Bioconversion of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast.

Water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate has been utilized as a substrate for ethanol production using Pichia stipitis NRRL Y-7124. Hydrolysate fermentability was considerable improved by boiling, and overliming up to pH 10.0 with solid Ca(OH)(2) in combination with sodium sulfite. The percent total sugar utilized and ethanol yield (Y(p/s)) for the untreated hydrolysate were 20.15+/-0.17% and 0.19+/-0.003 g(p) g(s)(-1), respectively, compared with 76.0+/-0.32% and 0.35 g(p) g(s)(-1), respectively for the treated material. The fermentation was very effective at an aeration rate of 0.02 v/v/m, temperature 30+/-0.2 degrees C and pH 6.0+/-0.2. However, the volumetric productivity (Q(p)) was still considerably less than observed in a simulated synthetic hydrolysate medium with a sugar composition similar to the hemicellulose acid hydrolysate. L-Arabinose was not fermented but assimilated. The presence of acetic acid in the hydrolysate decreased the ethanol yield and productivity considerably.

The influence of nutritional and environmental conditions on the accumulation of poly-beta-hydroxybutyrate in Bacillus mycoides RLJ B-017.

AIMS: To optimize the nutritional and environmental conditions for growth of and poly-beta-hydroxybutyrate (PHB) accumulation in Bacillus mycoides RLJ B-017. METHODS AND RESULTS: An isolate, identified as B. mycoides, was grown on different sources of carbon and nitrogen. Among these, sucrose, beef extract and di-ammonium sulphate were found to be the most suitable for growth and PHB accumulation. The overall maximum value of PHB (%) in cells, PHB yield (Yp/s) and productivities (Qp and qp) were 69.4 +/- 0.4% dry cell weight (DCW), 0.21 gp gS(-1), 0.104 +/- 0.012 gp l(-1) h(-1) and 0.03 gp gx(-1) h(-1), respectively when grown in a medium containing 20 gs l(-1) sucrose, supplemented with di-ammonium sulphate. The addition of beef extract increased the value of PHB (%) in cells, PHB yield and productivities by 17.58 +/- 0,3, 23.8, 19.23 +/- 0.3 and 13.8 +/- 0.2% , respectively. The overall maximum values of PHB (% DCW), PHB yield and productivities were obtained at pH 7.0 +/- 0 .1, temperature 30 +/- 0.5 degrees C, agitation 650 rev min(-1) and oxygen transfer rate 3.8 mmol O(2) l(-1) h(-1). CONCLUSIONS: Sucrose, glucose and fructose were found to be more suitable for cell growth and PHB accumulation, but sucrose was less expensive than glucose. Among the nitrogen sources, beef extract and di-ammonium sulphate promoted PHB synthesis. The accumulation of PHB was observed to be growth associated. SIGNIFICANCE AND IMPACT OF THE STUDY: Gram-positive bacteria have not been reported to accumulate large amounts of polyhydroxyalkanoate and hence have not been considered as potent candidates for industrial production. A number of Bacillus spp. have been reported to accumulate 9-44.5% DCW PHB. By comparison, Bacillus RLJ B-017 contained 69.4 +/- 0.4% DCW PHB. Therefore, this strain has been considered as a potent organism for industrial interest. A relatively high yield of PHB was obtained in this wild strain and PHB synthesis was independent of nutrient limitation. The conditions for the higher PHB yield and productivity will be optimized in the next phase using fed-batch culture.

Ethanol production from hardwood spent sulfite liquor using an adapted strain of Pichia stipitis.

