DESC:*** Complete Specification ***

“Medium, and method based thereon, for in-vitro production of entomopathogenic nematodes and their symbiotic bacteria”

Cross references to related applications: This complete specification is filed further to patent application No. 202221074142 filed on 21/02/2024 titled “Medium, and method based thereon, for in-vitro production of entomopathogenic nematodes and their symbiotic bacteria” and the entire content of the provisional specification filed therewith is incorporated herein in its entirety by way of reference.

Field of the invention
This invention relates to the methodologies and media involved in breeding or cultivation of invertebrates. More specifically, the disclosures herein are directed to an inventive liquid medium composition, and a method based on said medium composition, for in-vitro production of entomopathogenic nematodes along with their symbiotic bacteria.

Definitions and interpretations
Before undertaking the detailed description of the invention below, it may be advantageous to set forth definitions of certain words or phrases used throughout this patent document: the terms “include” and “comprise,” as well as derivatives thereof, mean inclusion without limitation; the term “or” is inclusive, meaning and/or; the phrases “associated with” and “associated therewith,” as well as derivatives thereof, may mean to include, be included within, interconnect, with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have, have a property of, or the like; and certain definitions are set forth for this document, as follows: -

(a) “CAS” shall refer to Chemical Abstracts Service, a division of the American Chemical Society which allocates unique “CAS numbers” to chemical substances which hence become unique identifiers of said chemical substances to provide a standardized global way to refer to them.
(b) “PTFE” refers to Polytetrafluoroethylene, a fluoropolymer also commonly known by its trade name, Teflon®.
(c) “IJ” refers to infective juveniles.

It shall be generally understood by the reader that although the present invention is described herein using these specific terms, these are used in a generic and descriptive sense only and are not intended to be limiting.

Background of the invention
Steinernema carpocapsae (common name roundworm) is an entomopathogenic nematode, that is, it is pathogenic to arthropods and, in symbiosis with certain bacteria, brings about rapid mortality, thus being valuable as an effective, safe, environmentally friendly, non-toxic biological insecticide for said arthropods.

Mode of action of Steinernema carpocapsae is typical. When in contact with insects, it enters the blood, body and cavities of said insects through the mouth, anus etcetera of the insects. When inside said host insects, symbiont bacteria hosted by Steinernema carpocapsae are released into the body of the host insects, which secrete antibiotics and / or chemicals capable of destroying the immune mechanism of said host insects, resulting in the rapid death of the host insects. This characteristic has enabled the utilization of Steinernema carpocapsae for killing insects harmful to plants such as crops to prevent damage to the plants. Hence, the production of Steinernema carpocapsae, preferably along with its symbiotic bacteria, is desirable in large quantities the world over.

A brief survey of prior art reveals various attempts at mass cultivation of nematodes. A variety of materials and methods have been proposed by various researchers. For example, solid culture methods using agar media, Styrofoam fragments, and liquid culture methods using liquid components have been studied. In-vivo method using Galleria mellonella as a host for infection and multiplication of entomopathogenic nematodes has also been proposed. However, these materials and methods are not capable of industrial-scale utility yet due to their inherently low yields, expensive media components and / or equipment, besides providing unstable cultures with low viability or resultant nematodes with reduced insecticidal efficacy and the indispensability of maintaining an insectary lab and skilled technicians for the same. Hence, there is a pressing need for the advent of means which can effectively overcome these shortfalls in the current state-of-art.

While there were many common art references researched by the inventor(s) in ensuring that the present invention is novel, the following patent prior art was identified as related to the present invention, and thus worthwhile to discuss in more detail in context of the present invention. One example is CN1115408C (issued to Han Richou Li Liying) which teaches a medium composition for cultivation of nematodes, said composition comprising soybean powder, 1% flour, 0.5% yeast extract paste, 1.2% powdered egg, 3% Semen Maydis oil and 92.3% water.

Another example is KR101187936B1 (filed by Kyeong-bon, Ecowin Co., Ltd.) which teaches a medium composition for cultivation of nematodes, said composition comprising whey powder, dry yeast, sodium chloride, egg powder, potassium hydrogen phosphate, cholesterol, restin and canola.

