Faculty Publications
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Item Optimization of culture medium for novel cell-associated tannase production from bacillus massiliensis using response surface methodology(2012) Belur, P.D.; Goud, R.; Goudar, D.C.Naturally immobilized tannase (tannin acyl hydrolase, E.C. 3.1.1.20) has many advantages, as it avoids the expensive and laborious operation of isolation, purification, and immobilization, plus it is highly stable in adverse pH and temperature. However, in the case of cell-associated enzymes, since the enzyme is associated with the biomass, separation of the pure biomass is necessary. However, tannic acid, a known inducer of tannase, forms insoluble complexes with media proteins, making it difficult to separate pure biomass. Therefore, this study optimizes the production of cell-associated tannase using a "protein-tannin complex" free media. An exploratory study was first conducted in shake-flasks to select the inducer, carbon source, and nitrogen sources. As a result it was found that gallic acid induces tannase synthesis, a tryptose broth gives higher biomass, and lactose supplementation is beneficial. The medium was then optimized using response surface methodology based on the full factorial central composite design in a 3 l bioreactor. A 2 3 factorial design augmented by 7 axial points (? = 1.682) and 2 replicates at the center point was implemented in 17 experiments. A mathematical model was also developed to show the effect of each medium component and their interactions on the production of cell-associated tannase. The validity of the proposed model was verified, and the optimized medium was shown to produce maximum cell-associated tannase activity of 9.65 U/l, which is 93.8% higher than the activity in the basal medium, after 12 h at pH 5.0, 30°C. The optimum medium consists of 38 g/l lactose, 50 g/l tryptose, and 2.8 g/l gallic acid. © The Korean Society for Microbiology and Biotexhnology.Item Determination of specific oxygen uptake rate of Photorhabdus luminescens during submerged culture in lab scale bioreactor(2013) Belur, P.D.; Inman, F.L.; Holmes, L.D.Photorhabdus luminescens, a bacterial symbiont of entomoparasitic nematodes, was cultured in a 10 L bioreactor. Cellular density and bioluminescence were recorded and volumetric oxygen transfer coefficient (kLa) and specific oxygen transfer rates were determined during the batch process. Exponential phase of the bacterium lasted for 20 h, showing a maximum specific growth rate of 0.339 h-1 in a defined medium. Bioluminescence peaked within 21h, and was maintained until the end of the batch process (48 h). The specific oxygen uptake rate (SOUR) was high during both lag and early exponential phase, and eventually reached a stable value of 0.33 mmol g-1 h-1 during stationary phase. Maintenance of 200 rpm agitation and 1.4 volume of air per volume of medium per minute (vvm) aeration, gave rise to a kLa value of 39.5 h-1. This kLa value was sufficient to meet the oxygen demand of 14.4 g L-1 (DCW) biomass. This research is particularly relevant since there are no reports available on SOURs of symbiotic bacteria or their nematode partners. The insight gained through this study will be useful during the development of a submerged monoxenic culture of Heterorhabditis bacteriophora and its symbiotic bacterium P. luminescens in bioreactors. © 2013 © 2013 Taylor & Francis.Item Multistrain probiotic production by co-culture fermentation in a lab-scale bioreactor(Wiley-VCH Verlag info@wiley-vch.de, 2016) Jangra, M.; Belur, P.D.; Oriabinska, L.B.; Dugan, O.M.Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co-culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co-culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co-culture fermentation in test tubes. Next, fermentation was carried out in a 3-L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co-culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Item Development of cost-effective media for the in vitro liquid culture of entomopathogenic nematodes(Brill Academic Publishers, 2022) Dunn, M.D.; Belur, P.D.; Malan, A.P.Summary Entomopathogenic nematodes (EPN) are successful biological control agents of a variety of soilborne insect pests. They have the potential to be mass-produced, using in vitro liquid culture technology, and can be formulated and sold as a biopesticide. To commercialise an EPN-based biopesticide successfully, the method of liquid mass production requires in-depth optimisation to reduce the cost of production and to increase yields, to make it affordable to the farming community. This study attempted to optimise the liquid culture protocol for the South African isolates, Steinernema jeffreyense and S. yirgalemense, by investigating the impact of cheaper medium ingredients on the recovery and yield of the liquid culture process. Studies were conducted by investigating alternative protein, lipid and nitrogen/yeast sources, compared to the more expensive laboratory-grade ingredients currently used. The results showed that egg yolk has no impact on the yield in the case of S. jeffreyense. However, for S. yirgalemense, egg yolk was shown to be a superior protein source to soy and insect-based protein in terms of nematode yield. Moreover, neither canola oil nor olive oil showed a significant difference in the yield of S. yirgalemense, with yeast extract being found to be the optimal nitrogen/yeast source. When comparing the yields with those in other liquid culture research on S. yirgalemense, yields have been successfully increased by 300%, with the cost of the nematode nutrient medium having decreased by 77%. Thus, it is imperative that, prior to a scale up to large bioreactors, the nutrient medium should be optimised to reduce the cost of production. © 2022 Copyright 2022 by Koninklijke Brill NV, Leiden, The Netherlands.