DESC:FIELD
The present disclosure relates to a microbial composition for the bioremediation of a geographical area contaminated with hydrocarbons.
DEFINITIONS
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.
Bioremediation: The term "bioremediation" refers to a managed or spontaneous process in which microbiological processes are used to degrade or transform contaminants to less toxic or non-toxic forms, thereby remedying or eliminating environmental contamination.
Treatment: The term "treatment” and grammatical variants thereof, used in the present disclosure, refers to any method, technique, or process which results in bioremediation of hydrocarbons.
Biosparging: The term “biosparging” refers to an in-situ remediation technology that uses indigenous microorganisms to biodegrade organic constituents in the saturated zone. In biosparging, air (or oxygen) and nutrients (if needed) are injected into the saturated zone to increase the biological activity of the indigenous microorganisms.
Bioventing: The term “bioventing” refers to a process of stimulating the natural in situ biodegradation of contaminants in soil by providing air or oxygen to existing soil microorganisms.
Bioslurping: The term “bioslurping” refers to the adaptation and application of vacuum-enhanced dewatering technologies to remediate hydrocarbon-contaminated sites.
Bioaugmentation: The term “bioaugmentation” refers to the practice of adding cultured microorganisms into the subsurface for the purpose of biodegrading specific soil and groundwater contaminants.
Biostimulation: The term “biostimulation” refers to the modification of the environment to stimulate the existing bacteria capable of bioremediation. This can be done by addition of various forms of rate limiting nutrients and electron acceptors, such as phosphorus, nitrogen, oxygen, or carbon (e.g. in the form of molasses).
Tilling: The term “tilling” refers to the turning over and breaking up of material for uniform mixing.
Geographical area: The term “geographical area” refers to soil or water present in land masses or water bodies that are contaminated with hydrocarbons, and for which the bioremediation process is to be carried out.
BACKGROUND
Oil discharge into the natural environment and aquatic ecosystems can cause serious global, ecological, and environmental problems. Industrial development has increased oily wastewater discharge into the environment. Operational and accidental crude spills which are increasing in frequency due to the ever increasing ocean transportation of petroleum crude and other petroleum products has, and will continue to result in, extensive ecological damage. Environmental contamination due to petroleum crude spillage poses a serious threat to the ecosystem. There has been an increasing concern over the accidental spillage of petroleum crude and petrochemical-derived hydrocarbon compounds during technological processes and transportation. Many of these hydrocarbons are considered to be a potential health hazard.
Several in-situ and ex-situ strategies have been developed for the bioremediation of contaminated soil and/or water. Physical treatment using absorbing material followed by incineration, for petroleum crude removal is one of the oldest and simplest methods. However, this method is not practical, as it must be used soon after the spillage has occurred. On the other hand, chemical treatments of the polluted soil/water using a chemical emulsifier are costly and hence are not economical. Further, these methods cannot remove petroleum crude completely from the polluted site.
For on-site treatment of contaminated soil, in-situ techniques such as bioventing / biosparging (done by sparging microorganisms, nutrients and air) or bioslurping (done by combining bioventing with simultaneous product recovery) are available, however the time required for these treatments is very long. Other in-situ techniques for bioremediation include the use of nutrients (biostimulation) or microorganisms (bioaugmentation) onto the contaminated sites. However, the results obtained by using these methods are not consistent.
Ex-situ bioremediation techniques, such as mixing of contaminated soils with non-contaminated soils (land farming) or organic waste (composting), although effective, are extremely slow.
Enhanced soil bioremediations have been carried out when contaminated soils are treated with nutrients and microorganisms under controlled process conditions in bioreactors, however, the cost of transporting the contaminated soil to the bioreactor site decreases its viability.
Another technique for petroleum crude-contaminated groundwater involves pumping of water to the remediation site, treating the contaminated water, and pumping back the treated water, using the ‘pump and treat’ strategy. Although this technique is effective, it requires the construction of withdrawal and injection wells, which is expensive and hence not commercially viable.
