Description:FIELD OF THE INVENTION

[0001] The present invention relates to a biodigestate liquid-based fermentative media composition and method for preparation thereof to develop an economical, eurytopic fermentative media having biodigestate liquid and carbon source that are macro and micro nutrient rich along with the scientific enrichment of microorganisms.

BACKGROUND OF THE INVENTION

[0002] Biodigestate, the byproduct of anaerobic digestion, contains essential nutrients like nitrogen, phosphorus, and organic carbon, making it a potential source for developing low-cost, nutrient-rich culture media. However, the process presents several challenges, including variability in the composition of biodigestate, which can affect the consistency and quality of the media. Additionally, the presence of inhibitory compounds, high moisture content, and the need for sterilization can complicate production. Producing fermentative, nutritive-rich media from residual biodigestate offers a sustainable approach to waste valorization, turning organic byproducts into valuable resources for microbial growth and fermentation processes.

[0003] Traditionally, there are commercially available synthetic or plant-based media, such as Nutrient Agar, Sabouraud Dextrose Agar, and Luria-Bertani (LB) medium. These media are widely used in microbiological research and industrial fermentation. However, these traditional media have several drawbacks. First, they are costly, as they often rely on expensive raw materials like peptones, agar, and specific carbon sources. Second, they are less environmentally sustainable, as they may require the use of non-renewable resources or generate significant waste. Furthermore, they also lack the nutrient diversity found in complex, waste-derived media, which could affect microbial growth and the production of desired metabolites in some fermentation processes.

[0004] US20210403859A1 discloses a growth media composition. The disclosure further relates to a method of producing the biomass at higher concentration by employing a growth media composition. The disclosure further relates to a method of producing value added product employing growth media composition. The growth media composition of the present disclosure is homogenous in nature and is self-sterilized. The present disclosure provides for enhanced productivity of the biomass and the value added products, respectively employing gaseous substrate. Although US’869 discloses a growth media composition. However, the cited art has certain limitation pertaining to utilization of biodigestates rich in macro- & micro- nutrient to form a fermentative nutritive rich media.

[0005] CN101238220A discloses a method for recycling of biomasses in fermentation processes, whereby the biomass obtained on production of fermentation products is treated by a particular method and recycled into the system. The prepared biomass can be used as a medium component on fermentation for the fermentative production of vitamins, in particular, vitamin B2. Although CN’220 discloses a method for recycling of biomasses in fermentation processes. However, the cited art has certain limitation pertaining to a fermentative nutritive rich media from biodigestates that are rich in macro- & micro- nutrient for proliferation of microorganisms.

[0006] Conventionally, many commercial medium exists in the market. However, there are certain limitations to the available medium such as utilization of biodigestates that is rich in macro- & micro- nutrient to be used as cultivation medium for solid and liquid biofertilizer production as a safe, economical fermentative nutritive rich media that encompasses the proliferation of microorganisms.

[0007] In order to overcome the aforementioned drawbacks, there exists a need in the art to develop a biodigestate liquid-based fermentative media composition by utilizing leftovers crop residues rich in macro- & micro- nutrient to be used as a primary enrichment media as well as metabolic inducer to increase the fermentation yield.

OBJECTS OF THE INVENTION

[0008] The principal object of the present invention is to overcome the disadvantages of the prior art.

[0009] An object of the present invention is to utilize macro & micro nutrient rich biodigestates to be used as a cultivation medium.

[0010] Another object of the present invention is to develop a safe, economical fermentative nutritive rich media from leftovers crop residues to support the proliferation of microorganisms.

[0011] Yet another object of the present invention is to develop a media having a stable pH and less impurities to be used as a primary enrichment media as well as metabolic inducer to increase the fermentation yield.

[0012] The foregoing and other objects, features, and advantages of the present invention will become readily apparent upon further review of the following detailed description of the preferred embodiment as illustrated in the accompanying drawings.

SUMMARY OF THE INVENTION

[0013] The present invention relates to a biodigestate liquid-based fermentative media composition and method of preparation thereof a media composition from biodigestate of leftovers crop residues that is rich in macro- & micro- nutrient to be used as a primary enrichment media to increase the fermentation yield.

