Characterization and statistical optimization of γ-PGA produced by Bacillus megaterium UP47 isolated from Pentaclethra macrophylla.

BACKGROUND: Gamma-polyglutamic acid (γ-PGA) is a microbially produced non-toxic peptide biopolymer which is gaining grounds in many biotechnological fields and has a wide range of applications. OBJECTIVES: In this study, the characteristics of γ-PGA produced by Bacillus megaterium isolated from an oil seed were determined, while the nutritional requirements of the bacterium were optimized using a predictive 15 factor-16 run Plackett-Burman experimental design. MATERIAL AND METHODS: The main effect of each factor, the interaction and quadratic effects of the factors on optimized production were determined from Box-Benkhen model using Dell Statistica v. 12 and 13 software. Bacillus megaterium UP47 produced the highest γ-PGA (16.33 g/L) out of 56 spore-forming Bacillus strains isolated from soil, water and fermented food samples. RESULTS: Hydrolysates of the produced γ-PGA had a retention factor which corresponded to the L-glutamic acid standard (retention factor (rf) 0.35), while high-definition fourier transform infrared (FT-IR) spectroscopic imaging showed characteristic peaks representative of the active bonds present in γ-PGA. The γ-PGA at a concentration as low as 50 mg/100 mL exerted antimicrobial inhibitions against test pathogens. A 2.00 w/v γ-PGA solution had 11 mm and 13 mm inhibition zones against Staphylococcus aureus and Shigella dysenteriae, respectively. A second order polynomial equation for prediction of γ-PGA was derived as: γPGA yield = 3316.061 - 449.708A + 9.036A2 - 139.813B + 3.095B2 - 7.699C - 0.164C2 + 13.116AB - 0.087AB2 - 0.248A2B + 3.781AC - 0.076A2C - 0.394BC. It showed an increase in γ-PGA yield with increasing L-glutamic acid and biotin, but a decrease with yeast extract. CONCLUSIONS: Bacillus megaterium UP47 had a maximum γ-PGA yield of 54 g/L and 62 g/L, respectively, from the Plackett-Burman and Box-Benkhen design, thereby resulting in an appreciable increase in polymer yield after the optimization process with a 95% confidence level.

Design and evaluation of the antimicrobial properties of ackee seed extract silver nanoparticle film formulations.

BACKGROUND: Plant-extract-reduced metal nanoparticles provide means of overcoming microbial resistance. Incorporating them into appropriate pharmaceutical formulations will enhance their portability and ease of administration. OBJECTIVES: To synthesize silver nanoparticles using methanol extracts of the seeds of Blighia sapida as capping agents and formulating the products in antimicrobial films. MATERIAL AND METHODS: Phytochemical screening of the methanol extract of Blighia sapida K.D. Koenig (ackee) seeds was performed and its antioxidant properties were determined using DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. The green synthesis of ackee seed extract silver nanoparticles (ASAgNPs) was accomplished with reacting 1 mM of aqueous silver nitrate (AgNO3) and the methanol extract in a flask; the bioreduction was performed at 37°C for 72 h. The resulting nanoparticles were lyophilized and characterized using UV-visible spectrophotometry, Fourier-transform infrared spectroscopy (FTIR) and photomicrography. The nanoparticles were further formulated into films using starch and carboxymethyl cellulose using the solvent evaporation method. The extract, biosynthesized nanoparticles and film formulations were screened for antimicrobial activity against several pathogens using the agar well diffusion method. RESULTS: The methanol seed extracts of the ackee fruit contained saponins, tannins, flavonoids, terpenoids, and anthraquinones. The extract exhibited significant antioxidant properties. The nanoparticles and film formulations had a broader range of activity against microbes than the plant extract, exhibiting significant activity against Escherichia coli ATCC 700728, Salmonella typhi ATCC 14028, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853. Activity was also observed with Candida krusei, C. albicans, and Penicillium sp. It is noteworthy that this last organism showed resistance to fluconazole. CONCLUSIONS: Ackee seed extract silver nanoparticles exhibited a synergistic antimicrobial activity against several pathogens. Film formulations of the nanoparticles retained this antimicrobial activity and allowed the product to be presented in a consumer-ready form.