Several commercially available bio-based products are used for bioremediation of petroleum crude. However, all the bio-based products show incomplete hydrocarbon/petroleum crude bioremediations due to decreased robustness under different environmental conditions. Moreover, the duration required for the near-complete petroleum crude bioremediation is very high for almost all the commercially available bioremediation products.
Another issue that refineries and oil terminals face is the huge quantities of oily sludge generated, which is created by the settling or sedimentation of particulate material in the crude oil storage tanks over an extended period of time. Conventionally, this sludge is collected periodically, subjected to oil extraction by physical methods and the residual material remains as a waste product. Since this sludge cannot be disposed because of environmental concerns, refineries and oil terminals allow it to be accumulated, thereby increasing the burden on the environment.
Therefore, there is felt a need for a method for bioremediation of hydrocarbons that mitigates the above mentioned drawbacks.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
Another object of the present disclosure is to provide a composition for bioremediation of a geographical area contaminated with hydrocarbons.
Still another object of the present disclosure is to provide a method for bioremediation of a geographical area contaminated with hydrocarbons.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates to a microbial composition for bioremediation of a geographical area contaminated with hydrocarbons. The microbial composition comprises at least one microorganism selected from the group consisting of fungi of the class Eurotiomycetes, fungi of the class Saccharomycetes, bacteria of the class Gammaproteobacteria, bacteria of the class Alphaproteobacteria, and bacteria of the class Actinomycetes. Typically, the microbial composition contains at least one fungus and at least one bacterium, and the proportion of the mass of the fungus to the mass of the bacterium can be in the range of 1:10 to 10:1. In an embodiment, the fungi belong to the genus Paecilomyces of the class Eurotiomycetes, and the bacteria belong to the genus Citrobacter of the class Gammaproteobacteria. In another embodiment, the microbial composition can further comprise at least one additive.
The present disclosure further provides a method for bioremediation of a geographical area contaminated with hydrocarbons using the microbial composition of the present disclosure. The method comprises adding and mixing the microbial composition to the geographical area and culturing the microorganisms inside the geographical area selectively, using, the hydrocarbon as a culturing medium. Typically, the soil or water contained in the geographical area can be periodically tilled or stirred, respectively, to accelerate the culturing step.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWING
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1a illustrates a graphical representation of petroleum crude bioremediation with concentration of petroleum crude (10g/100g of soil) against time (days) by Paecilomyces sp. at Day 1 to Day 60;
Figure 1b illustrates a graphical representation of petroleum crude bioremediation with concentration of petroleum crude (20g/100g of soil) against time (days) by Paecilomyces sp. at Day 1 to Day 80;
Figure 2 illustrates a graphical representation of petroleum crude bioremediation with percentage of petroleum crude (g/100g of soil) against time (days) by Citrobacter sp. at Day 1 to Day 33; and
Figure 3 illustrates a comparative graphical representation of bioremediation of sludge by different microbial compositions.
DETAILED DESCRIPTION
Petroleum or crude oil containing sludge is a complex mixture of different classes of hydrocarbons, such as saturates, aromatics, resins, and asphaltenes. Although most microorganisms are capable of utilizing hydrocarbons for growth, they usually have some preferences in terms of carbon chain length, branching, unsaturation, aromaticity, and the like and hence are not capable of degrading all fractions of crude oil.
Bioremediation is the use of microorganism(s) or microbial process to detoxify and degrade the environmental contaminants or attempt to accelerate the natural degradation rates by overcoming factors that limit the natural degradation of the environmental contaminants. Successful application of bioremediation is dependent on appropriate biodegradative microbes and environmental parameters. Despite the huge potential of microorganisms to degrade organic compounds under favourable conditions, a single species of microorganism cannot degrade all the components of petroleum crude. Currently, several microorganisms are known, each capable of degrading usually one or, at least, a few petroleum crude components at a time.
Therefore, the present disclosure envisages a microbial composition and a method for bioremediation of a geographical area contaminated with hydrocarbons, using the microbial composition. The microbial composition of the present disclosure is capable of providing desirable results within a short duration of time.
