EP4720256A1

EP4720256A1 - Microbial composition for the bioremediation of crude oil contamination, and a process for preparation thereof

Info

Publication number: EP4720256A1
Application number: EP23940544.2A
Authority: EP
Inventors: Ganesh Kumar A; Sujitha K; Sudhakar Tata; Kirubagaran R; Dharani G
Original assignee: National Institute Of Ocean Technology
Current assignee: National Institute Of Ocean Technology
Priority date: 2023-06-03
Filing date: 2023-09-11
Publication date: 2026-04-08
Also published as: WO2024252407A1

Abstract

The present disclosure proposes a microbial based composition for the bioremediation of polycyclic aromatic hydrocarbons. The microbial based composition is utilised for bioremediation of polluted/contaminated marine sites. The microbial based composition can be effectively utilised on sea surfaces at low pressure and deep-sea high pressure conditions for remediation of oil spills.

Description

MICROBIAL COMPOSITION FOR THE BIOREMEDIATION OF CRUDE OIL CONTAMINATION, AND A PROCESS FOR PREPARATION THEREOF

DESCRIPTION:

Field of the invention:

[0001] The present disclosure generally relates to the technical field of compositions for degrading polycyclic aromatic hydrocarbons (PAHs), and in specific relates a microbial based composition for the bioremediation of polycyclic aromatic hydrocarbon and a process for preparation thereof.

Background of the invention:

[0002] Oil is the most popular source of energy in all over the world. It is a mixture of various petroleum hydrocarbon and related compounds. PAHs and its byproducts containing aromatic hydrocarbon have contributed greatly to the industrial development of mankind, but on the other hand, they are the major cause of environmental pollution. Petroleum hydrocarbons cause serious environmental problems due to their resistance in degradation, bioaccumulation and toxicity to various biological systems.

[0003] Oil pollution in marine environment is mainly caused by leaks, breakage of storage tanks and transport accidents. As a consequence, high mortality rate will be observed and thus results in the destruction of marine ecosystem. Further it leads to emigration and extinction of some species that are low in marine population. In case of open sea, shoreline structures and coastal environments due to weathering, the viscosity of oil increases. During this period, methods like in situ burning, mechanical skimmers and chemical dispersants are used for cleaning up.

[0004] Typically, these mechanical methods like booms and barriers are used for the recovery of the spilled oil, but this will be effective and applicable only for immediate oil spill. And not effective when the oil reaches miles into the ocean. The use of synthetic chemicals is not biodegradable and leads toxicity to the marine environment. [0005] These contaminants are naturally removed by vaporization, photocatalysis and chemical reactions but takes considerable time. The chemical treatment methods have been mainly used, but it shows limitations in terms of treatment efficiency and cost. Due to the ineffective mechanical methods, and toxic effects of chemical dispersants, biological methods are preferred.

[0006] For example, many microorganisms are difficult to grow in sufficient quantities to be useful and subsequently deployed in marine production systems. These problems are exacerbated by reduced viability and activity due to stabilization prior to processing, compounding, storage, distribution, sporulation, transport, and application of feeder cells as a means of stabilization. Further, the microorganisms of the existing biological agents cannot provide effective bioremediation under extreme marine environmental conditions such as extreme pressure conditions, wide ranges of salinity, temperature, and pH.

[0007] Therefore, there is a need for a bacteria based composition for the bioremediation of PAHs polluted/contaminated marine sites.

Objectives of the invention:

[0008] The primary objective of the invention is to provide a bacterial based composition for bioremediation of polluted/contaminated marine sites.

[0009] Another objective of the invention is to provide a bacterial based composition that can be effectively utilised on the sea surface at ambient pressure and deep-sea high pressure conditions for the remediation of oil spills.

[0010] The other object of the invention is to utilize marine bacteria/ microorganism during the oil spills to neutralize the environmental pollutant through their metabolic process.

[0011] Another objective of the invention is to provide a bacterial based composition that effectively degrades the pollutants/contaminants under extreme marine environmental conditions such as wide ranges of pH, salinity, temperature and pressure. [0012] Another object of the invention is to provide a method for oil bioremediation comprising immobilization of bacterial strains on to the agro-residues.

[0013] Another object of the invention is to utilize organic or inorganic nutrients with nitrogen and phosphate fertilizers to increase the multiplication rate of microorganisms to enhance the degradation process.

[0014] Yet another object of the invention is to utilize biosurfactant to improve the availability of hydrocarbon for the bioremediation of oil spills in marine environment.