[0014] According to an embodiment of the present invention, a biodigestate liquid-based fermentative media composition comprises of i) biodigestate liquid in the range of 15-25% v/v, ii) jaggery in the range of 2-6% w/v, iii) gram flour in the range of 0.05-0.15% w/v, iv) sodium chloride (NaCl) in the range of 0.4% w/v, v) buffering agent in the range of 0.1% w/v, and vi) distilled water in the range of 72-80% w/v.

[0015] According to another embodiment of the present invention, a method for preparation of the fermentative media includes following steps: a) collection of the biodigestate liquid from compressed bio gas plant, b) taking the distilled water in a flask and adding the jaggery, gram flour, sodium chloride, and buffering agent to obtain a media solution, c) mixing the collected biodigestate liquid with the media solution to obtain the biodigestate liquid-based fermentative media.

[0016] While the invention has been described and shown with particular reference to the preferred embodiment, it will be apparent that variations might be possible that would fall within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:
Figure 1 illustrates a graphical representation of the optimization of carbon source (jaggery) as medium constituent;
Figure 2 illustrates a graphical representation of optimization of other medium constituents for w.r.t. HiMedia’s Nutrient Medium;
Figure 3 illustrates a pictorial representation of physical appearance of a) HiMedia’s Rose Bengal Agar (commercial medium) and b) biodigestate liquid-based fermentative media;
Figure 4 illustrates a pictorial representation of physical appearance of a) Control b) HiMedia’s Rose Bengal Agar (commercial medium) and c) biodigestate liquid-based fermentative media;
Figure 5 illustrates a pictorial representation of culture of bacteria (Paracoccus sp.s) on a) Nutrient Agar and b) biodigestate liquid-based fermentative media;
Figure 6 illustrates a pictorial representation of culture of Actinomycetes (Actinomyces sp.s) on a) Nutrient Agar and b) biodigestate liquid-based fermentative media;
Figure 7 illustrates a pictorial representation of culture of fungi (Aspergillus sp.) on a) Nutrient Agar and b) biodigestate liquid-based fermentative media;
Figure 8 illustrates a graphical representation of growth rate of Streptomyces badius in Nutrient medium and biodigestate liquid-based fermentative media;
Figure 9 illustrates a graphical representation of growth rate of Strenotrophomonas sp.s in Nutrient medium and biodigestate liquid-based fermentative media;
Figure 10 illustrates a graphical representation of growth rate of Azotobacter sp.s in Nutrient medium and biodigestate liquid-based fermentative media;
Figure 11 illustrates a a graphical representation of growth rate of Pseudomonas sp.s in Nutrient medium and biodigestate liquid-based fermentative media;
Figure 12 illustrates a graphical representation of growth rate of Bradyrhizobium sp.s in Nutrient medium and biodigestate liquid-based fermentative media;
Figure 13 illustrates a graphical representation of microbial count using plastic bottles as packaging material; and
Figure 14 illustrates a graphical representation of microbial count using glass bottles as packaging material.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The following description includes the preferred best mode of one embodiment of the present invention. It will be clear from this description of the invention that the invention is not limited to these illustrated embodiments but that the invention also includes a variety of modifications and embodiments thereto. Therefore, the present description should be seen as illustrative and not limiting. While the invention is susceptible to various modifications and alternative constructions, it should be understood, that there is no intention to limit the invention to the specific form disclosed, but, on the contrary, the invention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention as defined in the claims.

[0019] In any embodiment described herein, the open-ended terms "comprising," "comprises,” and the like (which are synonymous with "including," "having” and "characterized by") may be replaced by the respective partially closed phrases "consisting essentially of," consists essentially of," and the like or the respective closed phrases "consisting of," "consists of, the like.

[0020] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0021] The present invention relates to a biodigestate liquid-based fermentative media composition and method of preparation thereof for utilizing macro- and micro-nutrient-rich biodigestates to develop a nutritive-rich medium with minimal impurities for supporting the proliferation of microorganisms and enhancing fermentation yield.

