A. O. Ejiofor

Culture of Saccharomyces cerevisiae on hydrolyzed waste cassava starch for production of baking-quality yeast

Abstract A fermentation medium based on waste cassava starch hydrolysate and a four-phase feeding strategy for a fed-batch culture of Bakers yeast Saccharomyces cerevisiae are presented. Cassava starch isolated from the wastewater produced in processing of cassava mash into gari was liquefied with a thermostable 1.4-α- d -glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca 2+ at 80°C and pH 6.1–6.3 for one h. The liquefied material was saccharified with 1.4-α- d -glucan glucohydrolase (EC 3.2.1.3) at 55°C and pH 5.5 for two h. Over 98% of the starch was hydrolyzed; about 80.7% of the hydrolysate was glucose. The fermentation feeding profile which was based on a desired specific growth rare range of 0.18–0.23 h −1 , a biomass yield coefficient of 0.5 g g −1 , and a feed substrate concentration of 200 g l −1 was implemented manually using the cassava hydrolysate feed in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of the oxygen uptake rate, the carbon dioxide evolution rate, and the respiratory quotient. Off-line determinations of biomass, ethanol, and glucose were done, respectively, by dry weight, gas chromatography, and spectrophotometry. Cell mass concentrations of 50–58 g l −1 were achieved in all experiments within 28 h of which the last 15 h were in the fed-batch mode. The average biomass yields for the cassava and glucose media were identical at 0.49 g g −1 . No significant differences were observed between the leavening activities of the products of the test, the control media, and a commerical preparation of instant active dry yeast. Waste cassava starch hydrolysate was established as a suitable low cost replacement for glucose in the production of baking-quality yeast.

Physiological and molecular detection of crystalliferous Bacillus thuringiensis strains from habitats in the South Central United States.

Gram-positive, endospore-forming Bacillus thuringiensis-like strains were isolated from 95 of 413 samples collected at the 0–5 cm depth of noncultivated soils and stagnant or dried-up ponds as well as from dust from stored grain products in South Central United States. Based on the production of parasporal crystals, 25 isolates were identified as B. thuringiensis after examining 227 B. thuringiensis-like colonies. The greatest proportion of samples yielding B. thuringiensis were from the dust from grain storage. The sodium acetate selective medium, heat processing, and crystal staining used in the initial screening revealed diverse populations of B. thuringiensis, which were categorized into distinct crystal morphological groups. Sugar fermentation, antibiotic sensitivity, growth characteristics and PCR studies showed diversity among the isolates that were distributed among 25 of the 58 known strains. The most frequently isolated strains were kurstaki, aizawai, morrisoni, thuringiensis, sotto and kenyae that together represented more than 90% of the characterized isolates. PCR analysis using 30 family primer pairs for cry and cyt genes showed that the frequency of the cry1 gene (62%) was predominant followed by the cry2 genes (30%), and the rest (8%) were other cry gene types, such as cry3, cry4, cry10, cry11, cry14, cry15, cry20, cry24 and cry26. Both cyt1 and -2 genes were also detected. Several isolates showed PCR products on the gel that were not consistent with the expected sizes of nucleotides targeted by the primers. These were suggestive of nonspecific amplifications and were not used in the characterization process. Journal of Industrial Microbiology & Biotechnology (2002) 28, 284–290 DOI: 10.1038/sj/jim/7000244

A robust fed-batch feeding strategy for optimal parameter estimation for baker's yeast production

Parameter identification of structured models is often a problem in biotechnology, because the poor data situation and the number of unknown parameters only allow for inaccurate estimates. But often only a subset of all kinetic parameters of the model are of interest for production purposes, e.g. for fed-batch cultivation. These parameters should be estimated with a given accuracy. In addition, the experiments for information acquisition with respect to these parameters should be as simple as possible and should consider some practical restrictions. In this contribution a fed-batch feeding strategy is proposed to allow for an accurate estimation of yield and of critical growth rate of bakers yeast. The feeding also allows for economic and stereotyped use of staff and equipment and is therefore suitable for routine use in screening of strains and media. The overall pattern is similar to that one, usually used in production scale to minimize errors by limited model validity. After an initial phase for achieving a reproducible state three different growth rates are adjusted to cover the range of possible critical growth rates. From biomass and ethanol measurements yield and critical growth rate can be estimated with an accuracy of about 2.1%. The fermentation pattern ends up with a constant feeding rate to simulate a limited oxygen transfer rate and to allow for an uptake of residual sugar and ethanol before a dough test can be carried out. Beside experimental results simulations and sensitivity analyses are shown.