Conditions have been optimized for fermentation of pretreated hardwood spent sulfite liquor (HSSL) using an adapted strain of Pichia stipitis. The pretreatments, consisting of boiling and overliming with Ca(OH)2 of HSSL, to partially remove inhibitors, and adaptation of the yeast strain to HSSL, were both critical for a successful fermentation. Ethanol concentration was increased from 6.7 to 20.2 g l(-1) using adapted P. stipitis (A) and pretreated HSSL. The maximum ethanol yield (Yp/s) and productivity (Qp) were 0.41 g g(-1) and 0.44 g l(-1) h(-1), respectively, at an oxygen transfer rate of 2.0 mmol O2 l(-1) h(-1). The optimized results with this strain were compared to those of other xylose-fermenting yeasts and Saccharomyces cerevisiae (SSL-acclimatized) currently used at an industrial plant for the fermentation of spent sulfite liquor.

Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis.

Ethanol production was evaluated from wheat straw (WS) hemicellulose acid hydrolysate using an adapted and parent strain of Pichia stipitis. NRRL Y-7124. The treatment by boiling and overliming with Ca(OH)(2) significantly improved the fermentability of the hydrolysate. Ethanol yield (Yp/s) and productivity (Qp av) were increased 2.4+/-0.10 and 5.7+/-0.24 folds, respectively, compared to neutralized hydrolysate. Adaptation of the yeast to the hydrolysate resulted further improvement in yield and productivity. The maximum yield was 0.41+/-0.01 g(p) g(s)(-1), equivalent to 80.4+/-0.55% theoretical conversion efficiency. Acetic acid, furfurals and lignins present in the hydrolysate were inhibitory to microbial growth and ethanol production. The addition of these inhibitory components individually or in various combinations at a concentrations similar to that found in hydrolysate to simulated medium resulted a reduction in ethanol yield (Yp/s) and productivity (Qp av). The hydrolysate used had the following composition (expressed in g x l(-1)): xylose 12.8+/-0.25; glucose 1.7+/-0.3; arabinose 2.6+/-0.21 and acetic acid 2.7+/-0.33.

Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate.

AIMS: The objective of this study was to develop a mutant from Pichia stipitis NRRL Y-7124, tolerant of high concentrations of acetic acid and other inhibitory components present in acid hydrolysates, to improve ethanol yield and productivity. METHODS AND RESULTS: The mutant was developed through adaptation in acid hydrolysate supplemented with nutrients and minerals at 30 +/- 0.5 degrees C. When it was tested for its ability to ferment acid hydrolysate, it showed shorter fermentation time, better tolerance to acid and could ferment at lower pH. The ethanol yield (Yp/s) and productivity (Qp) were increased 1.6- and 2.1-fold, respectively. CONCLUSION: The development of a mutant and its tolerance to acetic acid present in hydrolysates is described. The selected mutant is capable of fermenting both hexoses and pentoses present in hydrolysate at lower pH in comparison with the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant could play a significant role in reducing environmental pollution by using sugars present in pulp mill effluent and, at the same time, could produce a marketable liquid fuel ethanol.

Growth-associated production of poly-3-hydroxybutyrate by Bacillus mycoides.

Bacillus mycoides strain RIJ B-017, a growth-associated poly-3-hydroxybutyrate (PHB) producer was grown on sucrose-containing media. PHB accumulated in cells up to 72% of dry cell mass. The overall maximum value of PHB yield (Yp/s) and productivities (Qp and qp) 250 mgp/gs, 120 mgp L-1 h-1 and 30 mgp gx-1 h-1, respectively, were obtained at 15 g/L sucrose. Differential scanning calorimeter heating curve showed two peaks, one at 95.9 degrees C and another at 165.4 degrees C with a shoulder around 154.6 degrees C. The viscosity-average molar mass in chloroform at 27 degrees C was 505 kDa. The carbon content of PHB was 55.4% of the mass.

Continuous ethanol production from pineapple cannery waste using immobilized yeast cells.

The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5. The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1). The volumetric ethanol productivity of the immobilized cells was ca. 11.5 times higher than the free cells. The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity. The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt. cell h(-1) and 2.6 g sugar g(-1) dry wt. cell h(-1), respectively, at a dilution rate of 1.5 h(-1).

Agar immobilized yeast cells in tubular reactor for ethanol production.

