1. Mass Production of the Beneficial Nematode Heterorhabditis
bacteriaphora and It’s Bacterial Symbiont Photorhabdus
luminescens on Solid Media Using Fermentation Technology
Mary T. Johnson; Devang N. Upadhyay; Leonard Holmes
Department of Chemistry and Physics, Biotechnology Research and Training Center, The
University of North Carolina at Pembroke, 115 Livermore Drive Pembroke, NC 28372
Abstract
The focus of this research study is to mass produce the entomopathogenic nematode (EPN),
Heterorhaditis bacteriophora and its symbiont bacteria Photorhabdus luminescens as a bio-
control agent (biopesticide) on a solid media surface. The process of growing these nematodes is
to upscale the surface area of a solid agar media, thus increasing the yield of the beneficial
nematodes. The solid agar media was adjusted to conditions of a two times nutrient broth and
agar concentration with a 1% lipid concentration which provided an ideal growing environment
for these nematodes to maintain vitality for an entire life cycle. The bacterial symbiont was then
inoculated by an in-vitro culture 24 hours prior to nematode inoculation and furthermore leading
to the inoculation of Heterorhaditis bacteriophora. The inoculated entomopathogenic nematodes
develop into the beginning of a 7-8 day life cycle. Once the nematodes developed into infective
juveniles, the hermaphrodites can begin to self-fertilize its eggs and reproduce new offspring.
Large amounts of new offspring then maximize in after approximately seven days post-nematode
inoculation. After harvesting, the nematodes are sanitized and stored for further use. As an initial
scale, the surface of a petri dish (56 cm²) is inoculated with approximately 500 nematodes per
cm², harvesting yields of approximately 8000 nematodes per cm² after 7 days. The scale-up
technology used in this study can be further improved by altering solid media concentrations to
optimize the environments of Heterorhaditis bacteriophora and Photorhabdus luminescens to
subsequently reach the objective of using a larger surface area for greater yield.
Keywords: Heterorhaditis baceriophora, Photorhabdus luminescens, entomopathogenic
nematodes, In-vitro culture, scale-up, endotokia

2. Mass production of H.bacteriophora 2
Introduction
Nematodes Used as Biological Control Agents:
Biological control agents are a necessity for maintaining a pest free environment in any
agricultural field. Entomopathogenic nematodes, also known as EPNs, have long been
recognized as an economically efficient way of controlling insect pests and provide many
advantages to using chemical insecticides (Inman 316). Heterorhabditis bacteriophora is
commonly considered one of the most efficient species of EPN’s and is used as a biopesticide for
large-scale commercial manufacturing in more than forty different countries [15].
Heterorhabditis bacteriophora is also considered the most versatile as it has the ability to control
many insect pests (Yoo 759). These microscopic nematodes are distinct in comparison to other
soil-dwelling parasites because of the evolutionary symbiotic bacteria that is passed and used to
infect their insect host. Symbiotic bacterial species such as Xenorhadbus and Photorhadbus can
be found only once a nematode has grown into the infective juvenile (IJ) phase in its life cycle.
Symbiotic Relationship between Heterorhabditis bacteriaphora and Photorhabdus
luminescens
Photorhabdus luminescens is a biphasic, gram negative, bioluminescent bacterium that
maintains a symbiotic relationship with Heterorhaditis bacteriophora providing a breeding
ground for nematode reproduction. The symbiotic bacterium metabolizes the haemolymph,
which produces favorable conditions for the nematodes to grow and without this bacteria, the
nematode reproduction will not happen (Strauch 369). This symbiotic bacteria can occur in two
phenotypic forms, but only one, known as phase I is needed to effectively kill any insect host
(Chavarria-Hernandez 145). The nematode then can expel the lethal bacteria from its foregut
into the insect host during the infective juvenile stage (Chavarria-Hernandez 580). Once the
bacteria sets into the insect host, death occurs within 48 hours and the nematodes begin to feed
on the symbiotic bacteria and the decomposing insect carcass as maturation progresses (Surrey
92). During nematode development, male and females mate and produce eggs that eventually
hatch into another generation of infectious juveniles. Surprisingly, this bacterium is extremely

