FIELD OF THE INVENTION:
This invention relates to a process for the bioconversion of lignite to humic acid.
This invention further relates to a process for the depolymerisation of lignite to yield humic acid in submerged condition by a newly isolated fungal strain belonging to Pleurotus genus.
BACKGROUND OF THE INVENTION:
Coal bioliquefaction, is a recently developed non-conventional mode of coal processing. It is hailed as a promising technology to combat the alarmingly rising environmental, economic and technological issues of concern. In nature, white-rot fungi, known lignin degraders, use plant matter as a source of nutrients, whereas the majority of microorganisms that live on plant matter do not degrade lignin. It is believed that lignin is a precursor of low-rank coals. A number of white-rot fungi are able to degrade low-rank coals as reported in the literature. Exploitation of several fungal strains for depolymerization of low rank coal has been reported by several researchers [Cohen MS, Gabriele PD. Degradation of coal by fungi Polyporous versicolor and Poria monticola. Appl Environ

Microbiol 1982;44:23-7; Hofrichter M, Fritsch W. Depolymerization of low rank coal by extracellular fungal enzyme systems-I: Screening for low rank coal depolymerizing activity. Appl Microbiol Biotech 1996;46:220-5; Hofrichter M, Fritsch W. Depolymerization of low rank coal by extracellular fungal enzyme systems-II: The ligninolytic enzymes of the coal humic acid-depolymerizing fungus Nematoloma frowardii. Appl Microbiol Biotech 1996;47:419-24; Ward B. Lignite-degrading fungi isolated from a weathered outcrop system. Appl Microbiol 1985;6:236-38; Ward B, Sanders A. Solubilization of coals by fungi. In: Symposium on biological processing of coal and coal-derived substances. Electric Power Research Institute, Palo Alto, California; 1989, p. 3/57-3/67; [58] Yuan HL, Yang JS, Wang FQ, Chem WX. Degradation and solubilization of Chinese lignite by Penicillium sp.P6. Appl Biochem Microbiol 2006;42(l):52-5; Tripathi RC, Jain VK, Tripathi PSM. Fungal biosolubilizations of Neyveli lignite into humic acid, energy sources,PartA: recovery, utilization and environmental effects. Energ Source 2010;32:72-82].
In U.S. patent 4960699, a method has been described to depolymerize an aqueous-soluble polymeric coal substrate extracted from coal by

chemical treatment which is then subjected to enzymatic treatment along with oxygen and hydrogen peroxide. The enzyme employed for this process is lignin peroxidase from Phanerochaete chrysosporium. Furthermore, a method to isolate the enzyme from mycelia-free, unconcentrated media of Phanerochaete chrysosporium culture has also been described. U.S. patent 5036013, discloses an improvement over US patent number 4960699 by detailing the process by which the soluble coal substrate is extracted from native coal. This process is entirely a chemical process. In addition down the line, U.S. patent 5084160 describes a process for isolating an extracellular product from Coriolus versicolor having a molecular weight less than 5000 dalton which is useful for biosolubilizing low rank coals. The substrate (low rank coal), for solubilization, is preferably pretreated by autoradiation/chemical means to enhance solubilization. In a similar fashion, U.S. patent 4882274 describes an extracellular product of Polyporus versicolor able to solubilize low rank coal.
But so far, the media employed for such studies were conventional media of high costs.

Moreover, the incubation period required for degradation was higher and the value-added product obtained by depolymerization was not properly quantified. It is therefore quite desirable to design a process which will lead to a faster depolymerization process resulting in high product yield. At the same time the process should be cost effective so that it can be economically viable for industrial implementation.
OBJECTS OF THE INVENTION;
It is therefore an object of this invention to propose a process for the bioconversion of lignite to humic acid, which is simple and cost-effective.
It is a further object of this invention to propose a process for the bioconversion of lignite to humic acid, which is fast.
Another object of this invention is to propose a process for the bioconversion of lignite to humic acid in very high yields.
These and other objects and advantages of the invention will be apparent from the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION:
According to this invention is provided a process for the bioconversion of lignite to humic acid.
In accordance with this invention, is proposed a process applying whole cells of a newly isolated fungal strain for depolymerization of untreated Indian lignite for production of humic acid in submerged condition. The fungal strain belongs to Pleurotus genus which is locally isolated and is hyperactive in laccase production. The yield of the targeted product, i.e. humic acid, in this process has been found to be highest reported till date. In addition to this, the incubation period required is also much lesser compared to existing methods. Moreover, the media designed for this purpose is unique as the newly designed media has unique composition which is of course minimal media where minimum carbon, nitrogen are present along with cost effective additives, and no growth factors have been selected. The medium comprises a basic medium to which additives have been added. The composition of the basic medium is as follows:
Sodium nitrate (NaNO3) 1.5 to 2.5 gm/l
Potassium dihydrogen phosphate 0.75 to 1.5 gm/1
Magnesium sulphate (MgSO4) 0.5 gm/1
Potassium chloride (KC1) 0.5 gm/1

