Julius Kola Oloke

Antimicrobial resistance of bacterial strains isolated from orange juice products

Forty samples of twenty brands of sachet orange juice products were examined microbiologically. All the products were contaminated with bacteria and yeasts. The organisms encountered include Saccharomyces cerevisiae, Saccharomyces sp, Rhodotorula sp, Bacillus cereus, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes and Micrococcus sp . The resistances of thirty bacterial strains isolated from orange juice products to the commonly used antibiotics were studied. About 66.67% of the isolates were resistant to augmentin and amoxycillin; 63.33% to cotrimoxazole, 56% to cloxacillin, and 23.33% to tetracycline. Resistances of 10, 6.67, and 3.33% were obtained for gentamicin, erythromycin and chloramphenicol respectively. Among the eight antibiotics tested, seven patterns of drug resistance were obtained. Six out of these are multiple-drug resistance with number of antibiotics ranging between 2 to 8. While MIC of amoxycillin ranged between 10-25mg/ml for the strains of E. coli, MIC of 10-20mg/ml was obtained for the strains of S. aureus . The MIC for cloxacillin was 0.1-1.0mg/ml for E. coli strains, and 0.01-1.0mg/ml for S. aureus strains. In all, ten strains of the bacterial isolates had evidence for the production of β-lactamases. Key words: Orange juice, antibiotics, resistance pattern, β-lactamase, microbiological standard. African Journal of Biotechnology Vol.3(6) 2004: 334-338

Rhizopus stolonifer LAU 07: a novel source of fructosyltransferase

Increasing awareness of the importance of fructooligosaccharides (FOS) as ingredients of functional foods has led to intensive search of new sources of fructosyltransferases (FTase), enzymes responsible for the conversion of sucrose to fructooligosaccharides. A local strain of Rhizopus stolonifer isolated from spoilt orange fruit with high fructosyltransferase activity (Ut) of 12.31–45.70 U mL−1 during a fermentation period of 24–120 h is herein reported. It showed low hydrolytic activity (Uh) in the range of 0.86–1.78 U mL−1 during the same period. FOS yield of 34 % (1-kestose, GF2, nystose, GF3) was produced by FTase obtained from a 72 h-old culture using 60 g of sucrose per 100 mL of the substrate. When the isolate was grown in a defined submerged medium, its pH dropped sharply from the intial value of 5.5 to 1.0 within 24 h, and this value was maintained throughout the fermentation. The biomass content ranged from 8.8 g L−1 at 24 h of fermentation to reach the maximum of 10 g L−1 at 72 h. It was reduced to 5.6 g L−1 at the end of 120 h of fermentation. This report represents the first reference to a strain of Rhizopus as a source of FTase for the production of FOS. The high Ut/Uh ratio shown by this isolate indicates that it may be a good strain for the industrial and commercial production of FOS. However, there is a need of further optimization of the bioprocess to increase the conversion efficiency of sucrose to FOS by the enzyme.

Novel optimal temperature profile for acidification process of Lactobacillus bulgaricus and Streptococcus thermophilus in yoghurt fermentation using artificial neural network and genetic algorithm.

The acidification behavior of Lactobacillus bulgaricus and Streptococcus thermophilus for yoghurt production was investigated along temperature profiles within the optimal window of 38–44 °C. For the optimal acidification temperature profile search, an optimization engine module built on a modular artificial neural network (ANN) and genetic algorithm (GA) was used. Fourteen batches of yoghurt fermentations were evaluated using different temperature profiles in order to train and validate the ANN sub-module. The ANN captured the nonlinear relationship between temperature profiles and acidification patterns on training data after 150 epochs. This served as an evaluation function for the GA. The acidification slope of the temperature profile was the performance index. The GA sub-module iteratively evolved better temperature profiles across generations using GA operations. The stopping criterion was met after 11 generations. The optimal profile showed an acidification slope of 0.06117 compared to an initial value of 0.0127 and at a set point sequence of 43, 38, 44, 43, and 39 °C. Laboratory evaluation of three replicates of the GA suggested optimum profile of 43, 38, 44, 43, and 39 °C gave an average slope of 0.04132. The optimization engine used (to be published elsewhere) could effectively search for optimal profiles of different physico-chemical parameters of fermentation processes.