Effects of media components and agricultural by-products on γ-polyglutamic acid production by Bacillus toyonensis As8.

BACKGROUND: Poly-γ­glutamic acid (γ­PGA) provides an environmentally friendly alternative to plastic materials which have widely polluted the environment. OBJECTIVES: The microbial production of γ­PGA, an amino acid biopolymer with glutamic acid subunits, was investigated using renewable agricultural residues in an attempt to find cheaper substitutes for conventional synthetic media components. MATERIAL AND METHODS: Bacteria which produce γ­PGA were isolated through depolymerizing Coix lacryma-jobi, a cellulosic grass, and the effects of various carbon and nitrogen sources, temperature, inoculant load, incubation period, and pH on γ­PGA yield were determined after submerged fermentation. Bacterial growth was measured turbidimetrically at 550 nm. The γ­PGA produced was characterized using Fourier transform infrared (FT-IR) spectroscopy and the polymer shape was determined using scanning electron microscopy (SEM). RESULTS: The best γ­PGA producer was molecularly identified as Bacillus toyonensis As8. The conditions which produced the highest γ­PGA yield were glucose, ammonium sulfate, 25°C, a pH of 5.5, and an incubation period of 48 h. This bacterium yielded the most γ­PGA (26.45 g/L) on cassava peels, while other agro-wastes (corn cob, sorghum leaves, Coix noir leaves, and rice bran) also supported bacterial growth with lower γ­PGA yields than conventional carbon sources. The wrinkled γ­PGA had absorbance peaks of hydroxyl, amide, carbonyl, and amine groups comparable with the ranges of those found in commercial γ­PGA. CONCLUSIONS: The use of agricultural by-products as fermentation substrates increased γ­PGA yield and may therefore be used as substitute components in γ­PGA production.

Characteristics of a ß-1,4-D endoglucanase from Trichoderma virens wholly applied in a palm-fruit husk-based diet for poultry layers.

The characteristics of an endoglucanase produced by a Trichoderma virens strain T9 newly isolated from a palm-fruit husk dump site, its physiological characteristics and enzyme production were studied. Whole cells of the depolymerizing-enzyme producing T. virens were applied to palm-fruit husk and bird performance characteristics when employed as poultry diet additive were considered. Endoglucanase activity in submerged fermentation was 1.6 nkat. Optimum activity was recorded at pH 6.0 and 55°C. The enzyme retained 50% residual glucanase activity at 70°C for 10 minutes. 1.0% Tween-80 and SDS yielded endoglucanase activity 2.15 times higher than the control. Activity was boosted by 20mM Ca(2+) (115.0%); 10mM K(+) (106.5%); and was totally inhibited by 1mM Hg(2+). The addition of T. virens-fermented palm-fruit husk with other layer feed components on the bird characteristics showed that change in bird weight between the control and test birds were not significantly different (p>0.05) but differed in terms of daily feed ingested (p<0.05). The feed to weight-gain ratio was best with the unmodified palm-fruit husk based diet (8.59). There was no significant difference in the egg weights from modified palm-fruit husk based diet and control (p>0.05). The shell thickness (0.64mm) and yolk content (23.61%) were highest in the microbially-modified husk diet. The alternative to maize based diets proffered by the application of T. virens-modified palm-fruit husk in poultry nutrition in terms of bird weight and feed to weight-gain ratio affords the poultry farmer an economic advantage and allows for a greater utilization of the maize in human diets.

Antibiotic resistance profile of Gram-negative bacteria isolated from Lafenwa abattoir effluent and its receiving water (Ogun River) in Abeokuta, Ogun state, Nigeria.

