AU2021103319A4 - A process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds - Google Patents

A process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds Download PDF

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AU2021103319A4
AU2021103319A4 AU2021103319A AU2021103319A AU2021103319A4 AU 2021103319 A4 AU2021103319 A4 AU 2021103319A4 AU 2021103319 A AU2021103319 A AU 2021103319A AU 2021103319 A AU2021103319 A AU 2021103319A AU 2021103319 A4 AU2021103319 A4 AU 2021103319A4
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soil
motor oil
used motor
biostimulants
oil
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Sanket Joshi
Hemen Sarma
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Sarma Hemen Dr
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    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22002—Papain (3.4.22.2)

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Abstract

The present invention generally relates to a process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds. The process comprises: collecting soil samples from a geographical location with used motor oil dissemination; screening and isolating number of heterotrophic bacteria using dilution plate technique; isolating single bacterial colonies in agar plates using the serial dilution technique with used motor oil as a carbon source; isolating morphologically different bacterial colonies and streaking on used motor oil (Carbon source) agar plate to obtain pure cultures of the isolates; and selecting single colonies with a clear zone indicating used motor oil catabolizing for preparation of pure culture for bioremediation of PAHs. 15 0 C0 0 M ddi do 10 Ia. -3 CL 5'1 fl .2 A c ;p 9 t