In accordance with one aspect of the present disclosure, there is a provided a microbial composition for bioremediation of a geographical area contaminated with hydrocarbons. The microbial composition comprises at least one microorganism selected from the group consisting of fungi of the class Eurotiomycetes, fungi of the class Saccharomycetes, bacteria of the class Gammaproteobacteria, bacteria of the class Alphaproteobacteria, and bacteria of the class Actinomycetes.
In an embodiment, the fungi belong to the genus Paecilomyces of the class Eurotiomycetes. In another embodiment, the bacteria belong to the genus Citrobacter of the class Gammaproteobacteria, which are capable of producing lipase and a biosurfactant and thereby aid in the bioremediation process.
In one embodiment, the microbial composition comprises at least one fungus and at least one bacterium. Typically, the proportion of the mass of the fungi to the mass of the bacteria can be in the range of 1:10 to 10:1. In one embodiment, the proportion of the mass of the fungi to the mass of bacteria is 1:1.
Typically, the fungi and the bacteria used in the microbial composition of the present disclosure can be isolated from refinery sites, especially petroleum crude refineries.
Typically, the microbial composition has a moisture content in the range of 1 wt% to 20 wt%.
Typically, the microbial composition of the present disclosure can be in a form selected from the group consisting of a solid mass, a semi-solid mass, a liquid, a spray, a tablet, a powder, a granule, a pellet, an emulsion, a suspension, a dispersion, and a gel.
The microbial composition of the present disclosure further comprises at least one additive. The additive can be selected from the group consisting of diluents (20 % to 30 % corn cob, 20 % to 30 % soya protein, 20 % to 30 % wheat bran), food waste materials ( 1 % to 2 % dehydrated fruit waste), binders (1 % to 2 % guar gum, 1 % to 2 % xanthan gum), gelling agents (1 % to 2 % potato starch), matrix forming agents (1 % to 2 % glutaraldehyde), dispersants (1 % to 2 % biosurfactant), pH modifiers (0.2 M to 0.5 M NaOH, 0.2 M to 0.5 M HCl), trace elements (100 ppm to 1000 ppm cobalt), stabilizers (1 % to 2 % glycerol), nutrients (1 % to 2 % urea), carriers (saw dust, soya hull), and combinations thereof.
In an embodiment of the present disclosure, the nutrient is a macronutrient or a micronutrient or a combination thereof.
The food waste material may contain nutrient sources known in the art such as conventional carbon, nitrogen, iron and phosphorous sources and the like depending on the type of microorganisms employed. In an embodiment of the present disclosure, the food waste material is organic waste, particularly, kitchen or agricultural waste materials.
The microbial composition can be in the form of a homogenized ready to use mixture or the microorganisms along with the additives may be packed separately, to be mixed just prior to use.
Typically, the microbial composition has a shelf life in the range of 10 months to 18 months, and can be stored in a place which is cool and shady and is away from direct sunlight, and used when required.
The present disclosure further provides a method for manufacturing the microbial composition. The method involves isolating the microorganisms and then cultivating the isolated microorganisms to manufacture the microbial composition of the present disclosure.
Soil samples are collected from different oil contaminated sites in the Hindustan Petroleum Corporation Limited Terminal, Bangalore and are used for isolating oil degrading microorganisms. The bacterial and fungal isolates obtained after the enrichment of the soil samples are screened for the ability to use crude oil as the only carbon source in minimal media agar plates. The strains are then grown in a medium containing 10 % crude oil. The strains that demonstrate >50 % oil degradation within fourteen days are selected for further study. These selected isolates are grown in 100 g soil mixed with 10 % crude oil under controlled temperature and humidity. conditions to identify the isolates. In one embodiment, the temperature is 30 °C and the relative humidity is 70 %.
The isolated bacterial and fungal cultures are then grown in a suitable medium to allow them to grow and multiply. The bacteria are then centrifuged to separate the bacteria from the nutrient medium, followed by gradual freeze drying or spray drying under sterile conditions to evaporate the water and obtain a dry, powdery batch of bacteria with a high degree of purity.
