• Microbiology and Biotechnology Letters
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• v.28 no.2
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• pp.80-86
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• 2000

To develop thermotolerant ethanol producing yeast strains, the protoplasts of Saccharomyces carlsbergensis having good fermentability at $30^{\circ}C$ and Kluyveromyces marxianus able to grow at $42^{\circ}C$ were fused. Under the optimal conditions for protoplast formation, the frequency of protoplast formation of S. carlsbergensis was 92 - 94% and that of K. marxianus was 98%. Fusion frequency between S. carlsbergensis and K. marxianus was $1.4\times10^{-6}-4.8$\times10^{-7}$. Among the 27 fusants obtained, 6 fusants were able to grow at $42^{\circ}C$. While the parental strains produced 3.2-3.4%(w/v) ethanol after 3 days from the fermentation medium containing glucose, fusants SK41-4 and SK53-22 produced 5.2%(w/v) ethanol in the same condition. The thermotolerance of SK53-22 was not high, but that of SK41-4 was quite high.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.222-229
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• 1994

In order to develop a method of economical production and to reduce energy-consumption in fuel alcohol production, we investigated the fermentation characters of two newly selected thermotolerant yeasts. The RA-74-2 showed stable and superior fermentability between 30 and $40^{\circ}C$ in 20% glucose media in comparison to the industrial strains. The optimum concentration of glucose for economical fermentation at $40^{\circ}C$ was 15-18%, and organic nitrogen was necessary for a satisfactory fermentation. The optimum pH was 4.0 and aeration was adversed for high temperature fermentation. Agitation was an important factor at $40^{\circ}C$ and the addition of magnesium ion 0.2% was required in this experiment. When the inoculum was increased, ethanol productivity as well as the speed of fermentation increased. On the other hand RA-912, which can grow at $48^{\circ}C$, showed similar fermentability between 30-$45^{\circ}C$ in 20% glucose media As the concentration of substrate decreased, fermentation ratio increased at $45^{\circ}C$ (45%, 65%, 95% fermentation ratio in 20%, 15%, 10% glucose media, respectively). Also, requirement of organic nitrogen and magnesium ion in RA-912 was similar in RA-74-2. The optimum pH for fermentation was 5.0, and the effects of agitation were enhanced at $37^{\circ}C$ than at $45^{\circ}C$. As the inoculum was increased, fermentation speed became more enhanced but the ethanol productivity was less affected. RA-912 showed fermentability with various substrates. Among the substrates used, inulin was the most promising substrate for the high-temperature fermentation. When 14.5% inulin was used as the substrate, 93% and 55% fermentation ratios were shown at $37^{\circ}C$ and $45^{\circ}C$, respectively.

• Microbiology and Biotechnology Letters
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• v.27 no.2
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• pp.145-152
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• 1999

Brettanomyces custersii CBS 5512 which has reported as a thermotolerant glucose-cellobiose co-fermentable yeast strain was mutated with UV and NTG to improve ethanol yield at higher than 4$0^{\circ}C$ B. custersii H1-23, H1-39, H1-55 and H1062 were finally selected for hyper-fermentable strains at higher than 4$0^{\circ}C$ from thermotolerant 7510 colonies through 5th selection. Among the selected strains, H1-39 mutant had better fermentability at 4$0^{\circ}C$ and 43$^{\circ}C$ from different concentrations of glucose. H1-39 and H1-23 mutants yielded more than 70% of the theoretical ethanol yield in 4 and 8% mixed sugars at above 4$0^{\circ}C$, which was 5-11% higher than those by original strain. Especially, H1-39 mutant had better fermentability in 4% mixed sugar. It showed 78.5% of the theoretical yield at 4$0^{\circ}C$ and 72.2% of the theoretical yield at 43$^{\circ}C$. On the other hand, theoretical yield of ethanol by H1-39 mutant in 8% mixed sugar at 4$0^{\circ}C$ and 43$^{\circ}C$ were 75.2% and 70.2%, respectively. Theses values increased up to 7-11% as compared to those by orginal strain. By the simultaneous saccharification and fermentation, ethanol production by H1-39 mutant increased up to more than 23% as compared to that by original strain.