Description

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A PROCESS FOR FORMULATION OF BIOSTIMULANTS AND THEIR APPLICATION IN SOIL TO ALLEVIATE POLYAROMATIC COMPOUNDS FIELD OF THE INVENTION
The present invention relates to the use of mustard oil cake hydrolysis products as biostimulant in the bioremediation of various different polyaromatic compounds in soil polluted by used motor oil. More specifically, the present invention relates to the formulation of biostimulants and their application in the soil to alleviate polyaromatic compounds. BACKGROUND OF THE INVENTION
Lubricating oils are widely used today for various purposes as it is required for the proper operation of automobile engines and other machinery including hydraulic motors, pumps, compressors, and electrical transformers. Used motor oil is characterized in a high concentration of aliphatic and aromatic hydrocarbons with chain lengths ranging from C15 to C50, as well as heavy metals that may contribute to a risk of chronic diseases, together with carcinogenicity and mutagenicity. As a result of their widespread use, such lubricants are contaminating terrestrial ecosystems at an alarming rate.
Thus, bioremediation of soils contaminated with used motor oil is critical. Bioremediation is a low-cost and environmentally friendly method of accelerating the cleaning process. In current years, using organic biostimulants derived from enzymatic hydrolysis has become a popular environmental technique for accelerating the degradation of organic compounds via enzymatic hydrolysis processes. These biostimulants contain a high concentration of proteins and amino acids and are easily absorbed by hydrocarbon-tolerant bacteria. This increases their biochemical activity and proliferation of the soil, hastening the degradation of toxins in the soil. Plant-derived protein hydrolysates (PHs) have gained popularity as plant biostimulants due to their ability to increase germination, productivity, and the quality of a wide range of horticultural and agronomic crops. The use of PHs can also help to mitigate the negative effects of abiotic plant stress caused by salinity, drought, heavy metals, and organic contaminants. Oil Cake is a by product of the mustard oil manufacturing process. It contains a significant amount of fiber (12.17 %) and protein (38.17 %).
The effectiveness of this biostimulant attained through enzymatic hydrolysis processes has been verified after application in contaminated soil. The goal of this innovation is to confirm the bioremediation efficiency of a biostimulant derived from mustard oil cake via enzymatic hydrolysis processes in soil contaminated by used motor oil. Moreover, also determined soil dehydrogenase activity after applying the said biostimulant to contaminated soil, as well as its influence.
In the view of the forgoing discussion, it is clearly portrayed that there is a need to have a process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds.
SUMMARY OF THE INVENTION
The present disclosure seeks to provide a process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds.
In an embodiment, a process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds is disclosed. The process includes collecting soil samples from a geographical location with used motor oil dissemination. The process further includes screening and isolating a number of heterotrophic bacteria using the dilution plate technique. The process further includes isolating single bacterial colonies on agar plates using the serial dilution technique with used motor oil as a carbon source. The process further includes isolating morphologically different bacterial colonies and streaking on used motor oil (Carbon source) agar plate to obtain pure cultures of bacterial isolates. The process further includes selecting single colonies with a clear zone indicating used motor oil catabolizing for preparation of pure culture for bioremediation of PAHs.
In an embodiment, petri dishes are supplemented with biostimulants derived from oil cake and a microbial consortium is further added.
In an embodiment, the soil is spiked with used motor oil at a rate of 1 percent (w/w) in bioremediation experiments for 49 days.
In an embodiment, a formulation of bacterial consortia isolated from soil contaminated with used motor oil spill, with bacterial strains used in the formulation, including Acinetobacterjunii HS 29, Brevibacillus reuszeri HS37, and Enterobacter asburiae HS22.
In an embodiment, dehydrogenase activity (g INTF g-1 h- 1) in soils amended with biostimulants derived from black mustard oil cake measured every seven days.
In an embodiment, the soil plates are incubated at 37 degree Celsius for 49 days.
In an embodiment, PAHs degradation is evaluated on 7, 28, and 49 days of the experiment, wherein the biostimulant and microbial consortium significantly reduced the concentration of PAHs with 3, 4, 5, and 6 aromatic rings by the end.
In an embodiment, the biostimulant is applied to the soil three times at a rate of 2% during the incubation cycle.
In an embodiment, PAH-degrading bacteria is isolated from soil and a consortium is formed.
In an embodiment, steps of enzymatic hydrolysis used to obtain biostimulants comprises performing enzymatic process on oil cake by maintaining 8 pH, 50-degree Celsius temperature, using papain purified from papaya latex and 10mg soluble in 1mL of water; and centrifuging the processed oil cakes to separate and obtain the biostimulant from the paste. An object of the present disclosure is to facilitate biological remediation of soils contaminated with used motor oil (organic toxic compounds).
Another object of the present disclosure is to use mustard oil cake hydrolysis products as biostimulant in the bioremediation of various different polyaromatic compounds in soil polluted by used motor oil.
Another object of the present disclosure is to describe the methods of enzymatic hydrolysis of oil industry cake for the formulation of biostimulant.
Another object of the present disclosure is to develop methods for PAH degrading the potential of bio-stimulants and their strength in environmental cleanup.
Yet another object of the present invention is to deliver an expeditious and cost-effective process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds.
To further clarify advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings. BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Figure 1 illustrates a flow chart of a process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds in accordance with an embodiment of the present disclosure; Figure 2 illustrates a process flow of enzymatic hydrolysis used to obtain biostimulants in accordance with an embodiment of the present disclosure; Figure 3 illustrates Table 1 depicts polycyclic aromatic hydrocarbons (mg kg- 1) used motor oil in accordance with an embodiment of the present disclosure; Figure 4 illustrates Table 2 depicts composition of amino acids in the Papain treated biostimulant produced from black mustard oil cake in accordance with an embodiment of the present disclosure;
Figure 5 illustrates Table 3 depicts dehydrogenase activity (pg INTF g-' h-1 ) in soils amended with biostimulants derived from black mustered oil cake in accordance with an embodiment of the present disclosure; Figure 6 illustrates Table 4 depicts creating a microbial consortium for the experiments in accordance with an embodiment of the present disclosure; and Figure 7 illustrates Table 5 depicts PAHs concentrations in control soil contaminated with used motor oil, and control soil contaminated with used motor oil amended with the biostimulator+ Microbial Consortia (mean i standard error, n=3) during the experimental period in accordance with an embodiment of the present disclosure.
Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present disclosure. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to "an aspect", "another aspect" or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.
Referring to Figure 1, a flow chart of a process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds is illustrated in accordance with an embodiment of the present disclosure. At step 102, the process 100 includes collecting soil samples from a geographical location with used motor oil dissemination.
At step 104, the process 100 includes screening and isolating number of heterotrophic bacteria using dilution plate technique. At step 106, the process 100 includes isolating single bacterial colonies in agar plates using the serial dilution technique with used motor oil as a carbon source.
At step 108, the process 100 includes isolating morphologically different bacterial colonies and streaking on used motor oil (Carbon source) agar plate to obtain pure cultures of the isolates. At step 110, the process 100 includes selecting single colonies with a clear zone indicating used motor oil catabolizing for preparation of pure culture for bioremediation of PAHs.
In an embodiment, petri dishes are supplemented with biostimulants derived from oil cake and a microbial consortium is further added.
In an embodiment, the soil is spiked with used motor oil at a rate of 1 percent (w/w) in bioremediation experiments for 49 days.
In an embodiment, formulation of bacterial consortia isolated from used motor oil spill soil, with bacterial strains used in the formulation including Acinetobacter junii HS 29, Brevibacillus reuszeri HS37, and Enterobacter asburiae HS22.
In an embodiment, dehydrogenase activity (g INTF g-1 h- 1) in soils amended with biostimulants derived from black mustered oil cake is measured every seven days.
In an embodiment, the soil plates are incubated at 37 degree Celsius for 49 days.
In an embodiment, PAHs degradation is evaluated on 7, 28, and 49 days of the experiment, wherein the biostimulant and microbial consortium significantly reduced the concentration of PAHs with 3, 4, 5, and 6 aromatic rings by the end.
In an embodiment, the biostimulant is applied to the soil three times at a rate of 2% during the incubation cycle.
In an embodiment, PAH-degrading bacteria is isolated from soil and a consortium is formed.
In an embodiment, the steps of enzymatic hydrolysis used to obtain biostimulants comprise performing an enzymatic process on oil cakes using papain purified from papaya latex and 10mg soluble in 1mL of water. This process is maintained in a pH of 8 at a temperature of 50 degrees Celsius and culminates in centrifuging the processed oil cakes to separate and obtain the biostimulant from the paste.
Figure 2 illustrates a process flow of enzymatic hydrolysis used to obtain biostimulants in accordance with an embodiment of the present disclosure. Biostimulant derived from oil cake through enzymatic hydrolysis processes with Papain (papaya proteinase I, is a cysteine protease). PAH-degrading bacteria are isolated from soil and a consortium is formed. The soil is contaminated with used motor oil at a rate of 1% (w/w) in the bioremediation experiments for a duration of 49 days. During the incubation cycle, the biostimulant is applied to the soil three times at a rate of 2 %. During the experiments, microbial consortia are used three times. The function of dehydrogenase and the concentration of polycyclic aromatic hydrocarbons (PAHs) in soil are investigated. The biostimulant's application to the polluted soil is steadily increased. PAHs degradation is assessed on 7, 28, and 49 days during the experiment. As a result, by the end of the experiment, the biostimulant had greatly reduced the concentration of PAHs with 3, 4, 5, and 6 aromatic rings. This degradation may be accelerated by the existence of various amino acids in the biostimulant, which are readily accessible to PAHs-tolerant soil microorganisms.
The current invention is useful in the workshop and the petrochemical industry (remediation of multiple polyaromatic hydrocarbons that disseminates in the soil environments). Bioremediation is emerging as a viable alternative in the fight against environmental pollution, and the current invention contributes to that goal. The present invention relates to the biological remediation of soils contaminated with used motor oil (organic toxic compounds). It specifically refers to a microbiological process based on the use of novel, non-genetically engineered, naturally occurring pollutant-degrading bacterial strains in conjunction with bio-stimulants for soil bioremediation or bio-restoration. Microbial consortia and oil cake hydrolysis products were developed in this method, in which, bacterial consortia metabolized aromatic hydrocarbons in the soil. The products (bacterial strains and bio-stimulants) obtained from the described process can also be used as a tool for the management of multiple PAH-polluted sites.
In one embodiment, a method of developing bio-stimulants has been described in which novel, non-genetically engineered, naturally occurring PAHs degrading bacterial strains are combined with biostimulants for the bioremediation or bio-restoration of soils in a laboratory experiment.
Table 1 shows polycyclic aromatic hydrocarbons (mg kg-1 ) used motor oil. Table 2 shows composition of amino acids in the Papain treated biostimulant produced from black mustard oil cake. Table 3 shows dehydrogenase activity (pg INTF g- 1 h- 1) in soils amended with biostimulants derived from black mustard oil cake. Table 4 shows creating a microbial consortium for the experiments. Table 5 shows PAHs concentrations in control soil contaminated with engine oil and control soil contaminated with used motor oil amended with the biostimulator
+ Microbial Consortia (mean i standard error, n=3) during the experimental period.
The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.
Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.

