Browsing by Author "Malan, A.P."

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A review of the in vitro liquid mass culture of entomopathogenic nematodes
(Taylor and Francis Ltd., 2021) Dunn, M.D.; Belur, P.D.; Malan, A.P.
Entomopathogenic nematodes (EPNs) are a safe insect biological control agent. Key to the implementation of EPNs as a biopesticide is their mass production in shake flasks and bioreactors. For commercial application, in vitro liquid culture is the predominant choice, due to the cost and scale of production, and to the ease of downstreaming. The in vitro liquid culture of EPNs begins in Erlenmeyer shake flasks to provide aeration and agitation. The initial liquid culture phase is followed by upscaling to 5-20-L desktop bioreactors and, thereafter, to 80-120-1000-L industrial-scale bioreactors. The ingredients of the liquid culture media, on which symbiotic bacteria and nematode develop, is of great importance for mass-culturing. The diet usually consists of essential nutrients that best replicate the constituency of the natural insect host, such as protein, carbohydrates and lipids. In addition, aeration and agitation are maintained, without causing shear damage due to the rotating impeller blades. Such factors, however, requires different parameters, depending on the EPN species involved, and, moreover, optimisation is required to obtain high yields and quality of infective juveniles. The objective of the current review is to assess the conditions required for optimal EPN production in liquid culture, and how the conditions might be optimised for South African EPN species. © 2020 Informa UK Limited, trading as Taylor & Francis Group.
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.
Effect of Glucose, Agar Supplementation and Bacterial Cell Density on the in vitro Liquid Culture of Steinernema jeffreyense
(Entomological Society of Southern Africa, 2021) Dunn, M.D.; Belur, P.D.; Malan, A.P.
Entomopathogenic nematodes (EPNs) of the family Steinernematidae are effective biological control agents against important pest insects. The in vitro liquid culture method of mass production is used to produce EPNs of high quantity, quality and with reduced cost by upscaling. The first step in liquid mass production is the use of shake flasks to obtain monoxenic infective juvenile (IJ) inoculum for optimisation purposes, followed by upscaling to a desktop fermenter. This study was undertaken to assess the impact of the addition of agar and glucose to the culture medium, as well as assessing the impact of bacterial cell density inoculum on IJ recovery and yield. Steinernema jeffreyense was cultured in 250 ml Erlenmeyer flasks, with the mutualistic bacterium Xenorhabdus khoisanae. In this study the impact of four different agar and glucose concentrations showed negligible impact on nematode recovery and yield. Different initial bacterial inoculum densities inoculated to the culture medium showed a low inoculum density of 2 % (6 × 106 cells) of the bacteria culture to be the optimal inoculum concentration. A bacterial growth curve for X. khoisanae in tryptic soy broth, showed 40-44 h to be the optimum time for introduction of the initial nematode inoculum into the complex medium. © 2021 Entomological Society of Southern Africa. All rights reserved.
In vitro liquid culture and optimization of Steinernema jeffreyense using shake flasks
(2019) Dunn, M.D.; Belur, P.D.; Malan, A.P.
Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml?1 after 14 days, in comparison to 75% and 99,875 IJs ml?1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml?1), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo. 2019, International Organization for Biological Control (IOBC).
In vitro liquid culture and optimization of Steinernema jeffreyense using shake flasks
(Springer editorial@springerplus.com, 2020) Dunn, M.D.; Belur, P.D.; Malan, A.P.
Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml?1 after 14 days, in comparison to 75% and 99,875 IJs ml?1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml?1), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo. © 2019, International Organization for Biological Control (IOBC).