[0015] Further objective of the invention is to provide an eco-friendly composition that decreases the level of pollution by releasing environmental friendly metabolites and intermediates that are non-toxic to the marine animals and birds.

[0016] Yet another objective of the invention is to provide a non-toxic composition that can be stored for long time without affecting the activity to degrade the crude oil contaminants.

[0017] Further objective of the invention is to provide an eco-friendly composition and a method for the degradation of several classes of polycyclic aromatic hydrocarbons and persistent organic and crude oil pollutants such as anthracene, phenanthrene, naphthalene, fluorene, perylene, phenol, toluene, benzene, petrol, spent engine oil, furnace oil, crude oil and diesel thereof.

Summary of the invention:

[0018] The present disclosure proposes a microbial based composition for the bioremediation of crude oil contamination and a process for preparation thereof. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later. [0019] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a microbial composition for the bioremediation of crude oil hydrocarbon contaminants.

[0020] According to an aspect, the invention provides a microbial composition for the bioremediation of crude oil hydrocarbon. The microbial composition comprises a bacterial consortium, at least one biodegradable carrier, at least one fertilizer and a microbial biosurfactant.

[0021] In specific, the bacterial consortium comprises a combination of bacterial strains such as Bacillus oceanisediminis IOS9, Kocuria flava IOS11, Nesiotobacter exalbescens COD22, Brevibacterium sediminis COD27 and Bacilus subtilis EB1. Each bacterial strain is mixed in an equal proportion by volume to obtain the bacterial consortium. Concentration of the bacterial consortium ranges from 10⁸ CFU/g to 10⁹ CFU/g to a total weight of the biodegradable carrier. The biodegradable carrier comprises at least one lignocellulosic material, wherein the lignocellulosic material is obtained from at least one of wheat bran, corn cob, rice bran, sweet sorghum, sawdust, and sugarcane bagasse from agricultural production.

[0022] The fertilizer comprises at least one of organic and inorganic fertilizer, wherein the inorganic fertilizer comprises either ammonium sulphate or urea or ammonium chloride or dipotassium hydrogen phosphate or potassium dihydrogen phosphate or ammonium dihydrogen phosphate. The concentration of the fertilizer ranges from 0.01% w/v to 0.1% w/v with respect to a total volume of the composition. The most preferable concentration of the fertilizer is 0.01% w/v. The concentration of the microbial biosurfactant ranges from 0.01% w/v to 0.1% w/v with respect to a total volume of an oil composition, most preferable concentration of the microbial biosurfactant is 0.01% w/v.

[0023] According to another aspect, the invention provides a method for the preparation of microbial composition. First, individual cultures are mixed to prepare a consortium. Next, the bacterial consortium is lyophilized/spray dried to obtain a dried consortium. Next, the biodegradable carrier is sterilized to obtain a sterilized biodegradable carrier. Next, the lyophilized consortium is added to the sterilized biodegradable carrier to obtain a first mixture. Next, the first mixture is incubated at a temperature for immobilizing the lyophilized consortium on the sterilized biodegradable carrier to obtain an immobilized consortium. In specific, the first mixture is incubated at the temperature ranges between 25°C to 35°C for 5 to 10 hours. Next, the fertilizer is added to the immobilized consortium to obtain a second mixture. Later a microbial biosurfactant is added to the second mixture to obtain the composition.

[0024] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.

Detailed description of drawings:

[0025] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0026] FIG. 1 illustrates a flowchart of a method for the preparation of a microbial composition, in accordance to an exemplary embodiment of the invention.

[0027] FIGs. 2A and 2B illustrate graphs depicting the effect of nitrogen source and phosphate source on crude oil/hydrocarbon degradation, in accordance to an exemplary embodiment of the invention.

Detailed invention disclosure:

[0028] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0029] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a microbial based composition for the bioremediation of crude oil/ hydrocarbon and process for preparation thereof. [0030] According to an exemplary embodiment of the invention, a microbial composition for bioremediation of crude oil/hydrocarbon. The microbial composition comprises a bacterial consortium, at least one biodegradable carrier, at least one fertilizer and a microbial biosurfactant. Addition of fertilizer is to enhance the bioremediation process. Further, addition of microbial biosurfactant improves the availability of hydrocarbons to increase the rate of biodegradation.

