Optimisation of the in vitro liquid culture protocol for Heterorhabditis bacteriophora

Summary Heterorhabditis bacteriophora is an entomopathogenic nematode that maintains a symbiotic relationship with Photorhabdus bacteria, and is a potent biocontrol agent against agricultural insect pests. The mass culture of these nematodes is a critical requirement for their use in commercial biocontrol application. In vitro liquid culture methods have been used successfully to mass produce H. bacteriophora , in both shake flasks and bioreactors. Although shake flask culture methods have achieved high yields, optimisation of in vitro liquid culture techniques for H. bacteriophora is essential for achieving high yields, shortened production time, and cost-effective production. Amendments to the culture medium (CM), such as trehalose powder and dimethyl sulfoxide (DMSO), showed no significant differences in yield from the control. To improve oxygen circulation in flasks the shaker rotational speed was increased from 140 rpm to 160 rpm. There were no significant differences in yield between the two rotational speeds. Additionally, this study highlights the issue of a second generation of nematodes in the CM, as unfertilised adults negatively impact on yields of infective juveniles and batch quality. Reducing the protein sources resulted in an increase in yield. Alternative yeast sources, the most expensive ingredient in the CM, were also compared, with 1451MG having a significantly higher yield compared to the standard laboratory grade yeast used in the culture media.