Claims (10)

WE CLAIM
1. A process for formulation of biostimulants and their application in soil to alleviate polyaromatic compounds, the process comprises:
Collecting soil samples from a geographical location with used motor oil dissemination; Screening and isolating number of heterotrophic bacteria using dilution plate technique; Isolating single bacterial colonies in agar plates using the serial dilution technique with used motor oil as a carbon source; Isolating morphologically different bacterial colonies and streaking on used motor oil (Carbon source) agar plate to obtain pure cultures of the isolates; and Selecting single colonies with a clear zone indicating used motor oil catabolizing for preparation of pure culture for bioremediation of PAHs.
2. The process as claimed in claim 1, wherein petri dishes are supplemented with biostimulants derived from oil cake and a microbial consortium is further added.
3. The process as claimed in claim 2, wherein the soil is spiked with used motor oil at a rate of 1 percent (w/w) in bioremediation experiments for 49 days.
4. The process as claimed in claim 1, wherein formulation of bacterial consortia isolated from used motor oil spill soil, with bacterial strains used in the formulation including Acinetobacter junii HS 29, Brevibacillus reuszeri HS37, and Enterobacter asburiae HS22.
5. The process as claimed in claim 1, wherein dehydrogenase activity (g INTF g-1 h- 1) in soils amended with biostimulants derived from black mustered oil cake is measured every seven days.
6. The process as claimed in claim 1, wherein the soil plates are incubated at 37 degree Celsius for 49 days.
7. The process as claimed in claim 1, wherein PAHs degradation is evaluated on 7, 28, and 49 days of the experiment, wherein the biostimulant and microbial consortium significantly reduced the concentration of PAHs with 3, 4, 5, and 6 aromatic rings by the end.
8. The process as claimed in claim 6, wherein the biostimulant is applied to the soil three times at a rate of 2% during the incubation cycle.
9. The process as claimed in claim 1, wherein PAH-degrading bacteria is isolated from soil and a consortium is formed.
10. The process as claimed in claim 1, wherein steps of enzymatic hydrolysis used to obtain biostimulants comprises:
Performing an enzymatic process on oil cakes using papain (10mg soluble in 1mL of water). This process is maintained in a pH of 8 at a temperature of 50 degrees Celsius and culminates in centrifuging the processed oil cakes to separate and obtain the biostimulant from the paste.
Figure 2
Figure 3
Figure 6
Figure 7
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