Yet another example is AU620833B2 (assigned to Biosys AB) which teaches a medium composition for cultivation of nematodes, said composition comprising 1-2% egg yolk, 0.1-0.5% yeast cells, 2-5% corn oil, and 1-3% soy flour.

Yet another example is US5694883A (assigned to Kubota Corp), which teaches a medium composition for cultivation of nematodes, said composition comprising 3% corn oil, 0.1% cholesterol and SGPY medium which is prepared by adjusting an aqueous solution of 0.6% soluble starch, 1.0% D-glucose, 1.5% bactopeptone, and 1.5% yeast extract to a pH of about 7.0 with sodium hydroxide, and adding 0.3% agar.

The above solutions, besides being limited in application, leave a lot to be desired while addressing the shortfalls mentioned in the foregoing narration, thus keeping the need for an effective medium composition for in-vitro production of entomopathogenic nematodes along with their symbiotic bacteria.

As the reader shall appreciate, there are a few solutions available in the state of art however they are not without problems or challenges that users encounter, hence presenting a few technical issues while designing media for Steinernema sp., including:
(a) Isolation and maintenance of symbiotic bacteria from Steinernema sp. and their preservation in phase I state.
(b) Screening of different carbon sources and nitrogen sources required to grow both the Steinernema sp. and their symbiotic bacteria
(c) Optimization of physical parameters like agitation RPM, temperature etcetera

Prior art therefore, does not list a single effective solution embracing all considerations mentioned hereinabove, thus preserving an acute necessity-to-invent for the present inventor/s who, as result of focused research, has come up with novel solutions for resolving all needs once and for all. Work of the applicant/s hereof, specifically directed against the technical problems recited hereinabove and currently part of the public domain including earlier filed patent applications, is neither expressly nor impliedly admitted as prior art against the present disclosures.

A better understanding of the objects, advantages, features, properties and relationships of the present invention will be obtained from the following detailed description which sets forth an illustrative yet-preferred embodiment of the present invention.

Objectives of the present invention
The present invention is identified in addressing at least all major deficiencies of art discussed in the foregoing section by effectively addressing the objectives stated under, of which:

It is a primary objective to establish a medium composition for in-vitro production of entomopathogenic nematodes along with their symbiotic bacteria.

It is another objective further to the aforesaid objective(s) to establish a method for production of the medium composition useful for in-vitro production of entomopathogenic nematodes along with their symbiotic bacteria.

It is another objective further to the aforesaid objective(s) to establish a method based on said medium composition, for in-vitro production of entomopathogenic nematodes along with their symbiotic bacteria.

The manner in which the above objectives are achieved, together with other objects and advantages which will become subsequently apparent, reside in the detailed description set forth below in reference to the accompanying drawings and furthermore specifically outlined in the independent claims. Other advantageous embodiments of the invention are specified in the dependent claims.

Attention of the reader is now requested to the detailed description to follow which narrates a preferred embodiment of the present invention and such other ways in which principles of the invention may be employed without parting from the essence of the invention claimed herein.

Detailed description
The present invention is directed at absorbing all advantages of prior art while overcoming, and not imbibing, any of its shortfalls, to thereby establish a liquid medium composition, and a method based on said medium composition, for in-vitro production of Steinernema carpocapsae along with its symbiotic bacteria Xenorhabdus nematophila.

According to a yet-preferred embodiment, the medium composition of the present invention comprises the following ingredients-

1) Sugar (Sucrose / Trehalose)
2) Yeast extract
3) Peptone
4) Vegetable oil
5) Sodium Chloride
6) Calcium chloride dihydrate,
7) Magnesium sulfate heptahydrate
8) Potassium chloride
9) Ferrous sulfate heptahydrate,
10) Glycerol / Cholesterol