To this basic medium the additives such as a carbon source, a nitrogen source, chelator, surfactants and metal ions are added. The carbon source may be selected from sugars such as sucrose, maltose etc., preferably sucrose; the nitrogen source is selected from urea, ammonium bicarbonate etc. preferably ammonium bicarbonate; the chelator used is selected from salicylic acid Triethanolamine, EDTA, preferably Triethanolamine; the surfactant used is selected from Triton-X, Tween 20, Tween 40, Tween 60 and SDS, preferably Triton X. Metal ions are copper and cobalt ions, preferably copper sulphate and cobalt chloride. No costly components like yeast extract, malt extract, beef extract, peptone was used in this medium composition.
The carbon source is used in 0.50 to 1.5 % w/v and nitrogen source are used in 0.25 to 1.5% (w/v). The chelators such as salicyclic acid or triethanolamine are used in 0.05 to 0.02% (v/v) whereas EDTA is used in 0.01 to 0.2% w/v. The surfactant is used in a proportion of 0.05 to 1.5 % v/v. The metal salts such as copper sulphate or cobalt chloride are added in 0.001 to 0.15 mM.
The fungal strain has been found to be a hyperactive laccase producer having an activity of 24,000 to 28,000 u/gds, which is probably

responsible for its fast depolymerization activity resulting in high product yield.
Non-pretreated Indian lignite has been used as substrate. The process does not require any pretreatment of the lignite. The lignite is taken in the medium and inoculated with the prepared innoculum. The pH of the medium is maintained between 3 to 8, preferably 6 to 7 and incubation is effected at a temperature between 25 to 40°C, for a period ranging from 7 to 30 days, preferably 15 to 28 days. The biomass and residual lignite is sepated by filtration and the humic acid is extracted. This process results in yield of 15 to 38% humic acid.
The process has been optimized for maximized solubilization and product yield.
The invention will now be explained in greater detail with the help of the following non-limiting example.
Example
Non-pretreated Indian lignite was taken in a medium with following composition.

methods. The media designed for this purpose is a novel one and cost-effective which may find its economically viable application in large scale.

WE CLAIM:
1. A process for the bioconversion of lignite to humic acid in submerged condition comprising the steps of providing lignite in a medium and inoculating with a prepared innoculum, comprising the fungal strain Pleurotus sp., incubating the medium for a predetermined period followed by separation of the biomass and residual lignite from the medium, followed by extraction of the humic acid from the medium.
2. The process as claimed in claim 1, wherein the medium comprises a basic medium with additives such as a carbon source, nitrogen source, chelator, surfactants, and metal ions.
3. The process as claimed in 2, wherein the basic medium comprises
Sodium nitrate (NaNO3) 1.5 to 2.5 gm/l
Potassium dihydrogen phosphate 0.75 to 1.5 gm/l
Magnesium sulphate (MgSO4) 0.5 gm/l
Potassium chloride (KC1) 0.5 gm/l
4. The process as claimed in claim 2, wherein the carbon source is a
sugar selected from sucrose, maltose etc, preferably sucrose.

5. The process as claimed in claim 1, wherein the nitrogen source is selected from ammonium bicarbonate, urea etc, preferably ammonium bicarbonate.
6. The process as claimed in claim 2, wherein the chelator is selected from triethanolamine, EDTA and salicylic acid, preferably triethanolamine.
7. The process as claimed in claim 2, wherein the surfactant is Triton-X, Tween-20, Tween-40, Tween-60 and SDS, preferably Triton-X.
8. The process as claimed in claim 2, wherein the metal ion is selected from copper salts and cobalt salts.
9. The process as claimed in claim 1, wherein the pH of the medium is maintained between 3 to 8, preferably 6 to 7.
lO.The process as claimed in claim 1, wherein the temperature of the medium is maintained in the range of 25 to 40°C.

11. The process as claimed in claim 1, wherein the incubation is effected for 7 to 30 days, preferably 15 to 28 days.