• Journal of Microbiology and Biotechnology
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• v.6 no.4
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• pp.286-291
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• 1996

A study was conducted to investigate the characteristics of segregation and alcohol fermentation of intergeneric fusants. The protoplast fusion of both Pichia stipitis CBS 5776 and Saccharomycess cerevisiae STV 89 was carried out. The fusion frequency was $5\times10^{-8}$ and among fusants selected, a fusant F5 showed the best results in ethanol production by sucrose and xylose fermentations. The performance of xylose fermentation by this fusant was better than that of P. stipitis CBS 5776 and fusant F5 exhibited sucrose fermentation patterns intermediate to the two parent strains. The fusant F5 was segregated into a pair of parental strains during the several culture passages. In the average, 91$%$ of colonies had a similar characteristics of P. stipitis while 7$%$ of colonies resembled S. cerevisiae. Only 2$%$ of colonies had the characteristics of the original fusants. At the sixth passage, all segregants resembled P. stipitis. From these results it is suggested that intergeneric protoplast fusion led to an integration of S. cerevisiae genes, rather than whole chromosomes, within the entire genome of P. stipitis.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.565-573
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• 2019

Production of biofuels and value-added materials from cellulosic biomass requires the development of a microbial strain capable of efficiently fermenting mixed sugars. In this study, the natural xylose fermenting yeast, Pichia stipitis, was evolved to simultaneously ferment cellobiose and xylose. Serial subcultures of wild-type P. stipitis in 20 g/l cellobiose were performed to increase the rate of cellobiose consumption. A total of ten rounds of the serial subculture led to the isolation of an evolved strain fermenting cellobiose significantly faster than the parental strain. The evolved strain displayed enhanced ethanol yield from 0 to 0.4 g ethanol/g cellobiose. The evolved P. stipitis simultaneously fermented cellobiose and xylose in batch fermentation. The genetic information of our evolved P. stipitis would be valuable in the development of a microbial host for the production of biofuels and biomaterials from cellulosic biomass.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.264-269
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• 2014

For the production of ethanol from seaweed as the source material, thermal acid hydrolysis and enzymatic saccharification were carried out for monosugars production of 25.5 g/l galactose and 7.6 g/l glucose using Gelidium amansii. The fermentation was performed with Pichia stipitis KCTC 7228 or Saccharomyces cerevisiae KCCM 1129. When wild P. stipitis and S. cerevisiae were used, the ethanol productions of 11.2 g/l and 6.9 g/l were produced, respectively. The ethanol productions of 16.6 g/l and 14.6 g/l were produced using P. stipitis and S. cerevisiae acclimated to high concentration of galactose, respectively. The yields of ethanol fermentation increased to 0.5 and 0.44 from 0.34 and 0.21 using acclimated P. stipitis and S. cerevisiae, respectively. Therefore, acclimation of yeasts to a specific sugar such as galactose reduced the glucose-induced repression on the transport of galactose.

• Journal of Life Science
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• v.29 no.3
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• pp.376-381
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• 2019

To construct useful yeast strain for bioethanol production, we improved yeast harboring various phenotypes by using yeast protoplast fusion method. In this study, S. cerevisiae BYK-F11 strain which have ethanol tolerance, thermotolerance and ${\beta}-glucanase$ activity and P. $stipitis{\Delta}ura$ strain which has xylose metabolism pathway were fused by genome shuffling. P. $stipitis{\Delta}ura$ strain was constructed for protoplast fusion by URA3 gene disruption, resulting in uracil auxotroph. By protoplast fusion, several fused cells were selected and BYKPS-F8 strain (fused cell) showing both karyotypes from two parent strains (S. cerevisiae BYK-F11 and P. $stipitis{\Delta}ura$ strain) among 22 fused cells was finally selected. Sequentially, various phenotypes such as ${\beta}-glucanase$ activity, xylose utility, ethanol tolerance, thermotolerance and ethanol productivity were analyzed. The BYKPS-F8 strain obtained ${\beta}-glucanase$ activity from BYK-F11 strain and 1.2 fold increased xylose utility from P. $stipitis{\Delta}ura$ strain. Also, the BYKPS-F8 strain showed thermotolerance at $40^{\circ}C$ and increased ethanol tolerance in medium containing 8% ethanol. In this fused cell, 7.5 g/l ethanol from 20 g/l xylose was produced and the multiple phenotypes were stably remained during long term cultivation (260 hr). It was proved that novel biological system (yeast strains) is easily and efficiently bred by protoplast fusion among yeasts having different genus.

• KSBB Journal
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• v.25 no.4
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• pp.351-356
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• 2010

To increase the production of ethanol by using sugar from lignocellulosic biomass, pentose and hexose have to be fermented simultaneously by yeast. The effects of mixed sugar and nitrogen on ethanol production by immobilized Pichia stipitis KCCM 12009 were investigated. When optimal mixed sugar and nitrogen concentration were 5% (Glucose/Xylose = 3:1) and 1%, respectively, ethanol concentration produced by immobilized P. stipitis was 19-20 g/L. In repeated fed-batch by immobilized P. stipitis, all glucose was consumed very quickly at 1-3% mixed sugar concentration. But, xylose consumption was decreased as the mixed sugar concentration increased. Also, ethanol (5.6 g/L) was stably produced and ethanol production rate was 0.13 g/$L{\cdot}h$ in immobilized cell reactor (ICR) with 1% mixed sugar (Glucose/Xylose = 3:1) as feeding media.

• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1434-1444
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• 2013

We established a two-step production process using immobilized S. cerevisiae and P. stipitis yeast to produce ethanol from seaweed (U. pertusa Kjellman) hydrolysate. The process was designed to completely consume both glucose and xylose. In particular, the yeasts were immobilized using DEAE-corncob and DEAE-cotton, respectively. The first step of the process included a continuous column reactor using immobilized S. cerevisiae, and the second step included a repeated-batch reactor using immobilized P. stipitis. It was verified that the glucose and xylose in 20 L of medium containing the U. pertusa Kjellman hydrolysate was converted completely to about 5.0 g/l ethanol through the two-step process, in which the overall ethanol yield from total reducing sugar was 0.37 and the volumetric ethanol productivity was 0.126 g/l/h. The volumetric ethanol productivity of the two-step process was about 2.7 times greater than that when P. stipitis was used alone for ethanol production from U. pertusa Kjellman hydrolysate. In addition, the overall ethanol yield from glucose and xylose was superior to that when P. stipitis was used alone for ethanol production. This two-step process will not only contribute to the development of an integrated process for ethanol production from glucose-and xylose-containing biomass hydrolysates, but could also be used as an alternative method for ethanol production.

• Microbiology and Biotechnology Letters
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• v.20 no.1
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• pp.91-95
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• 1992

The hydrolyzates of lignocellulosic biomass contain a mixture of glucose, xylose and cellobiose. The yeast which can produce ethanol efficiently from xylose and cellobiose was selected and its growth and ethanol formation behavior on each sugar and their mixture were investigated. Ethanol yields during batch culture of Pichia stipitis CBS 5776 were 0.4. 0.36 and 0.23 g/g substrate on glucose, xylose and cellobiose, respectively. Mixed sugar fermentation data indicate that glucose causes catabolite regulation on xylose and cellobiose utilization. However, xylose and cellobiose were utilized simultaneously. Ethanol yields on mixtures of sugars were generally additive for each of the substrates.

• KSBB Journal
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• v.26 no.4
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• pp.317-322
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• 2011

Ulva pertusa is one of the worst pollutant like a waste vinyl after agriculture and caused bad smell at seashore in Jejudo and south area of korean peninsular. For favorable environmental utilization of Ulva pertusa, it could be applied for ethanol production with its acid hydrolysate. The components of hydrolysate included fermentable sugar of glucose, xylose, mannose, galactose, and higher amounts of unfermentable rhamnose. Fermentable sugars were converted to ethanol with S. cerevisiae, also xylose to ethanol with P. stipitis, their maximun ethanol production at optimum conditions were 462 ${\mu}g$/mL and 475 ${\mu}g$/mL, respectively. While, rhamnose cannot be changed to ethanol with S. cerevisiae or P. stipitis, alone. Combination of S. cerevisiae and P. stipitis can convert rhamnose to ethanol, because P.stipitis degradaded rhamnose to pyruvate, and then S. cerevisiae convert to ethanol, at optimum conditions, ethanol reached to 782 ${\mu}g$/mL (30.24%) that is higher than that of 2 strain alone from 500 mg of dried Ulva pertusa contained 2586.45 ${\mu}g$/mL of reduced sugars. Ulva pertusa can be utilized for renewal energy insted of environmenatal enemy.

• KSBB Journal
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• v.4 no.1
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• pp.57-61
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• 1989

The feasibility of separating Pichia stipitis from a fermentation broth using a hollow fiber membrane was evaluated. The permeate flux was affected by such parameters as cell concentration, pH, content of antifoam agents, suction pressure, and recirculation rate. A minor effect of temperature on the flux loss was also observed. A microcomputer-aided backflush was proven effective in alleviating membrane fouling and allowing long term separation of P. stipitis from a fermentation broth.

• Journal of Life Science
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• v.27 no.12
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• pp.1403-1409
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• 2017

In this study, the xylitol dehydrogenase (XYL2) gene was expressed in Saccharomyces cerevisiae as a host cell for ease of use in the degradation of lignocellulosic biomass (xylose). To select suitable expression systems for the S.XYL2 gene from S. cerevisiae and the P.XYL2 gene from Pichia stipitis, $pGMF{\alpha}-S.XYL2$, $pGMF{\alpha}-P.XYL2$, $pAMF{\alpha}-S.XYL2$ and $pAMF{\alpha}-P.XYL2$ plasmids with the GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter to allow secretion. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$ strain and the xylitol dehydrogenase activity was investigated. The GAL10 promoter proved more suitable than the ADH1 promoter for expression of the XYL2 gene, and the xylitol dehydrogenase activity from P. stipitis was twice that from S. cerevisiae. The xylitol dehydrogenase showed $NAD^+$-dependent activity and about 77% of the recombinant xylitol dehydrogenase was secreted into the periplasmic space of the $SEY2102{\Delta}trp1/pGMF{\alpha}-P.XYL2$ strain. The xylitol dehydrogenase activity was increased by up to 41% when a glucose/xylose mixture was supplied as a carbon source, rather than glucose alone. The expression system and culture conditions optimized in this study resulted in large amounts of xylitol dehydrogenase using S. cerevisiae as the host strain, indicating the potential of this expression system for use in bioethanol production and industrial applications.