[0031] In specific, bacterial consortium comprises a combination of bacterial strains such as Bacillus oceanisediminis IOS9, Kocuria flava IOS11, Nesiotobacter exalbescens COD22, Brevibacterium sediminis COD27 and Bacilus subtilis EB1. Each bacterial strain is mixed in an equal proportion by volume to obtain the bacterial consortium. Concentration of the bacterial consortium ranges from 10⁸ CFU/g to 10⁹ CFU/g to a total weight of the biodegradable carrier. The biodegradable carrier comprises at least one lignocellulosic material. The lignocellulosic material is obtained from either wheat bran, corn cob, rice bran sweet sorghum, sawdust, and sugarcane bagasse from agricultural production.

[0032] The fertilizer comprises at least one of organic and inorganic fertilizer, wherein the inorganic fertilizer comprises either ammonium sulphate or urea or ammonium chloride or dipotassium hydrogen phosphate or potassium dihydrogen phosphate or ammonium dihydrogen phosphate. The concentration of the fertilizer ranges from 0.01% w/v to 0.1% w/v with respect to a total volume of the composition. The working concentration of the fertilizer is 0.01% w/v. The concentration of the microbial biosurfactant ranges from 0.01% w/vto 0.1% w/v with respect to a total volume of an oil composition, working concentration of the microbial biosurfactant is 0.01% w/v.

[0033] According to another exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of an exemplary method for preparation of the microbial composition. In specific, the proposed method is simple and scalable process to synthesize the microbial composition that is capable of degrading the oil. At step 102, a bacterial consortium is formed. At step 104, the bacterial consortium is lyophilized/spray dried to obtain a dried consortium. In specific, the bacterial consortium is dried and grounded to obtain a powdered consortium. [0034] At step 106, the biodegradable carrier is sterilized to obtain a sterilized biodegradable carrier. At step 108, the powdered consortium is added to the sterilized biodegradable carrier to obtain a first mixture. At step 110, the first mixture is incubated at a temperature for immobilizing the powdered consortium on the sterilized biodegradable carrier to obtain an immobilized consortium. In specific, the first mixture is incubated at the temperature varies between 25 °C to 35 °C for a time period of at least 5 to 10 hours. At step 112, the fertilizer is added to the immobilized consortium to obtain a second mixture. At step 114, a microbial biosurfactant is added to the second mixture to obtain the composition.

[0035] According to another embodiment of the invention, a mixture of bacteria consisting of 5 bacterial strains, capable of growing on crude oil as sole energy and carbon source, are isolated from deep sea samples. The bacterial strains Nesiotobacter exalbescens COD22, Brevibacterium sediminis COD27 are isolated from the deep-sea sediment samples that is collected at a depth of 2100 m from Bay of Bengal, Bacillus oceanisediminis IOS9 and Kocuria flava IOS11 are collected from the water sample at a depth of 3500 m, and the strain Bacilus subtilis EB1 is isolated from deep-sea sediment at a depth of 2191 m from Indian Ocean. These deep-sea strains are dried to obtain the bacterial consortium.

[0036] For instance, the characterization of oil degrading bacterial strains isolated from deep sea samples are given in table 1. The organisms for the preparation of the bacterial consortium are selected based on the hydrocarbon degradation potential are listed in the table 1. These oil degrading deep sea consortium that display substantial potential for hydrocarbon degradation can be applied for the effective bioremediation of oil spills.

[0037] Table 1:

[0038] In an embodiment, rice bran (RB) is used as the biodegradable carrier for immobilizing bacterial consortium. The RB is obtained from an agricultural industry. The RB is sun dried and stored at room temperature.1 gram of the RB is mixed in 100 mL of distilled water to obtain a sterile RB. After sterilization, the distilled water is discarded and 0.1 g of the bacterial consortium is added to the sterile RB to obtain the first mixture. The first mixture is incubated at a temperature of 28°C under stirring of 120 rpm for 5 hours for immobilizing the bacterial consortium onto the sterile RB. The immobilized consortium is stored at a temperature of 4°C until further use.

[0039] The rice bran with immobilized consortium of bacterial strains (0.1% w/w) is added to obtain a composition. The immobilized consortium is tested for bacterial growth. The obtained composition is then added to the sea water supplemented with 10% (v/v) crude oil. Cell dry weight, total count and hydrocarbon degradation percentage are recorded after 30 days, for free cells and immobilized cells. In specific, the bacterial growth is determined by two methods such as Total viable count (TVC) of bacterial cells and Cell dry weight measurements.