3. Mass production of H.bacteriophora 3
lethal to most soil dwelling insects but is completely safe for a large variety of plant and animal
species [15].
The Life Cycle of Heterorhabditis bacteriaphora
For every experiment, nematode and bacterial yields were determined using several
methods which included observation and monitoring of the nematodes during development
throughout inoculation by verifying the juvenile life stages (J1,J2,J3,J4, and infectious juveniles
[3]. During the 7-8 day incubation process, a lag phase is observed in the first 4-5 days and
growth is limited but as time progresses, a linear growth phase follows lasting for 2-3 days.
During this time, the amount of infective juveniles increases at a very rapid rate as they are
released from eggs. By the last day of incubation, the host mothers that had laid eggs are now
dead and the number of IJ’s are at an ultimate peak and reach the stationary phase [3]. At this
point, which can commonly described as endotokia, the nematodes are harvested at the peak of
their lifecycle in hopes of maintaining stability throughout the removal from the solid media.
Periodically during our research there were very low vitality rates after harvesting the
nematodes. This can be a result when harvesting takes place before endotokia has been fully
reached or if harvested too late after endotokia takes place and nutrients become limited to the
adult nematodes. Nematode growth percent yield can also be significantly low if the culture of
Photorhabdus luminescens was not in the appropriate phase or was not properly inoculated on
the total surface area of the media. This timely and often times problematic process is why
nematodes are often not studied to the full capacity.
Elements of Solid Media Concentration
In order to truly investigate the biological control potential of these nematodes, a large
scale of infective juveniles is required . In order to rear large numbers, artificial media was used
(Wouts 467). The use of artificial media has been implemented for years in the commercial
production of these nematodes, in which was later expanded for nematode production on a large-
scale (Surrey 92). The use of unsaturated fatty acids found in the olive oil throughout the media
concentration produced higher nematode yields and proved to be effective in providing an ideal
environment for both the symbiotic bacteria Photorhabdus luminescens and the
entomopathogenic nematode Heterohadbitis bacteriophora to reach maximum results.

4. Mass production of H.bacteriophora 4
Appropriate concentrations and what lipid based compound that serves most efficiently was
previously researched by our research team and also borrowed from other scientific studies. With
this previous research, we were able to enrich the medium concentration which can affect the
recovery rate of nematodes after harvesting and is another important element in vitality of
Heterohadbitis bacteriophora throughout this experiment.
Advantages of Researching Beneficial Nematodes
The capabilities of nematodes are an advantage to chemical pesticides because they do
not requiring safety equipment during application and can also eliminate any risks of water
contamination or pollution. Entomopathogenic nematodes can be produced by a variety of means
which include (but are not limited to) insect infection and grown on artificial media through solid
or liquid fermentation. Commercial entomopathogenic nematodes have been used for many
decades however are not considered competitive in the market when compared to chemical
insecticides because of the cost and quality of EPN’s. Research in the methods of these
entomopathogic nematodes has not been further explored because of problematic factors in
media composition in high-yield, short fermentation cycles, and having capabilities in recovering
an overall good quality product. In this study, we optimized media composition by maximizing
surface areas to gradually up-scale the total of nematodes harvested in hopes of creating a more
cost-efficient and effective way of harvesting nematodes to be redistributed into the industry
Materials and Methods
Isolation of Photorhabdus luminescens
Galleria mellonella is an 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. It is vital
that the culture of Photorhabdus lumiescens is maintained throughout the experiment. The
culture should also be evaluated periodically to maintain an appropriate RLU level that is vital
for nematode survival.
Sanitization of Heterorhabditis bacteriophora

5. Mass production of H.bacteriophora 5
For the first initial experiment, purchased repackaged nematodes were used as the first
generation growth cycle. In order to eliminate any bacterial contamination, a sanitation process is
used with a centrifuge to the prepackaged Heterorhabditis 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 for
inoculation.
Preparation of solid media
The following media concentrations were calculated to maintain a nutrient broth with a
2% agar concentration with a 1 % oil concentration and were modified for the appropriate
surface volume. Once the media has been autoclaved, it is then distributed to an appropriate
surface (petri dishes, small, medium and large trays) to be solidified in a sterile environment to
eliminate outside contamination that could factor in the vitality and growth of the nematodes.
Table 1: Media concentrations used in
experiment
Table 2: Amount of symbiotic bacteria
inoculated to each surface area
TotalMediaVolume 2XNutrientBroth 2%Agar 1%Oil pHLevel
SmallTray(400mL) 6.4g 8g 4mL 7.5
MediumTray(500mL) 8.0g 10g 5mL 7.5
LargeTray(600mL) 9.6g 12g 6mL 7.5
CookieSheet(800mL) 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