Fed-batch production of baker's yeast using millet (Pennisetum typhoides) flour hydrolysate as the carbon source

A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of bakers yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4-α-d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca2+, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4-α-d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h−1, biomass yield coefficient of 0.5 g g−1 and feed substrate concentration of 200 g L−1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L−1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g−1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.

Violacein induces p44/42 mitogen-activated protein kinase-mediated solid tumor cell death and inhibits tumor cell migration.

Microbial secondary metabolites have emerged as alternative novel drugs for the treatment of human cancers. Violacein, a purple pigment produced by Chromobacterium violaceum, was investigated in the present study for its anti-tumor properties in tumor cell lines. Clinically applicable concentrations of violacein were demonstrated to inhibit the proliferative capacity of tumor cell lines according to a crystal violet proliferation assay. The underlying mechanism was the promotion of apoptotic cell death, as indicated by poly(ADP ribose) polymerase cleavage and p44/42 mitogen-activated protein kinase signaling determined by western blot analysis. Collectively, this provided mechanistic evidence that violacein elicits extracellular-signal regulated kinase-induced apoptosis via the intrinsic pathway. The anti-malignant properties of violacein in the present study were further demonstrated by its inhibitory effects on brain tumor cell migration, specifically glioblastomas, one of the most invasive and therapeutically resistant neoplasms in the clinic. Additionally, solid tumors examined in the present study displayed differential cellular responses and sensitivities to violacein as observed by morphologically induced cellular changes that contributed to its anti-migratory properties. In conclusion, violacein is a novel natural product with the potential to kill several types of human tumor cell lines, as well as prevent disease recurrence by antagonizing cellular processes that contribute to metastatic invasion.

Determination of the plasmid size and location of d-endotoxin genes of Bacillus thuringiensis by pulse field gel electrophoresis

The genes encoding the d-endotoxins of Bacillus thuringiensis are located on plasmids ranging in size from 45 to 1000 kb. Plasmid size and variety are diagnostic features for characterizing subspecies of this aerobic spore-forming crystalliferous entomopathogen. Two of 25 B. thuringiensis isolates obtained from Middle Tennessee were characterized into subspecies on the basis of size, number, and varieties of plasmids they host using pulse field gel electrophoresis (PFGE). By using specific pulse angle (120°), field strength (5 V/cm), pulse time (26 h), and agarose concentration (1.2%) these DNA molecules were separated from other genomic DNA. The purified DNA product the agarose gel was tested for homology to 49 d-endotoxin gene using PCR.. The PFGE data for 5 of the 25 isolates collected in Tennessee showed distinct banding patterns. Two of the isolates had only 1 band whereas the others had more than 4 ranging from 45 to 1000 kb in size. Isolate 6 and 10 both yielded one 49 kb fragment that contained the cry1A gene. African Journal of Biotechnology Vol. 4 (7), pp. 580-585, 2005

Preparation of a flowable liquid bacterial insecticide based on Bacillus sphaericus

Fermenter-produced Bacillus sphaericus 2362 was formulated into a thick, dark flowable liquid concentrate containing 4.8×109 c.f.u./ml and charcoal as protector against ultraviolet light. The potencies of the formulation against L4Culex pipiens quinquefasciatus before and after storage for 2 years were 5714 and 5862 International Toxic Units (ITU), respectively, when compared with a standardized B. sphaericus from the WHO at 1000 ITU. In field trials, treatment at 1.01/ha gave 96 to 100% control of mosquito larvae. B. sphaericus could be re-isolated in 5% of the samples 9 months after application.