The aforementioned ingredients are admixed in an aqueous base and pH of the resulting admixture is adjusted to 7.2. According to the preferred embodiment hereof, the composition of the present invention is outlined in Table 1 below.
Ingredient Concentration (gL-1)
Sucrose 10.0
Yeast extract 10.0
Peptone 7.0
Vegetable oil 8.0
Sodium Chloride 4.0
Potassium Chloride 0.3
Calcium Chloride dihydrate 0.3
Magnesium Sulphate dihydrate 0.5
Ferrous Sulphate heptahydrate 0.02
Glycerol 0.2

Table 1

Approach / Role – based rationale for selection of ingredients: Among the aforesaid ingredients,
(a) Sucrose serves as a carbon source and energy source for Steinernema and their symbiotic bacteria. It helps to maintain the symbiotic bacteria of Steinernema in phase I (toxic phase) for the long term. Phase I bacteria is required to grow the Steinernema in in-vivo (insect body) and in-vitro (liquid or solid media) conditions. It also serves as an energy reserve for Steinernema as it metabolizes slowly. It protects the Steinernema from desiccation, cold and drought conditions.
(b) Yeast extract serves as a nitrogen source and feed and which supply as a source of amino acids and proteins.
(c) Peptone serves as a nitrogen source and feed. It provides a proteinaceous source for Steinernema sp. during growth in liquid media.
(d) Vegetable oil serves as a lipid source and energy source. Steinernema's body is made up of mostly lipids and vegetable oil helps as a lipid source.
(e) Sodium Chloride serves as a salt source required to grow and maintain isotonic conditions. And maintains the body electrolyte balance of nematodes and also servers as a macronutrient in maintaining Sodium potassium ratio.
(f) Potassium Chloride serves as a salt source required to grow and maintain isotonic conditions. And maintains the body electrolyte balance of nematodes and also serves as a macronutrient in maintaining Sodium potassium ratio.
(g) Calcium chloride serves as a calcium source. And maintains the body salt balance of nematodes and also serves as a macronutrient.
(h) Magnesium sulphate dihydrate serves as a magnesium source and Sulphur source. And maintains the body salt balance of nematodes and also serves as a macronutrient.
(i) Ferrous sulphate heptahydrate serves as an iron source required for hemoglobin synthesis.
(j) Glycerol serves as a humectant as Steinernema requires moisture to grow. Glycerol and vegetable oils also serve as an emulsifier for homogenized media.

Methodology of preparation of the medium: Yeast extract 10.0 g, peptone 7.0 g and then Sucrose 10.0 g were admixed initially. 400 ml of deionized water was poured into said admixture and mixed it till the medium got clear. 4.0 g sodium chloride, 0.3 g potassium chloride, 0.5 g magnesium sulphate dihydrate, 0.3 g calcium chloride dihydrate and then 0.02 g ferrous sulphate heptahydrate were added next. Thereafter, 200 ml of deionized water was added and mixed properly till the medium got clear. pH value was measured and adjusted to 7.2. Then, 8.0 g vegetable oil was added and volume was made up to 1 L. The medium so prepared was sterilized by autoclaving at 121°C for 25 min at 15 PSI. After sterilization the medium was allowed to cool at room temperature. Later on, in the laminar hood add, filter-sterilized 0.2 g Glycerol per 10 ml of sterile distilled water was added to the medium using a 0.2 micron filter and syringe to result in the medium of this invention.

Usage of the medium: Suitable quantity of the aforesaid medium is charged to a bioreactor, followed by inoculation with entomopathogenic nematodes along with their symbiotic bacteria. The resultant culture medium is incubated for a predefined period amidst optimized and controlled parameter setup to result in rapid proliferation of high-quality, viable, healthy and genetically-pure desired species of said entomopathogenic nematodes along with their symbiotic bacteria. It shall be appreciated that this method can be practiced around the year, as there is no environmental interaction or dependency whatsoever.

The aforementioned medium composition has been validated in independent scientific trials undertaken by the applicant named herein, for the mass cultivation of Steinernema carpocapsae carrying its symbiotic bacteria Xenorhabdus nematophila, particularly by following the underlying sequence of steps-
a) X. nematophila is cultivated in the aforesaid liquid medium for 48 hours;
b) Infective juveniles of S. carpocapsae are introduced to the broth of step a) and incubated for sufficient period to allow development of the infective juveniles of S. carpocapsae into amphimictic males and females and later on different stages of infective juveniles from parental nematodes

After the incubation, millions of infective juveniles of S. carpocapsae were recovered from the liquid medium, which could be then used for the formulation and application of pest control management.