In one embodiment of the present disclosure, the bacteria are coated with cryoprotectants before freeze drying.
In another embodiment of the present disclosure, the freeze dried bacteria are mixed with stabilizers and additives to protect the cryoprotectants from oxidation and provide a stable condition to assist the bacteria to be less susceptible to the undesirable environmental conditions.
In another embodiment of the present disclosure, the fungal culture is obtained by culturing the fungi in a fermenter. The fungi are then transferred to a sterile wheat bran and allowed to grow. The wheat bran mixture with the fungi is used for bioremediation experiments.
In an embodiment, the fungi and the bacteria are mixed thoroughly with the food or agricultural waste material for uniform distribution of the microorganisms and to prevent any agglomeration of mass or lump formation, to obtain the microbial composition.
In another embodiment, the fungi and the bacteria are mixed together and directly treated at the site of petroleum crude spillage followed by incubation for a time period in the range of 20 days to 80 days.
In accordance with another aspect of the present disclosure, there is provided a method for bioremediation of a geographical area contaminated with hydrocarbons. The method includes adding and mixing microbial composition of the present disclosure to the geographical area. The microorganisms are cultured inside the geographical area, selectively using the hydrocarbons as a culturing medium. Typically, the soil or water contained in the geographical area is periodically tilled or stirred, respectively, to accelerate the culturing step. Typically, the tilling and moistening can be carried out at an interval of 48 hours to 96 hours. The process of the present disclosure can further involve a step of adding the microbial composition at specific intervals to enhance the bioremediation of a geographical area contaminated with hydrocarbons.
The microbial composition of the present disclosure when used on actual tank sludge samples obtained from refineries results in 50 % to 58 % reduction in oil content within 28 days.
The oil degrading microbial composition of the present disclosure can be used in an open environment where the polluted soil is exposed to rain, and conditions, such as water-logging. Immobilization of the fungi can be done on solid supports, such as corn cob, soya hull, wheat bran, sawdust, and rice husk, to ensure that the microorganisms are retained in the soil and do not get washed off. These materials retain moisture, provide essential nutrients required for the growth and at the same time act as a physical support. Since mixing of the grown microbial culture with the solid support material may not be practical on a large scale, the microorganisms can be grown directly on the support material itself, so that it can be applied to the contaminated soil without any mixing.
The present disclosure provides a cost effective microbial composition for bioremediation of crude oil contaminated soil and oily sludge within a short span of time.
The present disclosure is further described in light of the following laboratory scale experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. These laboratory scale experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial/commercial scale.
Experimental details
Experiment-1: General method for preparing the microbial composition of the present disclosure
Step-1: Screening and isolation of microorganisms for preparing the microbial composition of the present disclosure
Soil samples were collected from oil contaminated sites in the Hindustan Petroleum Corporation Limited Terminal, Bangalore, India. Approximately 10 g of soil sample was inoculated into 250 ml Erlenmeyer flask containing 100 mL nutrient broth (NB) and incubated at 37 °C for 48 hours. Sixty bacterial and fungal isolates were obtained after the enrichment of these samples. On obtaining visible growth, the liquid culture was serially diluted using sterile saline and plated on nutrient agar plates which were then incubated at 37 °C for 48 hours. The colonies obtained on the nutrient agar were sub-cultured till pure isolates were obtained. The isolates were maintained on nutrient agar slants at 4 °C. Liquid cultures were obtained by inoculating a loopful of the pure culture from the slants into NB and incubating at 37 °C for 24 hours with agitation at 150 rpm. Serially diluted soil samples were also plated on minimal agar plates containing crude hydrocarbon so as to selectively isolate oil degrading strains from the environment. These strains were screened for the ability to use crude oil as the only carbon source in minimal media agar plates. The strains were then grown in Bushnell Haas medium containing 10 % crude oil and ten strains that demonstrated >50 % oil degradation within fourteen days were selected for further experiments. These selected isolates were grown in 100 g soil mixed with 10 % crude oil at at 30 °C and 70 % relative humidity, to identify the microorganisms.