Saccharomyces cerevisiae cells were immobilized in agar gel and used in a tubular reactor for conversion of cane molasses to ethanol at 30 degrees C, pH 4.5. Reactor was used in a continuous operation to test the operational stability and ethanol productivity. After 100 days of continuous fermentation at a dilution rate of 0.67 hr-1, some deactivation of cells was observed, but ethanol productivity was recovered by reactivating the cells by sparging air intermittently. It was found that intermittent reactivation during continuous operation was very important for satisfactory performance of the reactor. During operation, gel beads maintained their rigidity. Maximum ethanol concentration (94.9 g/L) was obtained with a feed containing 255 g/L reducing sugar, at a dilution rate of 0.2 hr-1. Maximum volumetric productivity (79.5 g ethanol /L/hr), specific ethanol productivity (0.58 g ethanol/g cells/hr), specific sugar uptake rate (1.12 g sugar/g cells/hr) and ethanol yield coefficient (0.43 g ethanol/g sugar) were obtained with a feed containing 195 g/L reducing sugar at a dilution rate of 1.33 hr-1.

Isolation and Screening of Yeasts That Ferment d-Xylose Directly to Ethanol.

Natural habitats of yeasts were examined for the presence of strains able to produce ethanol from d-xylose. Black knots, insect frass, and tree exudates were screened by enrichment in liquid d-xylose-yeast extract medium. These and each d-xylose-assimilating yeast in a collection from cactus fruits and Drosophila spp. were tested for alcohol production from this sugar. Among the 412 isolates examined, 36 produced more than 1 g of ethanol liter from 20 g of d-xylose liter, all under aerated conditions. Closer examination of the strains indicated that their time courses of d-xylose fermentation followed different patterns. Some strains produced more biomass than ethanol, and among these, ethanol may or may not be assimilated rapidly after depletion of d-xylose. Others produced more ethanol than biomass, but all catabolized ethanol after carbohydrate exhaustion. Ethanol production appeared best at low pH values and under mild aeration. Possible correlations between the nutritional profiles of the yeasts and their ability to produce ethanol from d-xylose were explored by multivariate analysis. d-Xylose appeared slightly better utilized by yeasts which rate poorly in terms of fermentation. The fermentation of d-glucose had no bearing on d-xylose fermentation. No specific nutritional trait could discriminate well between better d-xylose fermentors and other yeasts.

Aerobic Fermentation of D-Xylose to Ethanol by Clavispora sp.

Eleven strains of an undescribed species of Clavispora fermented D-xylose directly to ethanol under aerobic conditions. Strain UWO(PS)83-877-1 was grown in a medium containing 2% D-xylose and 0.5% yeast extract, and the following results were obtained: ethanol yield coefficient (ethanol/D-xylose), 0.29 g g (57.4% of theoretical); cell yield coefficient (dry biomass/D-xylose), 0.25 g g; maximum ethanol concentration, 5.9 g liter; maximum volumetric ethanol productivity, 0.11 g liter h. With initial D-xylose concentrations of 40, 60, and 80 g liter, maximum ethanol concentrations of 8.8, 10.9, and 9.8 g liter were obtained, respectively (57.2, 57.1, and 48.3% of theoretical). Ethanol was found to inhibit the fermentation of D-xylose (K(p) = 0.58 g liter) more than the fermentation of glucose (K(p) = 6.5 g liter). The performance of this yeast compared favorably with that reported for some other D-xylose-fermenting yeasts.

Effect of carbon and nitrogen sources on neutral proteinase production by Pseudomonas aeruginosa.

A strain of Pseudomonas aeruginosa from soil produced large quantities of extracellular neutral proteinase and could utilize several organic substances as carbon and nitrogen sources for enzyme production. The growth media required the presence of a high amount of phosphate when glucose was the carbon source. The intermediates of citric-acid cycle acids supported the proteinase production more than any other carbon sources. However, complex nitrogenous substances supported enzyme production more efficiently. Higher concentration of casamino acids suppressed the protinase synthesis.