Synergetic effect of rhamnolipid from Pseudomonas aeruginosa C1501 and phytotoxic metabolite from Lasiodiplodia pseudotheobromae C1136 on Amaranthus hybridus L. and Echinochloa crus-galli weeds

Rhamnolipid (Rh) is a biosurfactant produced by the bacterial Pseudomonas aeruginosa. This present study investigates rhizospheric strain C1501 of P. aeruginosa with an accession number KF976394 with the best production of rhamnolipid: a biosurfactant. The partially purified rhamnolipid from strain C1501 and Tween 80 was tested on mycelial growth of wild strain C1136. The enzyme activities involved in biodegradation, as well as necrosis induction on the tested weeds, were performed using scanning electron microscopy. It was observed that the different concentrations of rhamnolipid tested enhanced the dry mycelia weight yield of Lasiodiplodia pseudotheobromae which has been established to be producing a phytotoxic metabolite for killing weeds. It was observed that strain C1136 had a high level of cellulase and xylanase enzyme activities during this study. The scanning electron microscopy showed that the mutant strain of C1136 combined with 0.003% v/v of rhamnolipid enhances biodegradability and a high level of necrosis on the tested weeds compared with that on the untreated weeds. The highest CMCase activities and xylanase activities were obtained on the fourth day from the phytotoxic metabolite produced from the mutant strain of L. pseudotheobromae when combined with 0.003% v/v of rhamnolipid. This study has shown that rhamnolipid can serve as an adjuvant in order to enhance the penetrability of bioherbicide active ingredient for controlling weeds.

PRO-OPTIMIZER: A NOVEL WEB-ENABLED OPTIMIZATION ENGINE FOR MICROBIAL FERMENTATIONS

ABSTRACT Traditional strategies designed to determine the best combination of physico-chemical parameters to optimally drive a fermentation process suffer from large computational burden, or are unable to account for the interaction effects of the various process parameters. In this paper, we report a web-enabled engine for fermentation optimization using Genetic Algorithm. The setpoint values of the process parameters are concatenated into strings, called chromosomes and represent potential solution states. A population of such strings is formed. These chromosomes are evaluated for their fitness using an objective function or through experimentation. The best-fitted chromosomes are selected and undergo mutations and crossovers to produce the next generation. This procedure is repeated until the fitness value becomes constant and the best chromosome emerges. The system considers the interactive effects of all the process parameters. The effectiveness of this engine has been evaluated on the feeding strategy for the production of Saccharomyces cerevisiae DS2155, optimization of yogurt acidification process using Lactobacillus bulgaricus and Streptococcus thermophilus. Data revealed a feeding strategy with a biomass yield improvement of 0.53g/l on S. cerevisiae, and an acidification slope of 0.06117 compared to an initial of 0.0342, reducing the yogurt acidification time from six to two hours. This system will reduce the cost for fermentation process development.

Implementation Details of Computerized Temporary Immersion Bioreactor (TIB): A Fermentation Case of Pleurotos Pulmonarius

ABSTRACT Temporary immersion culture systems are emerging as alternative methods with advantages of culture in submerged medium, without the disadvantages of liquid environment. This paper details the implementation of computerized Temporary Immersion Bioreactor (TIB) from locally available resources. The hardware system was made up of the glass vessel unit, the oil-less air compressor, the valves and relays board which communicates with the control computer using RS232. The control software was developed using PHP and MySQL database structure. It offered the flexibility to run six TIBs simultaneously at different immersion frequencies. This system was used to culture Pleurotus pulmonarius at immersion frequencies of 1, 3 and 6 hours, thus high, medium and low immersion frequencies, respectively, for 8 days. The system frequently and timely controlled the states of organisms as immersed, aerated and drained according to the defined control strategy. Furthermore, downstream observations of the culture rheology revealed the culture broth being more turbid, turbid and less turbid with TIB operated at high, medium and low immersion frequency, respectively. In addition to the possibility of implementing a low-cost fully automated Temporary Immersion Bioreactors, these systems can be used to boost the range of metabolites of microorganisms considering the versatility of metabolic fluxes, by attempting various immersion frequencies.

Isolation, identification, characterization, and screening of rhizospheric bacteria for herbicidal activity

AbstractThe consistent application of agrochemical herbicides has been reported to impact negatively on human health, environment, and food safety, and facilitated the emergence of weed resistances. Rhizosphere bacteria (RB) of different crops were screened for antagonism against Amaranthus hybridus L. (pigweed) and Echinochloa crus-galli (L.) Beauv. (barnyard grass) using necrosis assay technique. A total of eight rhizosphere bacterial isolates (B1–B8) produced different degrees of leaf necrosis on target weeds with isolate B2 manifesting the most significant necrotic activity. The rhizospheric bacterium (B2) with the highest necrotic activity was identified using 16S rRNA sequencing technique and further investigated. Molecular, morphological, and biochemical characterizations confirmed B2 isolate to be Pseudomonas aeruginosa. On isolation with ethyl acetate, separation, defatting, purification, and flash chromatography, seven different fractions (fraction 1–fraction 7) were obtained out of which fraction 4 showed the highest necrotic activity in necrosis assay experiment. Preparative HPLC of fraction 4 resulted in a pure compound that completely inhibited seed germination and seedling development of pigweed and barnyard grass but remained non-antagonistic to other tested soil fungi used in this study. The result obtained from this present study consequently confirmed the antagonistic behavior of rhizosphere-inhabiting P. aeruginosa to the target weeds and qualified the suitability of bacterium as good alternative source of bioherbicide. Potential herbicidal formulation from P. aeruginosa will help reduce crop loss due to weed challenges while offering a partial solution to the use of agrochemicals and food security. ᅟ

