Mass Production of Heterorhabditis bacteriophora Using Solid State Fermentation Technology

Mary Tess Johnson

The University of North Carolina at Pembroke, NC 28372

Biotechnology Research and Training Center

Project Background

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.

Photorhabdus luminescens is a biphasic, Gram-negative, bioluminescent bacteria that maintains a very mutualistic relationship with the nematodes

Photorhabdus luminescens H. bacteriophora

Research Objective

The purpose 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, packaged and then stored for further use.

Variables

How to Produce P. luminescens?

Galleria mellonella is a insect considered to be a model host used for studies of entomoparasitic nematodes (EPNs)

If the larva is infected, the carcass should turn to a brownish-red color, reflecting the presence of P. luminescens.

Once infection is verified, P. luminescens is extracted from the intestinal tract of the Galleria to produce and grow multiple cultures.

VariablesWhat are the life stages of Heterorhabditis bacteriophora?

Developmental Stages: (J1, J2, J3 (IJ), J4)

Once endotokia has been verified, harvesting of nematodes can begin.

J4

Egg J1 Endotokia

MaterialsTotal Media Volume 2 X Nutrient Broth 2% Agar 1% Oil pH Level

Small Tray (400 mL) 6.4 g 8g 4 mL 7.5

Medium Tray (500 mL) 8.0g 10g 5mL 7.5

Large Tray (600 mL) 9.6g 12g 6mL 7.5

Cookie Sheet (800 mL) 12.8g 16g 8mL 7.5

Total Media Volume Total Surface Area P.lum Inoculated

Petri plates (30 mL) 56 cm² 30 µL

Small Tray (400 mL) 400 cm² 200 µl

Medium Tray (500 mL) 490 cm² 250 µL

Large Tray (600 mL) 742.5 cm² 400 µL

Cookie Sheet (800 mL) 1218 cm² 600 µL

Media Concentrations used throughout experiment.

Surface area and amount of P.lum added to solid media

Procedures Isolating P. luminescens: Transferred and isolated pure cultures to be used

during inoculation. Sanitization of H. bacteriophora: After approximately ten cycles of sanitation

using the centrifuge at 500 RPMs for five minutes, nematodes are sanitized with sterile water and decanted to reduce volume to a desired amount of 20 µl

Preparation of Solid Media: To create a baseline of growth, solid media of 2X nutrient broth and 2% agar is added to large surface area trays.

Inoculation of P. luminescens: To provide a optimal enviorment for Heterorhabditis bacteriophora.

Inoculation of sanitized H. bacterophora: Once nematodes have been sanitized with a hymine treatment (to sterilize and remove any external bacteria) they are added to the plates and grown.

Harvesting, Counting and Packaging:. Once nematodes are sanitized, they are added to solid media for redistribution.

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

B C

Counting Nematodes

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

Data/Observations

Significant increase in nematode growth of 16-25 times fold. Nematode count is increased as solid media surface area is increased as well

Conclusion

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.

Benefits of this Research Include: Withdraw from traditionally relied on insecticides High and more reliable efficacy with greater understanding of

products Desire for more environmentally sensitive growing.

Any Questions?