H.bacteriaphora Article

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

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


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.


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


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

Total 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


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.


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.


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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H.bacteriaphora Article