Step-2: Microbial Cultivation
Inoculum was prepared by inoculating a loopful of culture from fresh slants into 10 mL NB (Potato Dextrose Broth (DB was used for the cultivation of fungi), followed by incubation at 37 °C for 72 hours with agitation at 150 rpm. 1% (v/v) of this culture was then used to inoculate 500 ml Erlenmeyer flask containing 100 ml NB (casein hydrolysate, or yeast extract peptone dextrose (YEPD) broth can also be used) and incubated at 37 °C for 72 hours with agitation at 150 rpm.
Step-3: Product Blending
After the cells were cultivated in the medium, they were separated by centrifugation. The cell biomass was mixed with diluent/filler material and dried in tray dryers at 50 °C for 48 hours and packaged for further use.
Experiment-2: Culturing of Paecilomyces sp. in accordance with the present disclosure
A fungal isolate identified as belonging to the genus Paecilomyces, demonstrating the highest oil degradation of 65 % was selected for further experimentation. It was then cultivated in potato dextrose broth (PDB) at the 3L bioreactor scale at 30 °C for 4 days.
At Day 1 solid state culturing method using wheat bran was used for the production of Paecilomyces sp. The fungi were cultured in potato dextrose broth (PDB) in a 3L fermenter at 30 °C for 4 days. The resultant cell biomass was separated by centrifugation and the dry cell mass was used for the bioremediation experiments.
Alternatively, the fungi were then transferred to sterile wheat bran supplemented with nitrogen and carbon sources from 2 % dehydrated fruit waste and allowed to grow for a5 days at 30 °C. The fruit waste was soaked in water in a ratio ranging from 1:1 to 3:1 (depending on the initial moisture content of the waste), ground and subsequently added to sterile wheat bran solid culture, followed by incubation at 32 °C for 5 days. By Day 5 the fungi culture was ready for use in the microbial composition. The wheat bran mixture with the fungi was then used for the bioremediation experiments.
Experiment-3: Culturing of Citrobacter sp. in accordance with the present disclosure
On Day 1, Citrobacter sp. capable of producing lipase and a biosurfactant was inoculated in a round bottom flask containing 10 ml sterile nutrient broth. The inoculated Citrobacter sp. was then incubated by placing the flask on a shaker at 200 rpm for 24 hours at 30 °C.
On Day 2, 10 ml of the incubated inoculum from Day 1 was used as an inoculum for 100 ml nutrient broth and was incubated on a shaker at 200 rpm for 24 hours at 30 °C.
The process was repeated with 100 ml inoculums to reach final 1 L culture for bioremediation study. The cells were separated by centrifugation at 10,000 rpm and suspended in a 0.1 M phosphate buffer and used for the microbial composition.
Experiment-4a: Bioremediation of petroleum crude spilled soil using Paecilomyces sp. of the present disclosure
A control experiment was set up containing petroleum crude spilled soil (10g/100g), without any microorganism. Similarly, a test was also established containing the same quantity of petroleum crude spilled soil along with the Paecilomyces sp. (3 % w/w) obtained in experiment-2.
On Day 1 the concentration of the petroleum crude was determined using gravimetric analysis technique (Figure-1a, wherein the darker bar represents the bioremediation by Paecilomyces sp. and the lighter bar represents controlled bioremediation). The initial petroleum crude concentration in the soil at day 1 was found to be 10g/100g for both the control and the test.
Both the control and the test were then incubated for 60 days in similar well aerated and sunny conditions, with periodic checking for the petroleum crude concentration in both the control and the test.
The final petroleum crude concentration was then determined at the end of 60th day using gravimetric analysis technique (Figure-1a). The petroleum crude concentration in the control was found to be 7g/100g of the soil and that in the test was found to be 2.3g/100g of soil.