[0022] According to an embodiment of the present invention, a biodigestate liquid-based fermentative media composition comprises of: i) biodigestate liquid in the range of 15-25% v/v, ii) jaggery in the range of 2-6% w/v, iii) gram flour in the range of 0.05-0.15% w/v, iv) sodium chloride (NaCl) in the range of 0.4% w/v, v) buffering agent in the range of 0.1% w/v, and vi) distilled water in the range of 72-80% w/v.

[0023] The preferable value of: i) biodigestate liquid is 20% v/v, ii) jaggery is 4% w/v, iii) gram flour is 0.1% w/v, iv) sodium chloride (NaCl) is 0.4% w/v, v) buffering agent is 0.1% w/v, and vi) distilled water is 76% w/v.

[0024] The jaggery used herein is a source of carbon and micronutrients such as Iron, Magnesium, Manganese, Phosphorus, and vitamins.

[0025] The sodium chloride used herein maintains the optimum osmolarity.

[0026] According to another embodiment of the present invention, a method for preparation of the fermentative media includes following steps: a) collection of the biodigestate liquid from compressed bio gas plant, b) taking the distilled water in a flask and adding the jaggery, gram flour, sodium chloride, and buffering agent to obtain a media solution, c) mixing the collected biodigestate liquid with the media solution to obtain the biodigestate liquid-based fermentative media.

[0027] The biodigestate liquid-based fermentative media used herein is subjected to sterilization in an autoclave at 121oC for 20 minutes.

[0028] The buffering agents used herein are K2HPO4 and KH2PO4.

EXAMPLE

[0029] The method for preparation of the fermentative media includes following steps: a) collection of the biodigestate liquid from compressed bio gas plant, b) taking the distilled water in a flask and adding the 40g of jaggery, 1g of gram flour, 4g of sodium chloride, and 1g of buffering agent to obtain a media solution, c) mixing the collected biodigestate liquid with the media solution to obtain the biodigestate liquid-based fermentative media and sterilized in an autoclave at 121oC for 20 minutes.

CHARACTERIZATION

Optimization of Carbon source (jaggery)

[0030] The optimization of the carbon source that is jiggery here is done to improve the yield and productivity of processes, and reduce costs. Optimization of jaggery in the media was done by varying its concentration from 2-10 %. Result: Referring to Figure 1, a graphical representation of the optimization of carbon source (jaggery) as medium constituent is illustrated. The best yield was obtained at 4% of the carbon source (jaggery).

Optimization of other medium constituents for w.r.t. HiMedia’s Nutrient Medium

[0031] The medium constituent’s optimization is a process that involves systematically changing the concentration of individual components in a medium and observing the effect on cell growth and productivity to maximize metabolite yield. Result: Referring to Figure 2, a graphical representation of optimization of other medium constituents for w.r.t. HiMedia’s Nutrient Medium is illustrated. Quantity of each component of the medium was optimized to get the best yield.

Physical Appearance

[0032] Physical appearance was checked on solid media by performing culture on two petri plates, one using the HiMedia’s Rose Bengal Agar (commercial medium) and the other with media prepared herein to check the physical appearance. The appearance was also checked in the liquid media in conical flasks. Three flasks were prepared, one control, one with HiMedia’s Glucose Yeast Extract (GYE) broth and one with biodigestate liquid-based fermentative media.

[0033] Result: Referring to Figure 3, a pictorial representation of physical appearance of a) HiMedia’s Rose Bengal Agar (commercial medium) and b) biodigestate liquid-based fermentative media on solid media is illustrated. The physical appearance was observed through the prepared plates. Referring to Figure 4, a pictorial representation of physical appearance of a) Control b) HiMedia’s Rose Bengal Agar (commercial medium) and c) biodigestate liquid-based fermentative media on liquid media is illustrated. The physical appearance was observed through the conical flasks.

pH Test

[0034] pH is measured of HiMedia’s Rose Bengal Agar (commercial medium) and biodigestate liquid-based fermentative media. The electrode is rinsed with distilled water and blot with a Kim wipe. Submerge the electrode in a pH 7 buffer. Press the calibrate button on the pH meter and wait for the readout to stabilize. Place an empty beaker under the electrode. Take a reading one by one with each media. Result: pH of HiMedia’s Nutrient Medium was 6.8 and the pH of biodigestate liquid-based fermentative media was 6.5.

