Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.

Response of cellulase activity in pH-controlled cultures of the filamentous fungus Acremonium cellulolyticus.

Cellulase production was investigated in pH-controlled cultures of Acremonium cellulolyticus. The response to culture pH was investigated for three cellulolytic enzymes, carbomethyl cellulase (CMCase), avicelase, and beta-glucosidase. Avicelase and beta-glucosidase showed similar profiles, with maximum activity in cultures at pH 5.5-6. The CMCase activity was highest in a pH 4 culture. At an acidic pH, the ratios of CMCase and avicelase activity to cellulase activity defined by filter paper unit were high, but at a neutral pH, the beta-glucosidase ratio was high. The pH 6.0 culture showed the highest cellulase activity within the range of pH 3.5-6.5 cultures. The saccharification activity from A. cellulolyticus was compared to those of the cellulolytic enzymes from other species. The A. cellulolyticus culture broth had a saccharification yield comparable to those of the Trichoderma enzymes GC220 and Cellulosin T2, under conditions with the same cellulase activity. The saccharification yields from Solka floc, Avicel, and waste paper, measured as the percent of released reducing sugar to dried substrate, were greater than 80% after 96 h of reaction. The yields were 16% from carboxymethylcellulose and 26% from wood chip refiner. Thus, the A. cellulolyticus enzymes were suitable for converting cellulolytic biomass to reducing sugars for biomass ethanol production. This study is a step toward the establishment of an efficient system to reutilize cellulolytic biomass.

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.

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.

Microaeration enhances productivity of bioethanol from hydrolysate of waste house wood using ethanologenic Escherichia coli KO11.

This is the first study showing the successful application of waste house wood (WHW) to the pilot-scale production of bioethanol by hydrolysis using diluted acid and fermentation using the ethanologenic recombinant Escherichia coli KO11. The major sugars in the WHW hydrolysate were glucose, mannose and xylose; the percentages were approximately 35%, 35% and 20% (w/w), respectively. In anaerobic fermentation using a 5-l reactor in which the oxygen transfer rate (OTR) was 0 mmol/(l x h), KO11 consumed only 25% of the xylose in the WHW hydrolysate over the examined fermentation time of 100 h; however, hexoses such as glucose and mannose were consumed completely. Microaeration at an OTR of 4 mmol/(l x h) enhanced the xylose utilization ratio of KO11 to 100%, at which the ethanol concentration was 35.4 g/l and the ethanol yield was 0.42, although the maximum ethanol concentrations were 28.8 and 26.6 g/l at OTRs of 0 mmol/(l x h) and 15 mmol/(l x h), respectively. Moreover, this microaerobic fermentation at OTR of 4 mmol/(l x h) was applied to 1000-l scale bioethanol production using the WHW hydrolysate. The xylose utilization ratio reached 100% and the ethanol yield was determined to be 0.45 for a 63-h fermentation, which were comparable to those obtained from the laboratory-scale fermentation.

Efficient cellulase production by the filamentous fungus Acremonium cellulolyticus.

Cellulase production was investigated in a culture of a strain of Acremonium cellulolyticus. The medium components were optimized for the improvement of cellulase production. The maximum production of cellulolytic enzymes was obtained in a medium containing (grams per liter) 50 Solka Floc, 5 (NH4)2SO4, 24 KH2PO4, 4.7 potassium tartrate hemihydrate, 1.2 MgSO4.7H2O, 1 Tween 80, 4 urea, 0.01 ZnSO4.7H2O, 0.01 MnSO4.6H2O, and 0.01 CuSO4.7H2O, with a pH of 4.0. In the flask culture, 15.5 filter paper units (FPU)/mL of maximum cellulase activity was obtained, 17.42 FPU/mL in a 7-L bioreactor, and 13.08 FPU/mL in a 50-L scale bioreactor for 4-8 d at 30 degrees C. Average production rates were 1.94 FPU/mL.d in flasks, 2.86 FPU/mL.d in the 7-L bioreactor, and 2.56 FPU/mL.d in the 50-L bioreactor. Cellulase production on a small scale was successfully reproduced in the 50-L pilot scale bioreactor. Saccharification activity from A. cellulolyticus was compared with cellulolytic enzymes produced by other strains. The A. cellulolyticus culture broth had a comparable saccharification yield in comparison with those of other Trichoderma enzymes (GC220 or Cellulosin T2) under the same total cellulase activity. Its saccharification yield (percent of released reducing sugar to used dried substrate) was 60%, and its glucose content was 83%.

Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores.

Here we describe the functional relationship between YabG and transglutaminase (Tgl), enzymes that modify the spore coat proteins of Bacillus subtilis. In wild-type spores at 37 degrees C, Tgl mediates the crosslinking of GerQ into higher molecular mass forms; however, some GerQ multimers are found in tgl mutant spores, indicating that Tgl is not essential. Immunoblotting showed that spores isolated from a yabG mutant after sporulation at 37 degrees C contain only very low levels of GerQ multimers. Heat treatment for 20 min at 60 degrees C, which maximally activates the enzymatic activity of Tgl, caused crosslinking of GerQ in isolated yabG spores but not in tgl/yabG double-mutant spores. In addition, the germination frequency of the tgl/yabG spores in the presence of l-alanine with or without heat activation at 60 degrees C was lower than that of wild-type spores. These findings suggest that Tgl cooperates with YabG to mediate the temperature-dependent modification of the coat proteins, a process associated with spore germination in B. subtilis.

Bioconversion of waste office paper to gluconic acid in a turbine blade reactor by the filamentous fungus Aspergillus niger.

Gluconic acid production was investigated using an enzymatic hydrolysate of waste office automation paper in a culture of Aspergillus niger. In repeated batch cultures using flasks, saccharified solution medium (SM) did not show any inhibitory effects on gluconic acid production compared to glucose medium (GM). The average gluconic acid yields were 92% (SM) and 80% (GM). In repeated batch cultures using SM in a turbine blade reactor (TBR), the gluconic acid yields were 60% (SM) and 67% (GM) with 80-100 g/l of gluconic acid. When pure oxygen was supplied the production rate increased to four times higher than when supplying air. Remarkable differences in the morphology of A. niger and dry cell weight between SM and GM were observed. The difference in morphology may have caused a reduction of oxygen transfer, resulting in a decrease in gluconic acid production rate in SM.

Bioconversion of waste office paper to L(+)-lactic acid by the filamentous fungus Rhizopus oryzae.

L(+)-lactic acid production was investigated using an enzymatic hydrolysate of waste office automation (OA) paper in a culture of the filamentous fungus Rhizopus oryzae. In 4 d culture, 82.8 g/l glucose, 7 g/l xylose, and 3.4 g/l cellobiose contained in the hydrolysate were consumed to produce 49.1 g/l of lactic acid. The lactic acid yield and production rate were only 0.59 g/g and 16.3 g/l/d, respectively, only 75% and 61% of the results from the glucose medium. The low production rate from waste OA hydrolysate was elucidated by trials using xylose as the sole carbon source; in those trials, the lactic acid production rate was 7.3 g/l/d, only 28% that of glucose or cellobiose. The low lactic acid yield from waste OA hydrolysate was clarified by trials using artificial hydrolysates comprised of 7:2:1 or 7:1:2 ratios of glucose:cellobiose:xylose. For both, the lactic acid production rate of 17.4 g/l/d matched that of waste OA paper, while the lactic acid yield was similar to that of the glucose medium. This indicates that the production rate may be inhibited by xylose derived from hemicellulose, and the yield may be inhibited by unknown compounds derived from paper pulp.