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Mass Production of the Beneficial Nematode, Heterorhabditis bacteriophora, in Submerged Culture.

Floyd L. Inman, III*, Len Holmes

The goal of this work is to mass produce, in liquid media, a submerged culture of the beneficial nematode, Heterorhabditis bacteriophora. Initial shake flask cultures were scaled up to 10 L in a Sartorius-stedim Biostat B® Plus fermentation system. The culture conditions that were controlled by the system include pH, pO2, agitation, and temperature. Microscopic observations and nematode density counts were collected over a four week period to evaluate the growth and development of the nematodes in liquid culture. To evaluate nematode development, the nematode life cycles were used as markers. The system was inoculated with approximately 1,000 infective stage 3 juveniles (IJs) per mL of media and nematode development was observed within three days after inoculation.

Symbiotic Properties of the Entomopathogenic Bacterium, Photorhabdus luminescens, and its Nematode Host, Heterorhabditis bacteriophora.

Floyd L. Inman, III*, Len Holmes

Photorhabdus luminescens is a biphasic, Gram-negative, bioluminescent enteric bacterium that maintains a mutualistic relationship with its nematode host, Heterorhabditis bacteriophora. Not only does the bacterium maintain this close relationship, it also exhibits pathogenicity towards a diverse group of insects. Reports suggest that the phase I variant signals nematode development and that its virulence may provide an excellent breeding ground for nematode reproduction inside of the insect carcass. Initial investigations began when trying to culture the nematodes in liquid media. Reported liquid mediums were used and of those only one medium exhibited nematode growth and development. Investigational data suggests that strain specificity of the bacterium is indeed necessary for nematode reproduction. It was determined that more research was needed to understand how this bacterium can support the growth and development of the nematode. One investigation studied the antimicrobial pigment produced by P. luminescens and was shown that eleven species and strains of bacteria were determined to be sensitive to this pigment. It was then concluded that P. luminescens may have the capability to fight off many other competing microbes. Another investigation studied the effects of carbohydrate utilization on the stability and bioluminescence of the phase I variant. Results showed that trehalose, the “blood sugar” of insects, increased stability and bioluminescence of the phase I variant for seven days in liquid culture media. Genetic investigations are currently being performed on the isolated bacterial strain, P. luminescens ARB. Recent results show that this strain carries a plasmid that is approximately 29 kilobases in size and is in the process of being sequenced. Plasmid electrotransformation results of Escherichia coli DH10β suggest that this isolated plasmid may confer kanamycin resistance. Experimental studies of pathogenicity and symbiosis are currently being designed to utilize the electrotransformed E. coli as a bacterial replacement for P. luminescens ARB.