This clearly shows that the fungal composition of the present disclosure achieves about 70 % to 80 % bioremediation of the petroleum crude from the initial amount of petroleum crude in about 60 days. As illustrated in Figure-1a, the fungal composition achieves up to 50 % of petroleum crude bioremediation in the petroleum crude spilled area (soil) within 8 days to 12 days of incubation and the remaining petroleum crude bioremediation up to 80 % in 45 days to 60 days of incubation period.
Experiment-4b: Bioremediation of petroleum crude spilled soil using Paecilomyces sp. of the present disclosure
50 g of dry cell mass obtained in experiment-2 was mixed with 5 kg soil containing 20 % crude oil. This experimental setup was placed in a sunny well aerated place in the open and no temperature control was maintained. An identical experimental setup without fungus was also kept, as a control. 5 g soil sample was collected from both the setups every 7 days. In addition, the samples were thoroughly mixed/tilled on a weekly basis and the moisture content was also adjusted to 20 %. The residual oil content of the collected soil sample was estimated by standard gravimetric methods after extraction with hexane, and is illustrated in Figure-1b, wherein Line A represents the represents the bioremediation by Paecilomyces sp. and Line B represents controlled bioremediation). It is seen from Figure-1b that the control sample exhibited an oil degradation of 17 %, which could be attributed to the action of naturally occurring microorganisms in the soil and also, sunlight. The oil reduction observed in the test sample was 88.7 % in 70 days. As the same environmental and natural factors observed in the control experiment would be applicable, the effective oil degradation due to the fungi is 71.7 %.
Experiment-5: Bioremediation of petroleum crude spilled soil using Citrobacter sp. of the present disclosure
A control was established containing petroleum crude spilled soil (10g/100g), without any microorganism. Similarly, a test was also established containing the same quantity of petroleum crude spilled soil along the Citrobacter sp. (7 x 106 cells/g) obtained in experiment-3.
On Day 1 the concentration of the petroleum crude was determined using the gravimetric analysis technique as illustrated in Figure-2. The initial petroleum crude concentration in the soil at day 1 was found to be 10g/100g for both the control and the test.
Both the control and the test were then incubated for 33 days in similar well aerated and sunny conditions, with periodic checking for the petroleum crude concentration in both the control and the test.
The final petroleum crude concentration was then determined at the end of 33rd day using the gravimetric technique (Figure-2, wherein the lighter bar represents the bioremediation by Citrobacter sp. and the darker bar represents the controlled bioremediation). The petroleum crude concentration in the control was found to be 7g/100g of the soil and that in the test was found to be 4g/100g of soil.
This clearly indicates that Citrobacter sp. of the present disclosure achieves about 50 % to 60 % bioremediation of the petroleum crude from the initial amount of petroleum crude in about 33 days.
Experiment-6: Bioremediation of petroleum crude spilled soil using additives
Further enhancement of the process was tried by addition of various additives to overcome the nutritional deficiency of the soil, physically adsorb the crude oil, and emulsify the oil. NPK fertilizer, leaf waste, and soya hull were used to enhance the nitrogen content of soil while coco peat was used to adsorb and localize the oil content of soil. Culture broth containing biosurfactant was also used to emulsify the crude oil and enhance its utilization by the added fungi. The addition of the additives led to an overall increase of oil degradation by 0.2 % to 4%.
Experiment-7: Bioremediation of petroleum crude spilled soil using the microbial composition of the present disclosure
A control was established containing petroleum crude spilled soil (10g/100g), with Paecilomyces sp. only and with Citrobacter sp. only. Similarly, a test was also established containing the same quantity of petroleum crude spilled soil along with the microbial composition comprising Paecilomyces sp. to Citrobacter sp. The ratio of Paecilomyces sp. to Citrobacter sp. in the microbial composition was 1:1.
On Day 1 the concentration of the petroleum crude was determined using GC-FID chromatogram technique. The initial petroleum crude concentration on day 1 was found to be 10 g/100g in the soil for both the control and the test.