Pilot / bench scale studies:
Optimization of liquid culture medium for the mass production of Steinernema sp. along with its symbiotic bacteria was conducted in shake flask levels having total volumes of 100 ml to 2000 ml flasks.

The life cycle of S. carpocapsae involves the infection of infective juveniles (IJs) to the host, which then secretes Xenorhabdus nematophila bacterium to the haemocoel of the host. This bacterium releases the toxic compounds which kill the host. The IJs then develop to the fourth stage and then to adult males and females. Steinernema undergoes amphimictic reproduction. The female lays eggs after mating that hatch as first-stage juveniles and they moult to second, third and fourth-stage juveniles and males and females of second generation. Up to two to three generations can develop depending upon the food availability. After Nutrient Media depletion, the third stage juvenile arrests their growth and becomes non-feeding, free-living state infective (dauer) juveniles. This IJ then emerges from the host body and searches for a new host for the infection. In-vitro production of Steinernema involves the initial growth of their symbiotic bacteria i.e. Xenorhabdus sp. into the liquid culture medium. It is observed that only primary phase I Xenorhabdus bacteria is needed to create an essential environment required to grow Steinernema sp. Steinernema sp. feeds on the primary phase I cells of Xenorhabdus sp. bacteria. When this bacteria switches from phase I to phase II form due to unknown reasons Steinernema is unable to grow. Phase I form of Xenorhabdus is also essential to kill the host insect pest. For in-vitro production, initially, the phase I form of Xenorhabdus grows into a liquid culture medium and forms a suitable environment. After 48 hrs of growth of Xenorhabdus, approximately 400±50 IJs/ml IJs of Steinernema were inoculated into the pre-grown liquid medium. The production medium was kept at 26°C to 30°C preferably at 28±2 °C at 80 to 140 RPM preferably at 110±30 rpm. It was observed that Steinernema sp. IJs started to grow into adult males and females for 2-4 days. Then females produces stage one IJs, from stage one IJs matures to stage two IJs and then stage two to stage three IJs and finally to stage four which matures into adult male and females till 6-8 days. After copulation, females started to produce again stage one IJs which feeds on phase I Xenorhabdus cells and matured into stage two IJs, stage three IJs, stage four IJs and then to male and female. This cycle happens till the nutrient deficiency happens. Later on, after nutrient deficiency and low numbers of Xenorhabdus cells, Steinernema sp. IJs arrested their growth at the third stage IJs. The third stage is the only stage which is a non-feeding and free-living stage and infects the insect pests. It was observed that after the inoculation of approximately 400±50 IJs per ml into the liquid culture medium, the number of IJs was increased to 14±4 days and the final concentration of IJs reached about approximately 500000±50000 IJs per ml.

Scale up studies in fermenter:
Scale-up experimental studies were performed on a fermenter (Make: Scigenics India Pvt ltd., Chennai) having a total volume of 7 L and a working volume of 5.6 L. Medium used for fermentation was kept the same during the shake flask study elaborated above.

At a Higher scale propagation in fermenter the following parameters and conditions were maintained during the batch cycle of fermentation:
(a) Compressed air after being filtered through a series of PTFE filters of 5 microns, 10 micron and 0.2 microns were passed into the fermenter with a Airflow of 0.5-1.0±0.2 VVM with the help of a sparger placed inside the fermenter.
(b) Temperature was kept the same as in the shake flask level i.e. 26°C to 30°C preferably at 28±2 °C
(c) Shaking was done by the baffle and Rushton turbine impeller with RPM of 80 to 140 RPM preferably at 110±30 rpm range.
(d) In situ sterilization of media was performed in the fermenter at 121°C for 15 minutes.
(e) After sterilization media was cooled to the desired temperature.