6. Mass production of H.bacteriophora 6
Inoculation of Photorhabdus luminescens on solid media
The isolated P. luminescens bacteria is inoculated evenly to each surface area and grown
to be used as a nutrient that Heterorhabditis bacteriophora can feed off of once inoculated on the
solid media. A crucial part of the experiment is to grow an efficient amount of Photorhabdus
luminescens to maintain an appropriate amount of nematodes on the surface area.
Inoculation of sanitized Heterorhabditis bacteriophora
Before adding Heterohabditis bacteriophora to the solid media, it is necessary for
Photorhadus luminescens to grow for at least 24 hours after initial inoculation. Growth should be
identifiable by an evenly coated, light red film covering the entire surface area of the solid
media. If bacterial growth is not abundant during the inoculation of Heterorhabditis
bacteriophora then an environment to maintain the stability of the nematodes cannot be obtained
and will affect the vitality of the nematode population after harvesting. After Heterohadbitis
bacteriophora is inoculated, there is seven day incubation.
Harvesting, counting and packaging nematodes
Once nematodes have grown on the full surface area of the media for a full week,
nutrients begin to become scarce and removal of Heterohadbitis bacteriophora from the solid
media is necessary for survival. The removal process varies depending on the quantity but
involves little to anthing with the exception of distilled water. Once the distilled water is added
to the solid media, the surface nematodes are washed off with ease with gentle shaking. More
nematodes may be lodged into the agar of the media and can also be removed with soaking.
Once the new generation of Heterohadbitis bacteriophora is gathered from the media, a total
nematode count is made. This can be done through a dilution process and a graphical microscope
slide. That counted number is then used in a conversion calculation to find the total count for an
entire surface area.

7. Mass production of H.bacteriophora 7
An example taken from the smallest surface area, a collection of 8 petri dishes:
11 Nematodes / 0.1 mL / 100X Dilution
1,100 nematodes / 0.1 mL
11,000 nematodes / 1 ml X 325 mL of harvested volume
3,757,000 nematodes / 325 mL / 8 Petri plates
446,875 nematodes / Per Plate / 56 cm²
= 7,979 per cm² ≈ 8,000 nematodes per cm²
The total nematode count for 8 petri dishes was approximately 8,000 nematodes per cm².
This was used as the baseline count in our experiment as our aim was to up-scale in surface area
thus increase the nematode percent yield per cm². After counting, the nematodes are packaged
immediately and stored for further use in a temperature sensitive area.
Results
As demonstrated in Table 3 the total surface area was increased periodically which lead to the
nematode count collected also to increased dramatically. There was a significant increase in
nematode growth of approximately 16-25 times fold. Maintaining a consistent concentration had
a significant impact on the new juvenile population, as expected because nematode growth is
heavily influenced by media concentration.

8. Mass production of H.bacteriophora 8
Table 3: Demonstration of percent yield in nematode production over large scale solid media
Discussion
Nematode percent yield corresponds directly to composition and concentration of solid
media and served to efficiently produce over 20 times the original yield. Scale-up and separation
of nematodes from liquid media is typically viewed as an easier and more economical method
than scale-up and separation from solid media. Though solid media fermentation is more labor
intensive, the need for expensive bioreactors and laboratory equipment is obsolete. Our goal is to
use natural raw media products for nematode mass production with an easy and convenient way
for agriculturalist and also scale-up this process using larger surface area for greater yield then
traditional liquid cultures. Benefits of this research include the capabilities to withdraw from
traditionally relied on insecticides, providing a high and more reliable efficacy with greater
understanding of products being used and lastly possibly starting a desire for more
environmentally sensitive growing throughout our society. Additional research is required to
maximize nematode survival during the separation process and to assess the pathogenicity of
harvested nematodes to appropriate host insects.
Acknowledgments
A warm thank you to the Farm Bureau of Pembroke, North Carolina, the University of North
Carolina at Pembroke Chemistry and Physics Department, and the Biotechnology Center for
financial assistance of this research as well as providing efficient equipment and man power
required for such extensive research.
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