Mass Production of Heterorhabditis bacteriaphora Using Solid State Fermentation Technology

Mary T. Johnson; Devang N. Upadhyay; Leonard HolmesThe University of North Carolina at Pembroke; Biotechnology Research and Training Center

Introduction Discussion

Experimental Process

Acknowledgements

The focus of our research is to mass produce, on solid media, the beneficial nematode, Heterorhabditis bacteriophora that in essence will create a cost effective way to redistribute these nematodes for agricultural purposes. In order to do so, understanding the role of Photorhabdus luminescens, in the growth of its nematode host, Heterorhabditis bacteriophora is critical. Photorhabdus luminescens is a biphasic, Gram-negative, bioluminescent bacteria that maintains a very mutualistic relationship with the nematodes. It is critical to understand the stability requirements of this bacterial variant for nematode growth to be successful. The process of growing these nematodes is to each time upscale the surface area of a solid agar media to also increase the amount of nematodes produced. Once harvested from the media, these nematodes are sanitized and stored for further use.

Figure 7: Sanitation cycle of nematodes after harvesting before storage.

Thank you to Farm Bureau, the Biotechnology Center, and the University of North Carolina Chemistry and Physics Department for support of this research

Development stages of Nematodes

1)Isolation of Photorhabdus luminescens2)Sanitization of Heterorhabditis bacteriophora3 ) Preparation of solid media 2NA with 2% agar & 1 % lipid in in plates and trays having different surface area4) Inoculation of Photorhabdus luminescens on solid media 5) Inoculation of sanitized Heterorhabditis bacteriophora after 24 hours of bacterial growth & Incubation6) Harvesting, Counting and Packaging

Results

Figure: (A) Solid media before nematode inoculation (B) After nematode growth, 7 days incubation (C) Nematode Harvesting

A B C

J3 J4 Adult

Egg J1 Endotokia

H. bacteriophoraPhotorhabdus luminescens

Conclusion

Example of How to Count Nematodes11 Nematodes / 0.1 mL / 100X Dilution1,100 / 0.1 mL11,000 / 1 ml X 325 mL of harvested volume

3,757,000 / 325 mL / 8 Petri plates 446,875 / Per Plate / 56 cm² = 7,979 cm²≈ 8,000 cm²

A quick analysis of the data demonstrated throughout the graph can indicate a significant increase in nematode growth of 16-25 times fold. Nematode count is increased as solid media surface area is increased as well.

Our goal is to use natural media products during nematode growth to provide a easy and convenient way for agriculturalist to also grow nematodes at a large scale on solid media.