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
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)
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.
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
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.
Life-cycle complexes for beneficial nematodes
• Bacteria colonizes inside the infective
juveniles (IJs)
Tripartite interaction
• 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
Ref: Ehlers RU (2001) Mass production of entomopathogenic
nematodes for plant protection. Appl Microbiol Biotechnol (2001)
56:623–633
• Non-feeding IJ stage emerges into the soil
to forage for a new host
Development stages of Nematodes
IJ3
Egg
Young hermaphrodite
Adult
J1
Endotokia
Objective
 Mass production of EPN Heterorhabditis bacteriophora by Invitro 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
Outline of beneficial nematode culturing protocol
Challenges of the microbiology
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Phase variation of bacterial symbiont
Fecundity of nematode inoculum
Optimization of media composition
Agitation, dissolved oxygen and pH
Contamination
(t = 0)
(T = 24 hr)
SCALE-UP FOR PRODUCTION
5 Liter
10 Liter
30 Liter
Challenges in Production and Commercialization
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Consistency of mass production
Production capacity
Production costs
Pricing and shipping costs
Marketing
• UNCP Thomas Family Center for Entrepreneurship
• Website (http://sites.google.com/site/braveguardnematodes
• Two kinds of customers: Internet & regional
Regional Field Testing
References
• Ehlers, R., Lunau, S., Krasomil K. and Osterfeld, K. 1998. Liquid culture of the
entomopathogenic nematodebacterium-complex Heterorhabditis
megidis/Photorhabdus luminescens. BioControl 43: 77–86
• Ehlers, R.U. 2001. 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): 316-324.
doi:10.1007/s12088-012-0270-2
• Shapiro-Ilan, D. and Gaugler, R. 2002. 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. 2013. Mass
production of the beneficial nematode Steinernema carpocapsae utilizing a fedbatch culturing process. ESci J. Plant Pathol. 02 (01): 52-58
• Yoo, S., Brown, I. and Gaugler, R. 2000. Liquid media development for
Heterorhabditis bacteriophora: lipid source and concentration. Appl microbial
Biotechnol. 54: 759-763
THANK YOU!