[0040] In case of immobilized conditions, RB is mixed in phosphate buffer and vortexed. The released cells are collected by centrifugation followed by cell mass measurement by standard plate count method, using Zobell Marine Agar (ZMA) plates. The total count of free cells is enumerated using plating on ZMA and incubated at 30°C for 48 h.

[0041] To study the cell dry weight, samples are vortexed for a time period of 5 minutes in the presence of Triton X-100 as desorbing agent to dislodge the immobilized cells and obtain a broth. The broth is centrifuged for a time period of 15 min at 13,000 rpm, followed by drying to a constant weight at a temperature of 80°C. [0042] At a particular time interval, the sample from the biodegradation experiment is withdrawn and to that equal volume of dichloromethane is added to obtain a mixture. The mixture is kept in a magnetic stirrer for complete absorption of crude oil with dichloromethane. After 1 hour, DCM layer is separated and crude oil is retrieved using rotary vacuum evaporator.

[0043] Oil weight is measured and the percentage of degradation is calculated using the formula:

Initial weight of oil - Observed weight of oil

% of degradation = - , . . , - — - - — - - x 100

Initial weight of oil

[0044] Gas chromatography mass spectrometry (GC-MS) analysis: The change in total hydrocarbons of the crude oil samples is studied through GC-MS analysis. The Agilent technologies instrument (GC 7890 A, 240-MS/4000, USA) under external ionization mode using fused silica column HP-5 MS column (30mx0.32mmx0.25 pm).

[0045] For instance, table 2 depicts Cell dry weight, total viable count, total hydrocarbons and degradation percentage after 30 days of biodegradation experiment.

[0046] Table 2:

[0047] From table 2, it is evident that the percentage degradation of crude oil by the immobilized mixture of bacterial strains reached a maximum of 81%, whereas the percentage degradation of crude oil by the mixture of bacterial strains without immobilization on carrier material is 72%. The percentage degradation of crude oil by the individual strains is still lower in the range of 63% to 71%. Therefore, the mixed bacteria immobilized on RB have a stimulating effect on oil utilization and showed a high percentage of microbial growth and oil degradation.

[0048] According to another embodiment of the invention, FIG. 2A refers to a graph 200 depicting effect of nitrogen source on crude oil degradation. FIG. 2B refers to a graph 202 depicting effect of phosphate source on crude oil degradation. Different nitrogen and phosphate sources, such as ammonium sulfate, urea, ammonium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and ammonium dihydrogen phosphate, in amount of 0.01% (w/v) are added to the second mixture for bioremediation of oil spill.

[0049] The effect of different nitrogen and phosphorus sources on growth of immobilized cultures is recorded after 30 days of bioremediation process as shown in the FIGs. 2A and 2B. The experimental results of all analyses are expressed as mean ± standard deviation from three individual replicates.

[0050] It is evident that the efficacy of immobilized consortium of bacterial strains for growth and degradation of oil is improved by addition of these readily available nitrogen sources. Further, the addition of phosphorus source to the medium increased the viable count of microorganisms during the biodegradation analysis, leading to increased efficacy of oil degradation. [0051] According to another embodiment of the invention, emulsification activity of biosurfactant with different grades of hydrocarbons and oil is determined. The microbial biosurfactant at a concentration of 0.01% with respect to the total volume of different hydrocarbons (kerosene, xylene, toluene, benzene, diesel and petrol) and oils such as spent engine oil, crude oil, cedar wood oil and furnace oil are tested for calculating the emulsification index (E24%). The emulsification index is determined by the substrate mixed with the PBS dissolved biosurfactant solution equal volumes and kept for incubation at 24h. Emulsification activity is estimated by using formula:

Height of the emulsified layer

Emulsification activity (£’24%) = X 100

Total height of the liquid medium

[0052] For instance, table 3 depicts Emulsification index of crude biosurfactant in the presence of different hydrocarbon.

[0053] Table 3:

[0054] According to another embodiment of the invention, bioremediation of crude oil with the microbial composition is observed. The effect of biosurfactant at a concentration of 0.01% (weight of biosurfactant/weight of oil) is observed by adding the biosurfactant in the second mixture to obtain the microbial composition. Later, the degradation experiment is carried out. Determination of bacterial growth is carried out by total viable cell count and hydrocarbon degradation percentage is recorded after 30 days of experiment. The maximum rate of degradation around 98% is observed for dipotassium hydrogen phosphate and urea along with biosurfactant.