Impurity test

[0035] The impurity of HiMedia’s Nutrient Medium and biodigestate liquid-based fermentative media is accessed through an analytical method. Result: The impurity of HiMedia’s Nutrient Medium was observed to be 3.63 and biodigestate liquid-based fermentative media was 3.12.

Growth promotion test

[0036] Growth promotion test is performed by culturing bacteria, actinomycetes and fungi on Nutrient Agar and biodigestate liquid-based fermentative media separately. Result: Referring to Figure 5, a pictorial representation of culture of bacteria (Paracoccus sp.s) on a) Nutrient Agar and b) biodigestate liquid-based fermentative media is illustrated. Referring to Figure 6, a pictorial representation of culture of Actinomycetes (Actinomyces sp.s) on a) Nutrient Agar and b) biodigestate liquid-based fermentative media is illustrated. Referring to Figure 7, a pictorial representation of culture of fungi (Aspergillus sp.) on N a) Nutrient Agar and b) biodigestate liquid-based fermentative media is illustrated. The medium tested for growth proved to be effective for providing enhanced growth of different microorganism as compared to the commercial medium.

Comparative microbial growth of biofertilizer cultures

[0037] Validation of the medium was done by the comparative microbial growth assessment of formulated biodigestate liquid-based fermentative media with respect to the established HiMedia’s Medium. Result: Referring to Figure 8, a graphical representation of growth rate of Streptomyces badius in Nutrient medium and biodigestate liquid-based fermentative media is illustrated. Referring to Figure 9, a graphical representation of growth rate of Strenotrophomonas sp.s in Nutrient medium and biodigestate liquid-based fermentative media is illustrated. Referring to Figure 10, a graphical representation of growth rate of Azotobacter sp.s in Nutrient medium and biodigestate liquid-based fermentative media is illustrated. Referring to Figure 11, a graphical representation of growth rate of Pseudomonas sp.s in Nutrient medium and biodigestate liquid-based fermentative media is illustrated. Referring to Figure 12, a graphical representation of growth rate of Bradyrhizobium sp.s in Nutrient medium and biodigestate liquid-based fermentative media is illusatred. The growth rate of different populations was compared in Nutrient medium and biodigestate liquid-based fermentative media. The growth in the biodigestate liquid-based fermentative media was found be good in comparison with the commercially available Nutrient medium.

Shelf stability study

The shelf stability of the prepared media was checked in glass and plastic bottles individually for a certain time period. Effect of media type on the survival rate of microbial population compared in two types of packaging material used. Result: Referring to Figure 13, a graphical representation of microbial count using plastic bottles as packaging material is illustrated. Referring to Figure 14, a graphical representation of microbial count using glass bottles as packaging material is illustrated. The effect of media type on the survival rate of microbial population in two types of packaging materials was observed to be effective for promoting the growth of the microbial population as per the microbial count.

[0038] Although the field of the invention has been described herein with limited reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention, will become apparent to persons skilled in the art upon reference to the description of the invention. , Claims:1) A biodigestate liquid-based fermentative media composition, comprising:

i) biodigestate liquid in the range of 15-25% v/v;

ii) jaggery in the range of 2-6% w/v;

iii) gram flour in the range of 0.05-0.15% w/v;

iv) sodium chloride (NaCl) in the range of 0.4% w/v;

v) buffering agent in the range of 0.1% w/v; and

vi) distilled water in the range of 72-80% w/v.

2) The composition as claimed in claim 1, wherein said jaggery is used as a source of carbon and micronutrients such as Iron, Magnesium, Manganese, Phosphorus, and vitamins.

3) The composition as claimed in claim 1, wherein said sodium chloride is used to maintain optimum osmolarity.

4) A method for preparation of said fermentative media as claimed in claim 1, comprising the steps:
a) collection of said biodigestate liquid from compressed bio gas plant;
b) taking said distilled water in a flask and adding said jaggery, gram flour, sodium chloride, and buffering agent to obtain a media solution;
c) mixing said collected biodigestate liquid with said media solution to obtain said biodigestate liquid-based fermentative media.

5) The method as claimed in claim 4, wherein said biodigestate liquid-based fermentative media is subjected to sterilization in an autoclave at 121oC for 20 minutes.