Improvement of bacterial hydrogen production by ATP in mixed organic compounds extracted from Rhodobacter sphaeroides aerobically cultured under dark conditions.

The aim of this study was to increase the hydrogen production of recombinant Escherichia coli harboring HupSL hydrogenase by supplementing physiologically activating compounds extracted from Rhodobacter sphaeroides cultured under anaerobic dark condition after treating them with dimethyl sulfoxide, and the 0.5% extracts contained 4×10(-8)M ATP, which was 100-fold higher than that in the extracts from E. coli. In addition, it was found that the hydrogen production from recombinant E. coli harboring HupSL hydrogenase isolated from R. sphaeroides was doubled under anaerobic conditions when it was supplemented by the extracts from R. sphaeroides cultured aerobically in dark conditions, and this also showed consistent pattern with the increased level of HupSL hydrogenase expression. Therefore, we conclude that the mixed organic compounds extracted from R. sphaeroides have an ATP which enhances the hydrogen production by increasing the amount of HupSL hydrogenase.

Fermentation and evaluation of Klebsiella pneumoniae and K. oxytoca on the production of 2,3-butanediol.

Klebsiella is one of the genera that has shown unbeatable production performance of 2,3-butanediol (2,3-BD), when compared to other microorganisms. In this study, two Klebsiella strains, K. pneumoniae (DSM 2026) and K. oxytoca (ATCC 43863), were selected and evaluated for 2,3-BD production by batch and fed-batch fermentations using glucose as a carbon source. Those strains' morphologies, particularly their capsular structures, were analyzed by scanning electron microscopy (SEM). The maximum titers of 2,3-BD by K. pneumoniae and K. oxytoca during 10 h batch fermentation were 17.6 and 10.9 g L(-1), respectively; in fed-batch cultivation, the strains showed the maximum titers of 50.9 and 34.1 g L(-1), respectively. Although K. pneumoniae showed higher productivity, SEM showed that it secreted large amounts of capsular polysaccharide, increasing pathogenicity and hindering the separation of cells from the fermentation broth during downstream processing.

Effect of glucose on xylose utilization in Saccharomyces cerevisiae harboring the xylose reductase gene.

We have constructed recombinant Saccharomyces cerevisiae JH1 harboring a xylose reductase gene (xyl1) isolated from Pichia stipitis. However, JH1 still utilizes glucose more easily than xylose. Therefore, in this study, we characterized the effect of a glucose supplement on xylose utilization, the expression level of xylose reductase as a recombinant gene in JH1, and the expression levels of two hexose transporters (Hxt4 and Hxt7) due to co-fermentation of different concentrations of glucose and xylose. Co-fermentation using 20 g/l of glucose increased xylose consumption up to 11.7 g/l, which was 7.9-fold that of xylose fermentation without a glucose supplement. In addition, we found xyl1 mRNA levels dramatically increased as cells grew under co-fermentation conditions with supplementary glucose; this result is consistent with a significant decrease in the xylose concentration 48 h after cultivation. In addition, the expression levels of Hxt4 and Hxt7 were strongly activated by the presence of glucose and xylose; in particular, Hxt7 showed a 2.9-fold increased expression relative to that of recombinant S. cerevisiae JHM with only a backbone vector, pYES2. The results of this study suggest that xylose utilization would be improved by activation of hexose transporters induced by glucose (rather than xylose) reductase expression.

Enhanced enzymatic saccharification of barley straw pretreated by ethanosolv technology.

The fermentable sugars in lignocellulosic biomass are derived from cellulose and hemicellulose, which are not readily accessible to enzymatic saccharification because of their recalcitrance. An ethanosolv pretreatment method was applied for the enzymatic saccharification of barley straw with an inorganic acid. The effects of four process variables (temperature, time, catalyst dose, and ethanol concentration) on the barley straw pretreatment were analyzed over a broad range using a small composite design and a response surface methodology. The yield of the residual solid and composition of the solid fraction differed as ethanosolv conditions varied within the experimental range. A glucan recovery, xylan recovery, and delignification were 85%, 14%, and 69% at center point conditions (170°C, 60 min, 1.0% (w/w) H(2)SO(4), and 50% (w/w) ethanol), respectively. Ethanosolv pretreatment removed lignin effectively. Additionally, the highest enzymatic digestibility of 85.3% was obtained after 72 h at center point conditions.

Specific expression patterns of xyl1, xyl2 and xyl3 in response to different sugars in Pichia stipitis.

The effects of two different sugars (glucose and xylose) on the expression levels and patterns of xylose reductase (xyl1), xylitol dehydrogenase (xyl2) and xylulokinase (xyl3) genes were analyzed using Pichia stipitis. A significant increase in mRNA levels of xyl1 was observed after 6 hours growth in culture conditions using xylose as a sole carbon source, but expressions of the three genes were not influenced by normal culture media with glucose. In addition expression levels of xyl2 and xyl3 were not observed during the entire culture period during which xylose was added. It also was found that the expression level of xyl1 increased as a function of the xylose concentration (40, 60, 80 g/l) used in this study, indicating that xyl1 expression sensitively responded to xylose presence in the culture media. Although the induced level of xyl2 increased slightly after 48 hours in the xylose-supplemented culture conditions, the expression level of xyl2 was not observed in the xylitol-supplemented culture conditions. Finally, considering the expression of each gene in response to glucose or xylose, the absolute expression levels of the three genes indicate that xyl1 is induced primarily by exposure to xylose.

Isolation and characterization of ethanol-producing Schizosaccharomyces pombe CHFY0201.

Ethanol-producing yeast strain, CHFY0201 was isolated from soil in South Korea using an enrichment technique in a yeast peptone dextrose medium supplemented with 5% (w/v) ethanol at 30 degrees C. The phenotypic and physiological characteristics, as well as molecular phylogenetic analysis based on the D1/D2 domains of the large subunit (26S) rDNA gene and the internally transcribed spacer (ITS) 1+2 regions suggested that the CHFY0201 was novel strain of Schizosaccharomyces pombe. During shaking flask cultivation, the highest ethanol productivity and theoretical yield of S. pombe CHFY0201 in YPD media containing 9.5% total sugars was 0.59 +/- 0.01 g/l/h and 88.4 +/- 0.91%, respectively. Simultaneous saccharification and fermentation for ethanol production was carried out using liquefied cassava (Manihot esculenta) powder in a 5 l lab-scale jar fermenter at 32 degrees C for 66 h with an agitation speed of 120 rpm. Under these conditions, S. pombe CHFY0201 yielded a final ethanol concentration of 72.1 +/- 0.27 g/l and a theoretical yield of 82.7 +/- 1.52% at a maximum ethanol productivity of 1.16 +/- 0.07 g/l/h. These results suggest that S. pombe CHFY0201 is a potential producer for industrial bioethanol production.

Continuous ethanol production from cassava through simultaneous saccharification and fermentation by self-flocculating yeast Saccharomyces cerevisiae CHFY0321.

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h(-1). Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h(-1). At dilution rates in the range of 0.014 h(-1) to 0.042 h(-1), high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h(-1), the ethanol volumetric productivity and ethanol yield were 3.58 g/L x h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.