1. In-vitro mass production of the beneficial nematode Heterorhabditis bacteriophora using liquid culture fermentation technology
Len Holmes, Michael Menefee and Floyd Inman III
Biotechnology Research and Training Center
University of North Carolina at Pembroke
Pembroke, NC 28372

2. Agricultural Biological Control Products
Biocontrol: Reduction of pest populations by natural enemies; involves active human role.
Natural enemies include: Parasitoids, predators, microbes & beneficial nematodes.
Targets: animals, weeds and disease.
During last century hundreds of exotic control agents have been used.
(North Carolina State University)

3. How Biocontrol Directly Benefits Agriculture
Controls insect or weed pests to manageable levels (potentially permanent).
Reduces hazard of chemical use and acute or long-term impact on humans and non-target organisms.
No potential resistance build-up as with chemical inputs.
No delay between application and harvesting.
Residue-free products from farms.
No phytotoxic effects on young plants (abortion of flowers).
Public is more accepting of biological control than chemical agents.

4. Barriers to Adoption of Biocontrol Approaches
I. Necessity of educated management and planning. II. More complete and accessible information on risks to environment and non-target organisms is needed. III. Knowledge on insect pest range and biology of insect host(s). IV. Abiotic and biotic factor interactions with biocontrol agent. V. Knowledge of synchronization of host and it natural enemies. VI. Knowledge of dispersal technology

5. Beneficial Nematodes How they are beneficial?
Environmentally-friendly substitute for chemical insecticide
Photorhabdus luminescens bacteria are symbiotically associated with the beneficial nematode Heterorhabditis bacteriophora
Nematode recovery in liquid culture can vary considerably based upon many factors such as: bacterial phase variant, media formulation, bacterial density, etc.

6. Life-cycle complexes for beneficial nematodes
Bacteria colonizes inside the infective juveniles (IJs)
IJs live in the soil until they invade a susceptible insect host, seeking the hemolymph
IJs release the bacteria into the insect’s hemolymph
The bacteria participate in overcoming the insect’s defense system and killing the host
Nematode growth and reproduction take place
Nutrient limitation in the host insect cadaver
Non-feeding IJ stage emerges into the soil to forage for a new host
Tripartite interaction
Ref: Ehlers RU (2001) Mass production of entomopathogenic nematodes for plant protection. Appl Microbiol Biotechnol (2001) 56:623–633

7. Development stages of Nematodes
IJ3
Young hermaphrodite
Adult
Egg J1
Endotokia

8. Objective
Mass production of EPN Heterorhabditis bacteriophora by In-vitro liquid culture fermentation technology using three different bioreactor working volumes of 5, 10 and 30 liters and scale-up.
In vivo culture
In vitro: solid culture
In vitro: liquid culture

9. Outline of beneficial nematode culturing protocol

10. Challenges of the microbiology
Phase variation of bacterial symbiont
Fecundity of nematode inoculum
Optimization of media composition
Agitation, dissolved oxygen and pH
Contamination
(t = 0)
(T = 24 hr)

11. Scale-up for ProDUCtion
5 Liter
10 Liter
30 Liter

13. Challenges in Production and Commercialization
Consistency of mass production
Production capacity
Production costs
Pricing and shipping costs
Marketing
UNCP Thomas Family Center for Entrepreneurship
Website (
Two kinds of customers: Internet & regional
Regional Field Testing

14. References
Ehlers, R., Lunau, S., Krasomil K. and Osterfeld, K Liquid culture of the entomopathogenic nematodebacterium-complex Heterorhabditis megidis/Photorhabdus luminescens. BioControl 43: 77–86
Ehlers, R.U Mass production of entomopathogenic nematodes for plant protection. Appl. Microbiol. Biotechnol. 56: 623–633.
Inman, F.L. III and L.D. Holmes. 2012a. Mass production of the beneficial nematode Heterorhabditis bacteriophora and its bacterial symbiont Photorhabdus luminescens. Indian J. Microbiol. 52(3): doi: /s
Shapiro-Ilan, D. and Gaugler, R Production technology for entomopathogenic nematodes and their bacterial symbionts. Journal of Industrial Microbiology & Biotechnology. 28: 137 –146
Upadhyay, D., Kooliyottil, R., Mandjiny, S., Inman III, F. and Holmes, L Mass production of the beneficial nematode Steinernema carpocapsae utilizing a fed-batch culturing process. ESci J. Plant Pathol. 02 (01): 52-58
Yoo, S., Brown, I. and Gaugler, R Liquid media development for Heterorhabditis bacteriophora: lipid source and concentration. Appl microbial Biotechnol. 54:

15. THANK YOU!