• Journal of Life Science
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• v.13 no.1
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• pp.110-117
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• 2003

A thermotolerant yeast strain, Saccharomyces cerevisiae KNU5377 (abbreviated as KNU5377, was exposed to inorganic acids including sulfuric, nitric and hydrochloric acid. As a stressor, each inorganic acid is very easily dissociated in water, resulting in lowering environmental pH. When compared with a reference S. cerevisiae ATCC24858, KNU5377 could overcome such a severe condition containing a final 0.4% concentration of sulfuric acid or nitric acid to grow at the overnight culture, but this reference could not. Additionally, this strain showed a surprisingly strong tolerance by surviving despite of exposure to the regime of 0.35% of hydrochloric acid for over 90 min and also to 0.6% of sulfuric acid for 30 min. On the contrary, both strains could not survive against a final 0.45% concentration of nitric acid. This strain KNU5377 could produce ethanol of 3% in 2 days by using the fermentation medium containing a final 0.3% concentration of sulfuric arid. Moreover, change into a final 0.2% concentration of sulfuric acid caused this strain to enhance fermentation productivity up to about 4.5% even at $40^{\circ}C$. In exposure to a final 0.2% of sulfuric acid for 60 min, trehalose was most accumulated within 30 min in KNU5377, and this suggested a cellular defense system led by this disaccharide was profitable for this strain to lead to no morphological changes.

• Journal of the Korea Organic Resources Recycling Association
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• v.5 no.1
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• pp.1-13
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• 1997

The fermentative conversion of food wastes into feed was investigated by seeding of mixed inoculum YM (Youngjin Environmental co.), and thermotolerant yeast Kl. marxianus. For 6 days' fermentaion, the fermentation method of 2 days' aerobic followed by 4 days' anaerobic was better for the production of organic acids and increasing total microbial population than 6 days' continuous aerobic or anaerobic fermentation. By seeding YM, the total microbial count increased about 100 times of the control group. In addition, Kl. marxianus seeding together with YM increased total viable cell count, but did not increase yeast count significantly.

• Korean Chemical Engineering Research
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• v.51 no.6
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• pp.709-715
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• 2013

The waste from beer fermentation broth (WBFB) has been found an excellent and inexpensive resource for bioethanol production. We tried to evaluate the saccharification and fermentation capabilities of WBFB to confirm its effectiveness for bioethanol production. The saccharification potentials of the WBFB were evaluated at various temperatures (30, 40, 50, 60 and $70^{\circ}C$). It was found that the saccharification capabilities increased with temperature and highest reached maximum at $60^{\circ}C$ and $70^{\circ}C$ after 4h. Ethanol production from a mixture of WBFB and chemically defined media (CDM) without addition of any microbial species confirmed the fermentation capabilities of WBFB. Simultaneous saccharification and fermentation were performed using WBFB, starch solution and CDM as culturing media. The maximum yield of bioethanol production was obtained at $30^{\circ}C$. The saccharifying enzymes and the yeast cells present in WBFB were essential factors for the production of bioethanol from WBFB without any additional enzymes or microbial cells.

• Journal of the Korea Organic Resources Recycling Association
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• v.13 no.4
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• pp.100-106
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• 2005

Sugar beet stillages were used as a substrate for the production of single cell protein by the thermotolerant yeasts Candida rugosa, Kluyveromyces marxianus and C. utilis. The biomass production increased in accordance with the increase of pH-value, but protein content decreased. C. rugosa showed the highest crude protein production as 3.68g/l and C. utilis 2.9g/l, Kl. marxianus 2.30g/l, respectively. The rate of COD reduction in stillage versus crude protein production of C. rugosa showed the highest value as 0.35~0.39g/l as a good strain for single cell protein production using sugar beet stillages.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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• pp.43-51
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• 2002

The cloning and expression of $\beta-glucosidase$ II, encoded by the gene ${\beta}glu2$, from thermotolerant yeast Pichia etchellsii into Escherichia coli is described. Cloning of the 7.3 kb BamHI/SalI yeast insert containing ${\beta}glu2$ in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid into E. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At $50^{\circ}C$, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of $0.14\;h^{-l}$. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.304-312
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase ${\delta}$ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'${\rightarrow}$5' exonuclease activity. The resulting $HpPOL3^*$ gene encoding the error-prone proofreading-deficient DNA polymerase ${\delta}$ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a $URA3^-$ mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of $HpPOL3^*$. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

• Microbiology and Biotechnology Letters
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• v.48 no.2
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• pp.179-184
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• 2020

The MBY/L6793 strain showing the highest ethanol yield of 0.48 ± 0.00 g ethanol/ g glucose was isolated from Senecio cruentus. Its ethanol yield was approximately 1.5 times that of the MBY/L6986 isolated from Callistephus chinensis. The strain was identified as Hanseniaspora opuntiae by sequence analysis of the 18S rRNA gene, and the sequenced gene was registered to the GenBank (MN859968). When grown at 40℃, the strain produced 3.82 ± 0.98 g ethanol from 20 g glucose and 10.05 ± 0.06 g ethanol from 60 g glucose, corresponding to approximately 2.45 and 5.74 times, respectively, compared to the control strain H. opuntiae KCCM50747. The MBY/L6793 strain was deposited to KCTC (Korean Collection for Type Culture) as KCTC37025.