Inoculation was done after sterilization, the symbiotic bacterial inoculation having 1-3% v/v of inoculum was inoculated into the fermenter by transferring aseptically. After 8-10 hours of incubation IJ’s were transferred aseptically at the rate of 400±50 IJ’s/ml. IJ’s were inoculated and incubated at 26°C to 30°C preferably at 28±2 °C at 80 to 140 RPM preferably at 110±30 rpm and the batch was sampled periodically to check for the appearance, size, growth stage, count, cross-contamination and finally harvested after the lag phase of fermentation is achieved usually at 14±4 days.

Steinernema sp. required their symbiotic bacteria in the phase I state to grow into a liquid culture medium. After performing various trials it can be concluded that the symbiotic relation between bacteria and nematode relation is only possible with phase-I bacteria. Various media combinations were formulated but success was obtained only in the above stated media only with maximum yield of final nematode. Using this optimized nutrient media it was possible to successfully achieve 500000±50000 IJs per ml.

Sourcing of chemical and biological ingredients
All chemical ingredients included in the media composition hereof have been sourced from standard sources according to their respective CAS numbers as listed in Table 2 below.
Ingredient Source CAS No.
Sucrose Loba Chemie, India 57-50-1
Yeast extract HiMedia, India 8013-01-2
Peptone HiMedia, India 68990-09-0
Sodium Chloride Loba Chemie, India 7647-14-5
Potassium Chloride Loba Chemie, India 7447-40-7
Calcium Chloride dihydrate Loba Chemie, India 10043-52-4
Magnesium Sulphate dihydrate Loba Chemie, India 22189-08-8
Ferrous Sulphate heptahydrate Loba Chemie, India 7782-63-0
Glycerol Sisco Research Laboratories, India 56-81-5

Table 2

All biological ingredients included in this invention have been sourced from India using sustainable harvesting practices while keeping a core outlook for their ethical sourcing, cultivation, and conservation of said resources which preserves biodiversity, protects endangered species and supports the livelihoods of local communities. Lone ingredient in this line, Vegetable oil (brand: Fortune) Refined Soyabean Oil was sourced from the local market in Pune, Maharashtra, India.

Industrial Applicability
Steinernema carpocapsae is a species of entomopathogenic nematode, meaning it's a type of roundworm that parasitizes insects and it is often sold as a biological control agent in the form of IJs. It is widely used in biological pest control to manage a variety of insect pests in agriculture, horticulture, and forestry. Hence industrial production thereof is of immense global commercial importance. Applicability of the method and media composition as provided herein above for other entomopathogenic nematodes along with their symbiotic bacteria is being studied and it is added intention of the present invention to cover such embodiments and their localizations for such cogent applications.

In both the shake flask level as well as in the fermenter level we have scaled up from 100 ml to 5000 ml using fermenter by using this optimized nutrient media it is thus possible to produce Steinernema sp. IJs in mass quantities for the end use as a biological insecticide in agriculture.

Reduction to practice: The inventive product of this invention has been reduced to practice by the Applicant hereof using materials and processes which are subject to stringent quality assurance and control standards, and thus typified by the following advantageous features scoring above prior art-

As may become abundantly clear to the reader, traditional microbiological and media formulation practices are generic and unable to spell out the exact and precise selection, potentization, formulation and usage of the composition of the present invention as proposed herein. To best extent surveyed, no document exists which foretells the specific composition conveyed herein, thus proving distinctness and original contribution of the inventor over the hitherto available traditional knowledge.

