Lab-scale <i>in vitro</i> Mass Production of the Entomopathogenic Nematode <i>Heterorhabditis bacteriophora</i> Using Liquid Culture Fermentation Technology

Abstract

Abstract:The objective of this research is to develop fermentation methodology for the production of the biocontrol agent Heterorhabditis bacteriophora. Deployment of this organism will reduce the use of chemical insecticides which threaten the environment. This study shows how to produce the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora and its bacterial symbiont Photorhabdus luminescens utilizing an in vitro, monoxenic liquid culture. EPNs were cultured in three different bioreactor working volumes of 1.5, 4 and 7 liters with initial nematode inoculation concentrations of approximately 2x10 3 /mL. Liquid nematode media was conditioned with the bacterial symbiont 24 hours prior to nematode inoculation. Within three days after inoculation, infective juveniles (IJs) developed into self-fertilizing hermaphrodites and eventually produced IJ offspring. Maximum nematode densities were obtained seven days post-nematode inoculation. All three working volumes (1.5, 4 and 7 liters) produced final yields of 4.6x10 4 ± 2000 IJs/mL, 4.2x10 4 ± 2200 IJs/mL and 3.9x10 4 ± 2000 IJs/mL, respectively. In vitro scale-up technology can be further optimized for production of this biocontrol agent by improving media formulation, process parameters, bioreactor design and inoculation times that will maximize nematode yield.

Mass Production of the Beneficial Nematode Steinernema carpocapsae Using Solid State Fermentation

“…Johnson et al (2016) reported 25 fold yield of the beneficial nematode Heterorhabditis bacteriophora by inoculating 900 nematodes per cm 2 of solid media surface which correlates closely with this report [17]. reported 19 folds of the entomopathogenic nematode Heterorhabditis bacteriophora using liquid culture fermentation technology [18]. However, in terms of absolute production, solid state fermentation yielded 110 maximum fold, but relatively small final yield.…”

Biological Control Technology Utilizing Heterorhabditis bacteriophora and Steinernema carpocapsae

“…Another challenge in EPN research is consistency. Published yields of IJs vary considerably with average counts between 20,000 to 40,000 per mL Upadhyay, 2015). The quality of infective juveniles (IJs) also depends on the method of production, media composition, and successful infection during inoculation.…”

“…Furthermore, Phase I variants also produce a wide range of antimicrobials that prevent other organisms from invading the insect cadaver. By doing so, the bacterial symbionts produce an ideal breeding ground for its nematode partner (Upadhyay, 2015). EPNs inhibit pests through a symbiotic nematodebacterium mechanism.…”

Inter-generational Consequences for Growing C.elegans in Liquid

“…Entomopathogenic nematodes such as Steinernema [21,22] and Heterorhabditis [23,24] are grown in liquid cultures as means of mass production of a biocontrol agent. However, liquid-grown entomopathogenic nematodes were found to have lower dauer stage recovery rates and pathogenicity [21,55,56], and much effort was invested in optimizing the different aspects of their mass production in liquid cultures [22].…”

“…Liquid media was also used to study how swim exercise affects the worm's physiology [20]. In addition to C. elegans, other nematodes, such as the entomopathogenic species Steinernema [21,22] and Heterorhabditis [23,24] are routinely grown in liquid cultures, and recently, growth in liquid medium of different Pristionchus species was shown to generate striking phenotypic differences [25] (see also discussion).…”