Both the control and the test were then incubated for 30 days in similar conditions with periodic checking for the petroleum crude concentration in both the control and the test.
The final petroleum crude concentration was then determined at the end of the 30th day using the GC-FID chromatogram technique. The petroleum crude concentration in the control was found to be 7g/100g of the soil and that in the test was found to be 5g/100g of soil.
This clearly indicates that the microbial composition comprising Paecilomyces sp. to Citrobacter sp. exhibits a synergistic effect for the bioremediation of the petroleum crude from the initial amount of petroleum crude in about 30 days. It is clear from the above data that the microbial composition achieves up to 50 % of petroleum crude bioremediation in the petroleum crude spilled area (soil) within 30 days incubation period.

Experiment-8: Bioremediation of sludge using the microbial composition of the present disclosure
Sludge samples were procured from a refinery and an experiment was set up similar to that in experiment-7, in which three different microbial formulations were tested, namely, Citrobacter sp. only, Paecilomyces sp. only, and a combination of Citrobacter sp. and Paecilomyces sp. The results obtained are illustrated in Figure-3. Figure-3 illustrates a graphical representation of sludge bioremediation with percentage bioremediation against different microbial compositions, wherein A represents control sludge, without any microorganism, B represents bioremediation of sludge by bacterial species belonging to the genus Citrobacter, C represents bioremediation of sludge by fungal species belonging to the genus Paecilomyces, and D represents bioremediation of sludge by the microbial composition comprising Paecilomyces sp. to Citrobacter sp. It is seen from Figure-3 that all the three combinations (B, C, and D) were able to achieve similar bioremediation of sludge.
TECHNICAL ADVANCEMENTS
The microbial composition of the present disclosure described herein above has several technical advantages including, but not limited to, the realization of:
- a microbial composition for enhanced bioremediation of a geographical area contaminated with hydrocarbons;
- a microbial composition for bioremediation of a geographical area contaminated with hydrocarbons within a shorter duration of time;
- a simple and economic microbial composition for the bioremediation of a geographical area contaminated with hydrocarbons; and
- an environmental friendly method for the bioremediation of a geographical area contaminated with hydrocarbons.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications to the formulation of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this invention.
The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

,CLAIMS:WE CLAIM:
1. A microbial composition for bioremediation of a geographical area contaminated with hydrocarbons, said composition comprising at least one microorganism selected from the group consisting of fungi of the class Eurotiomycetes, fungi of the class Saccharomycetes, bacteria of the class Gammaproteobacteria, bacteria of the class Alphaproteobacteria, and bacteria of the class Actinomycetes.
2. The microbial composition as claimed in claim 1, wherein said composition contains at least one fungus and at least one bacterium, and the proportion of the mass of said fungus to the mass of said bacterium is in the range of 1:10 to 10:1.
3. The microbial composition as claimed in claim 2, wherein the proportion of the mass of said fungi to the mass of said bacteria is 1:1.
4. The microbial composition as claimed in claim 1, wherein said fungi belong to the genus Paecilomyces of the class Eurotiomycetes.
5. The microbial composition as claimed in claim 1, wherein said bacteria belong to the genus Citrobacter of the class Gammaproteobacteria.
6. The microbial composition as claimed in claim 1, wherein said microbial composition further comprises at least one additive.
7. The microbial composition as claimed in claim 6, wherein said at least one additive is selected from the group consisting of diluents, food waste materials, binders, gelling agents, matrix forming agents, dispersants, pH modifiers, trace elements, stabilizers, carriers, and combinations thereof.
8. The microbial composition as claimed in claim 1, wherein the moisture content of said microbial composition is in the range of 1 wt% to 20 wt%.
9. A method for bioremediation of a geographical area contaminated with hydrocarbons, using the microbial composition as claimed in claim 1, said method comprising adding and mixing said microbial composition to said geographical area, and culturing said microorganisms inside said geographical area, selectively, using, said hydrocarbon as a culturing medium, wherein the soil or water contained in said geographical area is periodically tilled or stirred, respectively to accelerate the culturing step. .