp13 (Nasmyth and Tatchell 1980) was used. Four plasmids containing an overlapping region of 4.1 kb were isolated and characterized by restriction endonuclease mapping. Southern analysis of genomic digests prepared with different restriction enzymes confirmed the same pattern for the chromosomal sequences. Transformants with the isolated plasmids had a phosphoglucose isomerase activity increased by a factor of 7. The cloned sequence hybridized to a constitutively synthesized 2.2 kb RNA in Northern analysis. The coding region includes a 2.05 kb EcoRI fragment common to all four inserts. A fragment including part of the PGI1 region was subcloned into vector YRp7 and used to induce integration at the PGI1 locus. Genetical and Southern analysis of stable transformants showed that single as well as tandem integration took place at this locus. This showed that the PGI1 gene had been isolated. Finally, and in contrast to the results of Kempe et al. (1974a, b) who reported three isoenzymes in yeasts, only one copy of the PGI1 gene per genome was found in several laboratory strains tested by Southern analysis.     

Isolation and characterization of a mutant of Saccharomyces cerevisiae defective in phosphoenolpyruvate carboxykinase

A mutant of Saccharomyces cerevisiae lacking phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) was isolated. The mutant did not grow on gluconeogenic sources except glycerol. The mutation was recessive and apparently affected the structural gene of the enzyme. Intracellular levels of metabolites related to the metabolic situation of the enzyme were not significantly affected after transfer of the mutant from a medium with glycerol to a medium with ethanol as carbon source. In these conditions only AMP decreased 3 to 5 times. A search for mutants affected in the other gluconeogenic enzyme, fructose 1,6 bisphosphatase, remained unsuccessful.Abbreviation PEPCK phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32)     

Combining induced mutation and protoplasting for strain improvement of Aspergillus oryzae for kojic acid production

By combining induced mutation, using NTG and UV irradiation, and protoplasting of a wild type strain of Aspergillus oryzae ATCC 22788, a hyper-producing strain was obtained that accumulated 41 g kojic acid l(-1) in shake-flasks, which was 100-fold higher than that in the wild type strains. Similar production of kojic acid was obtained in 5 l stirred-tank fermentations.     

好氧反硝化菌Defluvibacter lusatiensis str.DN7的分离鉴定和异养硝化性能

从稳定运行处理竹子加工废水的生物接触氧化反应器中分离得到一株好氧反硝化菌DN7,其72 h NO3-降解率达99.4％.细胞显微镜观察显示,菌株为革兰氏阴性小杆菌,大小为0.5 μm×1.5 μm,菌落为乳白色.通过生理生化特性及16S rDNA同源性分析,初步推断该菌株为根瘤菌中的Defluvibacter lusatiensis str.碳源、C/N、硝酸盐初始浓度、溶解氧(DO)、pH对DN7反硝化性能影响的结果表明:菌株对柠檬酸钠、葡萄糖等小分子有机物的利用较好;C/N为9时,脱氮率达99.0％;硝酸盐浓度低于138.48 mg·L-1情况下,DN7脱氮率在96％以上,且亚硝酸盐浓度均在1.0mg·L-1以下;菌株DN7对DO不敏感,中性偏碱性环境有利于DN7反硝化反应的进行;DN7具有良好的异养硝化性能,72 h铵氮降解率达84.7％.