As will be realized further, the present invention is capable of various other embodiments and that its several components and related details are capable of various alterations, all without departing from the basic concept of the present invention. Accordingly, the foregoing description will be regarded as illustrative in nature and not as restrictive in any form whatsoever. Modifications and variations of the system and apparatus described herein will be obvious to those skilled in the art. Such modifications and variations are intended to come within ambit of the present invention, which is limited only by the appended claims. ,CLAIMS:1] A process for preparing a medium for the in-vitro production of entomopathogenic nematodes and their symbiotic bacteria, comprising:
(a) Admixing Yeast extract 10.0 g, peptone 7.0 g and then Sucrose 10.0 g to form an initial admixture;
(b) Pouring 400 ml of deionized water into the initial admixture and mixing the same until the admixture turns clear;
(c) Adding 4.0g sodium chloride, 0.3g potassium chloride, 0.5g magnesium sulphate dihydrate, 0.3g calcium chloride dihydrate and 0.02g ferrous sulphate heptahydrate and 200 ml of deionized water to the solution of step (b) and mixing it till the medium turns clear.
(d) Adjusting the pH of the solution of step (c) to 7.2;
(e) Adding 8.0 g vegetable oil and making up volume up to 1 liter using deionized water;
(f) Sterilizing the solution at step (e) by autoclaving at 121°C for 25 min at 15 PSI;
(g) Allowing the sterilized solution of step (f) to cool at room temperature; and
(h) Under laminar flow, adding filter-sterilized 0.2 g Glycerol per 10 ml of sterile distilled water to the medium of step (g) using a 0.2 micron filter and syringe to result in the medium for the in-vitro production of entomopathogenic nematodes and their symbiotic bacteria.

2] The process for preparing a medium for the in-vitro production of entomopathogenic nematodes and their symbiotic bacteria as claimed in claim 1, wherein the entomopathogenic nematode is Steinernema carpocapsae.

3] The process for preparing a medium for the in-vitro production of entomopathogenic nematodes and their symbiotic bacteria as claimed in claim 1, wherein the symbiotic bacteria is Xenorhabdus nematophila.

4] A medium for in-vitro production of entomopathogenic nematodes and their symbiotic bacteria prepared by the process of claim 1, consisting of-
(a) 10.0 gL-1 Sucrose;
(b) 10.0 gL-1 Yeast extract;
(c) 7.0 gL-1 Peptone;
(d) 8.0 gL-1 Vegetable oil;
(e) 4.0 gL-1 Sodium Chloride;
(f) 0.3 gL-1 Potassium Chloride;
(g) 0.3 gL-1 Calcium Chloride dihydrate;
(h) 0.5 gL-1 Magnesium Sulphate dihydrate;
(i) 0.02 gL-1 Ferrous Sulphate heptahydrate; and
(j) 0.2 gL-1 Glycerol.

5] A medium for in-vitro production of entomopathogenic nematodes and their symbiotic bacteria prepared by the process of claim 1, consisting of-
(a) 10.0 gL-1 Trehalose;
(b) 10.0 gL-1 Yeast extract;
(c) 7.0 gL-1 Peptone;
(d) 8.0 gL-1 Vegetable oil;
(e) 4.0 gL-1 Sodium Chloride;
(f) 0.3 gL-1 Potassium Chloride;
(g) 0.3 gL-1 Calcium Chloride dihydrate;
(h) 0.5 gL-1 Magnesium Sulphate dihydrate;
(i) 0.02 gL-1 Ferrous Sulphate heptahydrate; and
(j) 0.2 gL-1 Cholesterol.

6] A process for in-vitro production of entomopathogenic nematodes along with their symbiotic bacteria using the medium composition of either claim 4 or claim 5, the process comprising the steps of-
(a) Charging a suitable quantity of said medium to a bioreactor;
(b) Inoculating the medium charged into the bioreactor with entomopathogenic nematodes along with their symbiotic bacteria;
(c) Incubating the inoculated medium for a predefined period amidst optimized and controlled parameter setup to result in rapid proliferation of high-quality, viable, healthy and genetically-pure desired species of said entomopathogenic nematodes along with their symbiotic bacteria.

7] The process for in-vitro production of entomopathogenic nematodes along with their symbiotic bacteria as claimed in claim 6, wherein the optimized and controlled parameter setup consists of-
(a) Airflow of 0.5-1.0 ± 0.2 VVM using Compressed air filtered through a series of PTFE filters of 5 microns, 10 micron and 0.2 microns;
(b) Temperature ranging between 26°C to 30°C, 28±2 °C in particular; and
(c) Achieving mass homogenization using baffle and Rushton turbine impeller with RPM of 80 to 140 RPM preferably at 110±30 rpm range.