[0055] Further, effective degradation is observed at a wide range of temperature from 10 - 45°C. Microorganisms in the consortium have the ability to survive in wide ranges of salinity, pH and hydrostatic pressure, and could be adapted to catalyze many reactions in a broad physiological condition.

[0056] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, bacterial based composition is disclosed for the bioremediation of oil polluted/contaminated marine sites.

[0057] The bacteria based composition can be effectively utilised on sea surfaces at ambient pressure and deep-sea high pressure conditions for the remediation of oil spills. The marine bacteria/ microorganisms are utilized to neutralize the environmental pollutant during marine oil spills through their metabolic process. The bacteria based composition effectively degrades pollutants/contaminants under extreme marine environmental conditions such as wide ranges of pH, salinity, temperature and pressure

[0058] The organic or inorganic nutrients with nitrogen fertilizers and phosphate fertilizers are utilised to increases the multiplication rate of microorganisms to enhance the degradation process. The biosurfactant is utilised to improve the availability of hydrocarbons for bioremediation of oil spills in marine environment. The eco-friendly composition decreases the level of pollution by releasing environmental friendly metabolites and intermediates that are non-toxic to marine animals and birds.

[0059] The non-toxic composition can be stored for long time without affecting the activity to degrade oil/hydrocarbons. The eco-friendly composition and a method is utilised for the degradation of several classes of polycyclic aromatic hydrocarbon and persistent organic pollutants such as anthracene, phenanthrene, naphthalene, pyrene, spent engine oil, crude oil, fluorene, perylene, phenol, toluene, benzene, petrol, furnace oil and diesel thereof.

[0060] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.

Claims

CLAIMS: I / We Claim:

1. A microbial composition for bioremediation of crude oil/hydrocarbon, comprising: a bacterial consortium; at least one biodegradable carrier; at least one fertilizer; and a microbial biosurfactant, whereby, the microbial composition is utilized for the bioremediation of polycyclic aromatic hydrocarbons in marine environment.

2. The microbial composition as claimed in claim 1, wherein the bacterial consortium comprises a combination of bacterial strains such as Bacillus oceanisediminis IOS9, Kocuria flava IOS11, Nesiotobacter exalbescens COD22, Brevibacterium sediminis COD27 and Bad I us subtil is EB1.

3. The microbial composition as claimed in claim 2, wherein each bacterial strain is mixed in an equal proportion by volume to obtain the bacterial consortium.

4. The microbial composition as claimed in claim 1, wherein concentration of the bacterial consortium ranges from 10⁸ CFU/g to 10⁹ CFU/g of a total weight of the at least one biodegradable carrier.

5. The microbial composition as claimed in claim 1, wherein the biodegradable carrier comprises at least one lignocellulosic material, wherein the lignocellulosic material is obtained from at least one of wheat bran, corn cob rice bran sweet sorghum, sawdust, and sugarcane bagasse.

6. The microbial composition as claimed in claim 1, wherein the fertilizer comprises at least one of organic and inorganic fertilizer, wherein the inorganic fertilizer comprises at least one from ammonium sulfate, urea, ammonium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and ammonium dihydrogen phosphate.

7. The microbial composition as claimed in claim 1, wherein concentration of the fertilizer ranges from 0.01% w/v to 0.1% w/v with respect to a total volume of the composition, wherein the working concentration of the each fertilizer is 0.01% w/v.

8. The microbial composition as claimed in claim 1, wherein the concentration of microbial biosurfactant ranges from 0.01% w/v to 0.1% w/v with respect to a total volume of an oil composition, wherein the working concentration of the microbial biosurfactant is 0.01% w/v.

9. A method for preparation of a microbial composition, comprising: forming a bacterial consortium; lyophilizing/spray drying the bacterial consortium to obtain a dried consortium; sterilizing at least one biodegradable carrier to obtain a sterilized biodegradable carrier; adding the lyophilized consortium to the sterilized biodegradable carrier to obtain a first mixture; incubating the first mixture at a temperature for immobilizing the dried consortium on the sterilized biodegradable carrier to obtain an immobilized consortium; and adding at least one fertilizer to immobilized consortium to obtain a second mixture; and adding a microbial biosurfactant to the second mixture to obtain the composition for the bioremediation of oil spill.

10. The method for preparation of a microbial composition as claimed in claim 9, wherein the first mixture is incubated at the temperature varies between 25°C to 35°C for a time period of 5 to 10 hours.