Untreated abattoir effluent constitutes potential reservoir for transmission of pathogenic strains of multiple antibiotic-resistant bacteria by pollution of surface and ground water sources. This study was carried out to determine the antibiotic resistance and extended spectrum ß-lactamase (ESBL) production profiles of Gram-negative bacteria isolated from effluent collected from Lafenwa municipal abattoir and its receiving surface water, Ogun River, in Abeokuta, Ogun state, Nigeria. Twelve effluent and 18 water samples were collected for this study. Total heterotrophic and coliform counts were estimated, bacterial identification was performed using standard culture-based procedures, whilst antibiotic resistance profiles of isolated bacteria against five antibiotics (ceftazidime, cefpodoxime, cefotaxime, ertapenem and amoxicillin-clavulanate) and detection of ESBLs were done using disk diffusion and double-disc synergy tests. A total of 54 Gram-negative bacteria were isolated, including Salmonella spp. (9), Escherichia coli (15), Klebsiella spp. (7), Shigella spp. (5), Pseudomonas spp. (12) and Enterobacter spp. (6). Both Enterobacteriaceae and Pseudomonas isolates (31% and 66.6%, respectively) were resistant to all selected antibiotics except ertapenem (98% susceptibility). Overall, 77% isolates had multiple antibiotic resistance index (MARI) values, but none of the antibiotic-resistant isolates showed evidence of ESBL production. The presence of multiple antibiotic-resistant isolates in the effluent and receiving water of Lafenwa abattoir suggests a major risk to public health and food safety. Current methods of waste disposal at the abattoir are unacceptable and greatly reduce the qualities of the processed meat and contaminate the environment. There is a need for improved abattoir waste management and water treatment strategies.

Production and characterization of polyhydroxyalkanoic acid from Bacillus thuringiensis using different carbon substrates.

Polyhydroxyalkanoic acids (PHA) are biodegradable microbially synthesised polymers which can act as alternatives to conventional petrochemical based plastics. Bacterial isolates from soil and sewage samples were screened for the ability to accumulate PHA using Sudan Black B dye, and growth conditions of the best PHA accumulating bacterium was optimized. The PHA produced was characterized by FTIR spectroscopy. Eight PHA-producing bacteria were isolated and isolate SBC4 which had the highest percentage accumulation of 30% was genotypically identified as Bacillus thuringiensis and selected for further studies. Glucose and ammonium sulfate supplementation afforded the optimum cultural conditions for cell growth and PHA accumulation yielding 0.33g/l and 0.40g/l respectively. B. thuringiensis SBC4 utilized corn bran, corn cob and wheat bran for PHA production, with corn cob yielding the highest PHA content of 21.05%. The optimum pH, temperature and incubation time for PHA productions were 7.0, 37°C and 48h. The use of all the optimum conditions in a newly modified medium increased the PHA content, with an overall increase in PHA accumulation from 30% to 44.96% equivalent to an increase of 50% in PHA yield. The presence of major peaks such as CO stretching, CH vibrations, COC stretching confirmed PHA production.

Characteristics of a â-1, 4-D endoglucanase from Trichoderma virens wholly applied in a palm-fruit husk-based diet for poultry layers

The characteristics of an endoglucanase produced by a Trichoderma virens strain T9 newly isolated from a palm-fruit husk dump site, its physiological characteristics and enzyme production were studied. Whole cells of the depolymerizing-enzyme producing T. virens were applied to palm-fruit husk and bird performance characteristics when employed as poultry diet additive were considered. Endoglucanase activity in submerged fermentation was 1.6 nkat. Optimum activity was recorded at pH 6.0 and 55ºC. The enzyme retained 50% residual glucanase activity at 70ºC for 10 minutes. 1.0% Tween-80 and SDS yielded endoglucanase activity 2.15 times higher than the control. Activity wasboosted by 20mM Ca2+ (115.0%); 10mM K+ (106.5%); and was totally inhibited by 1mM Hg2+. The addition of T. virens -fermented palm-fruit husk with other layer feed components on the bird characteristics showed that change in bird weight between the control and test birds were not significantly different (p>0.05) but differed in terms of daily feed ingested (p<0.05). The feed to weight-gain ratio was best with the unmodified palm-fruit husk based diet (8.59). There was no significant difference in the egg weights from modified palm-fruit husk based diet and control (p>0.05). The shell thickness (0.64mm) and yolk content (23.61%) were highest in the microbially-modified husk diet. The alternative to maize based diets proffered by the application of T. virens -modified palm-fruit husk in poultry nutrition in terms of bird weight and feed to weight-gain ratio affords the poultry farmer an economic advantage and allows for a greater utilization of the maize in human diets.