American Journal of Bioscience and Bioengineering
Volume 3, Issue 6, December 2015, Pages: 203-207
Received: Dec. 8, 2015;
Accepted: Dec. 17, 2015;
Published: Dec. 30, 2015
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Devang Upadhyay, Sartorius Stedim Biotechnology Laboratory, Biotechnology Research and Training Center, the University of North Carolina at Pembroke, Pembroke, USA
Sivanadane Mandjiny, Department of Chemistry and Physics, the University of North Carolina at Pembroke, Pembroke, USA
Rebecca Bullard-Dillard, School of Graduate Studies and Research, the University of North Carolina at Pembroke, Pembroke, USA
Meredith Storms, College of Arts & Sciences, the University of North Carolina at Pembroke, Pembroke, USA
Michael Menefee, Thomas Family Center for Entrepreneurship, the University of North Carolina at Pembroke, Pembroke, USA
Leonard D. Holmes, Sartorius Stedim Biotechnology Laboratory, Biotechnology Research and Training Center, the University of North Carolina at Pembroke, Pembroke, USA
The objective of this research is to develop fermentation methodology for the production of the biocontrol agent Heterorhabditis bacteriophora. Deployment of this organism will reduce the use of chemical insecticides which threaten the environment. This study shows how to produce the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora and its bacterial symbiont Photorhabdus luminescens utilizing an in vitro, monoxenic liquid culture. EPNs were cultured in three different bioreactor working volumes of 1.5, 4 and 7 liters with initial nematode inoculation concentrations of approximately 2x103/mL. Liquid nematode media was conditioned with the bacterial symbiont 24 hours prior to nematode inoculation. Within three days after inoculation, infective juveniles (IJs) developed into self-fertilizing hermaphrodites and eventually produced IJ offspring. Maximum nematode densities were obtained seven days post-nematode inoculation. All three working volumes (1.5, 4 and 7 liters) produced final yields of 4.6x104 ± 2000 IJs/mL, 4.2x104 ± 2200 IJs/mL and 3.9x104 ± 2000 IJs/mL, respectively. In vitro scale-up technology can be further optimized for production of this biocontrol agent by improving media formulation, process parameters, bioreactor design and inoculation times that will maximize nematode yield.
Leonard D. Holmes,
Lab-scale in vitro Mass Production of the Entomopathogenic Nematode Heterorhabditis bacteriophora Using Liquid Culture Fermentation Technology, American Journal of Bioscience and Bioengineering.
Vol. 3, No. 6,
2015, pp. 203-207.
Salma J, Shahina F (2012) Mass production of eight Pakistani strains of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae). Pakistan Journal of Nematology 30 (1): 1-20.
Surrey M, Davies R (1996) Pilot-Scale Liquid Culture and Harvesting of an Entomopathogenic Nematode, Heterorhabditis bacteriophora. Journal of Invertebrate Pathology 67: 92–99.
Shapiro-Ilan D, Gaugler R (2002) Production technology for entomopathogenic nematodes and their bacterial symbionts. Journal of Industrial Microbiology & Biotechnology. 28: 137 –146.
Patterson W, Upadhyay D, Mandjiny S, Bullard-Dillard R, Storms M, Menefee M, Holmes L (2015) Attractant Role of Bacterial Bioluminescence of Photorhabdus luminescens on a Galleria mellonella Model. American journal of life sciences 3(4): 290-294.
Strauch O, Ehlers R (1998) Food signal production of Photorhabdus luminescens inducing the recovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture. Applied Microbiology and Biotechnology 50: 369-374.
Gerdes E, Upadhyay D, Mandjiny S, Bullard-Dillard R, Storms M, Menefee M, Holmes L (2015) Photorhabdus Luminescens: Virulent Properties and Agricultural Applications. American Journal of Agriculture and Forestry 3(5): 171-177.
Yoo S, Brown I, Gaugler R (2000) Liquid media development for Heterorhabditis bacteriophora: lipid source and concentration. Applied Microbiology and Biotechnology 54: 759-763.
Ehlers RU (2001) Mass production of entomopathogenic nematodes for plant protection. Applied Microbiology and Biotechnology 56: 623-633.
Gaugler R (1997) Alternative paradigms for commercializing biopesticides. Phytoparasitica 25: 179-182.
Ehlers RU, ShapiroIlan DI (2005) Mass production 65-78pp. In: Nematode as biological control agent. Grewal, Ehler and Shapiro-Ilan, CAB International, Wallingford, UK.
Lacey L, Grzywacz D, Shapiro-Ilan D, Frutos R, Brownbridge M, Goettel M (2015) Insect pathogens as biological control agents: Back to the future. Journal of Invertebrate Pathology 132: 1–41.
Wouts W (1981) Mass production of the entomopathogenous nematode Heterorhabditis bacteriophora (Nematode: Heterorhabditidae) on artificial media. Journal of Nematology 13: 467-469.
Shapiro-Ilan D, Han R, Dolinksi C (2012) Entomopathogenic Nematode Production and Application Technology. Journal of Nematology 44(2): 206–217.
Neves JM, Teixeira JA, Simoes N, Mota M (2001) Effect of airflow rate on yield of Steinernema carpocapsae Az 20 in liquid culture in an external-loop airlift bioreactor. Biotechnology and Bioengineering 72: 369–373.
Strauch O, Stoessel S, Ehlers R (1994) Culture conditions define automictic or amphimictic reproduction in entomopathogenic rhabditid nematodes of the genus Heterorhabditis. Fundamental & Applied Nematology 17(6): 575-582.
Kooliyottil R, Upadhyay D, Inman III FL, Mandjiny S, Holmes LD (2013) A Comparative Analysis of Entomoparasitic Nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae. Open Journal of Animal Science 3(4): 326-333.
Jessen P, Strauch O, Wyss U, Luttmann R, Ehlers RU (2000) Carbon dioxide triggers dauer juvenile recovery of entomopathogenic nematodes (Heterorhabditis species.) Nematology 2: 310–324.
Jeffke T, Jende D, Matje C, Ehlers RU, Berthe-Corti L (2000) Growth of Photorhabdus luminescens in batch and glucose fed batch culture. Journal of Applied Microbiology and Biotechnology 54: 326–330.
Inman FL, Holmes LD (2012b) The effects of trehalose on the bioluminescence and pigmentation of the phase I variant of Photorhabdus luminescens. Journal of Life Science 6: 119-129.
Upadhyay D, Kooliyottil R, Mandjiny S, Inman III F, Holmes L (2013) Mass production of the beneficial nematode Steinernema carpocapsae utilizing a fed-batch culturing process. ESci Journal of Plant Pathology 02(01): 52-58.
Akhurst RJ (1980) Morphological and functional dimorphism in Xenorhabdus spp. bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis. Journal of general microbiology 121: 303–309.
Inman FL, Holmes LD (2012a) Mass production of the beneficial nematode Heterorhabditis bacteriophora and its bacterial symbiont Photorhabdus luminescens. Indian Journal Microbiology 52(3): 316-324.
Ehlers R, Lunau S, Krasomil K, Osterfeld K (1998) Liquid culture of the entomopathogenic nematode bacterium-complex Heterorhabditis megidis/Photorhabdus luminescens. Bio Control 43: 77–86.
Friedman M, Langston S, Pollitt S (1992) Mass production in liquid culture of insect killing nematodes. Patent WO1989004602A1.