1. Mass Production of Heterorhabditis bacteriaphora Using Solid
State Fermentation Technology
Mary T. Johnson; Devang N. Upadhyay; Leonard Holmes
The 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 luminescens
2)Sanitization of Heterorhabditis bacteriophora
3 ) Preparation of solid media 2NA with 2% agar & 1
% lipid in in plates and trays having different surface
area
4) Inoculation of Photorhabdus luminescens on solid
media
5) Inoculation of sanitized Heterorhabditis
bacteriophora after 24 hours of bacterial growth &
Incubation
6) 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 Nematodes
11 Nematodes / 0.1 mL / 100X Dilution
1,100 / 0.1 mL
11,000 / 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.