Environmental fate and effects of granular pesta formulation from strains of Pseudomonas aeruginosa C1501 and Lasiodiplodia pseudotheobromae C1136 on soil activity and weeds

This work investigated the effect of variably formulated pesta granules containing wild and UV mutated Pseudomonas aeruginosa and Lasiodiplodia pseudotheobromae on the rate of CO2 evolution, organic carbon content, enzymatic activity (acidic and alkaline phosphatase, dehydrogenases, urease and protease) and representative soil microorganisms in the soils using different assay techniques. After the 35th day period of experiment, the pesta granule formulation BH4 showed the best evolution of CO2 (824 ± 6.2 mg CO2 kg-1 soil hr-1) as against control treatment (689 ± 3.7 mg CO2 kg-1 soil hr-1). Enzymes activities, organic carbon content of 3.8% on the 15th day of study and stable representation of microorganisms that include actinomycetes, fungi, heterogenous as well as soil nitrogen-mediatory bacteria were equally at their maximum level BH4 treatments. The phytotoxic assay showed no inhibitory effect on Solanum lycopersicum seeds and seedlings compared to the observed growth inhibition on the tested weeds (Amaranthus hybridus and Echinocholoa crus-galli) which corresponds with positive control glyphosate treatment. The glyphosate treated soil had the least critical results on parameters investigated during the study. The order of bioherbicidal activity is BH4>BH2>BH6>BH3>BH1>BH5>positive control. Results from this study confirmed the target efficacy of variably formulated pesta granules which is sustainable, cheap, ecologically suitable and recent. This is in addition to recognizing the microbial-derived formulations as characteristically potent alternative to chemical herbicides utility in agrosystems practice. Further study of the underlining factor responsible for the bioherbicidal performances of the variably formulated pesta granules and field trials are critical for their future commercialization.

Compatibility study using hybridization procedure among Pleurotus genotypes and authentication by enzyme expression and ITSR of rDNA

Cross compatibility and authentication of hybrid genotypes among species of Pleurotus were studied using lignocellulotic enzymes production and Internal transcribed spacer (ITS) of rDNA. Schematic procedure of hyphal anastomosis hybridization between the species of Pleurotus was employed in this study. Formation of clamp connexion, changes in morphological hyphal arrangement and general increased in the enzymes production by the hybrid strains over their wild types is an evidence for the compatibility of crossed genotypes. Highest produced enzyme was manganese peroxidase by hybrid LN 97 with 3.25 Uml–1, followed by laccase with 3.06 Uml–1 obtained in LN 91 (hybrid). Lignin peroxidase was the least enzyme produced, but with better performance in hybrid LN 97 (2.75 Uml–1). Dendrogram of relationship among the genotypes revealed two major clusters. Cluster II contained singleton (LN 95), hybrid strain which totally separated from both parents. Most strains clustered away from their parents, authenticating the hybrids produced. Current study shows the Pleurotus employed for this investigation are cross compatible with high expression of heterosis in hybrid strains relative to the parents on the basis of enzymes produced. This technique could serve as a good tool in breeding programs for Pleurotus strain improvement.

Microbes in Irrigation Water and Fresh Vegetables Potential Pathogenic Bacteria Assessment and Implications for Food Safety

Irrigated fresh vegetables are a potential portal for dissemination of human pathogens that may be associated with irrigation water source. This study investigated the quality of irrigation water and the fresh vegetables produced in regard to pathogenic bacteria found at a selected vegetable farm in southwestern Nigeria. Enumeration and characterization of total heterotrophic bacteria and potential pathogens (mostly enteric bacteria) were done with international procedures. Susceptibility of the potential pathogens to antibiotics was by disk diffusion techniques, while plasmid profile was by polymerase chain reaction–based DNA fingerprinting. The mean values for total heterotrophic bacteria in irrigation water and fresh vegetable samples were, however, statistically comparable (P = .86). The corresponding comparison for coliform also showed no significant difference (P = .07). The isolated pathogenic bacteria genera from the irrigation water and fresh vegetable samples include Citrobacter, Enterobacter, Escherichia, Klebsiella, Pseudomonas, and Salmonella. In sum, 67.2% of the isolates were obtained from irrigation water samples, compared with 32.8% in the vegetable samples; 54.1% of the isolates were resistant to >1 antibiotic with a multiple antibiotic resistance index >0.2, suggesting an antibiotic pressurized environment. Resistance to ampicillin was very high (98.36%), whereas resistance to ciprofloxacin was very low (0.0%); 27.9% of the multiresistant enteric isolates harbored ≥1 plasmids. There is a possibility of increased numbers of pathogens on irrigated vegetables as a direct consequence of poor irrigation water quality in the study site. The use of pathogen-free irrigation water and good agricultural practices has the potential to eliminate microbial hazards in fresh vegetables.