WO2016170492A1

WO2016170492A1 - Treatment of a coal component

Info

Publication number: WO2016170492A1
Application number: PCT/IB2016/052265
Authority: WO
Inventors: Ashton Keith COWAN; Gerald Oghenekume EDEKI; Lwazikazi MADIKIZA; Lerato Mary SEKHOHOLA; Michelle Louise ISAACS
Original assignee: Rhodes University
Priority date: 2015-04-22
Filing date: 2016-04-21
Publication date: 2016-10-27
Also published as: ZA201707229B

Abstract

A biological process for treating a coal component includes inoculating the 5 coal component with a bacterial component comprising at least one bacterial strain selected from the group consisting of the following bacterial strains deposited with the Microbial Culture Collection (MCC), Maharashtra, India on 21 October 2013 (KC620476), 4 August 2014 (KC700328 and KC620473), 20 October 2015 (KC620474, KC620478, KC620475), 25 February 2015 10 (KC758162, KC700329, and KC620477) and 8 April 2015 (KC700330). The coal component is allowed to biodegrade and to transform into a particulate material. The bacterial strain thus facilitates the biodegradation and transformation of the coal component.

Description

TREATMENT OF A COAL COMPONENT

THIS INVENTION relates to the treatment of a coal component. More particularly, it relates to a biological process for treating a coal component.

The conversion of coal into simpler products has been studied for many years. Various methods have been employed for the breakdown of coal into simpler products. Some of these methods include thermal and chemical processes which use high temperatures and pressures. It has been documented that the severity of the operating conditions may commonly include temperatures in excess of 427^QC. These processes also require significant capital investment. Biological processes have also been used in the breakdown of coal into simpler products and this method is generally more acceptable due to the fact that little or no temperature/pressure increase is required, it is cost effective and there is no alteration to the physical structure of the environment.

The solubilization of coal by microorganisms has been studied by various authors, with fungal biosolubilization taking the lead (Reiss, 1992; Hofrichter et al., 1997; Yuan et al., 2006; Yin et al., 2009 and 201 1 ). Experiments using fungal strains have shown that biosolubilized liquids can be produced when these strains are cultured on the surface of lignite coal in the presence of humid air (Cohen et al., 1982 and Yin et al., 2009). The liquids produced from the solubilized coal surfaces are highly polar, water soluble and of moderately high molecular weight having a high degree of aromaticity.

Spectroscopic evidence for the use of coal as a substrate by a bacterial strain of Pseudomonas isolated from a mixed enrichment culture growing in the presence of finely ground bituminous coal has been found (Fakoussa et al, 1983). In this publication, two factors were suggested as responsible for the solubilization of coal: the production of enzymes by the bacteria, and the formation of surface active agents. An inability to detect loss in mass of coal during and after this experiment became a major flaw. The solubilization of coal by bacteria was also subsequently detected (Strandberg et al, 1988). This publication suggested that the process of coal solubilisation by the bacterium Streptomyces setonii 75Vi2 was a non-enzymatic process.

An object of the present invention is thus to provide means whereby a coal component can effectively be biodegraded and transformed. This is provided by the process of the invention.

Thus, according to the invention, there is provided a biological process for treating a coal component, the process including

inoculating the coal component with a bacterial component comprising at least one bacterial strain selected from the group consisting of the following bacterial strains deposited with the Microbial Culture Collection (MCC), Maharashtra, India on 21 October 2013 (KC620476), 4 August 2014 (KC700328 and KC620473), 20 October 2015 (KC620474, KC620478, KC620475), 25 February 2015 (KC758162, KC700329, and KC620477) and 8 April 2015 (KC700330) under the accession numbers as given in Table A:

TABLE A

MCC Accession Number Genbank Accession Number

MCC 0034 KC620473

MCC 0021 KC620474

MCC 0027 KC620475

MCC 0016 KC620476

MCC 0042 KC620477

MCC 0022 KC620478

MCC 0039 KC758162

MCC 0033 KC700328

MCC 0041 KC700329

MCC 0062 KC700330 and

allowing the coal component to biodegrade and to transform into a particulate material, with the bacterial strain thus facilitating the biodegradation and transformation of the coal component.

The product, i.e. the particulate material, may in particular be a soil-like material which is high in humic (or a humic-like substance) and fulvic acid.

The bacterial component may comprise a consortium of two or more of the bacterial strains listed in Table A above. In other words, the bacterial component or inoculum may comprise a consortium of different bacterial strains selected from Table A. The bacterial strains may be enriched with a coal medium, e.g. a waste coal medium. More particularly, the bacterial strains may be as specified in Table B. They may be sourced from diesel contaminated soil or a coal slurry:

TABLE B

MCC Accession Genbank Closest Related Source

Numbers asseccion Bacterial

numbers strain/species

KC620473 Bacillus massiliensis Diesel

MCC 0034

contaminated soil

MCC 0021 KC620474 Seratia Diesel

nematodiphila contaminated soil

MCC 0027 KC620475 Proteus penneri Diesel

contaminated soil

MCC 0016 KC620476 Exiguobacterium Diesel

aurantiacum contaminated soil

MCC 0042 KC620477 Mycobacterium Diesel

aurm contaminated soil

MCC 0022 KC620478 Proteus mirabilis Diesel

contaminated soil MCC 0039 KC758162 Bacillus flexus Diesel

contaminated soil

MCC 0033 KC700328 Citrobacter freundii Coal slurry

MCC 0041 KC700329 Escherichia colli Coal slurry

MCC 0062 KC700330 Bacillus subtilis Coal slurry

Thus, the consortium may comprise one of the following combinations of bacterial strains/specie:

(I) KC620473+ KC620475

(ii) KC620473+ KC620474

(iii) KC620473+ KC620476

(iv) KC620473+ KC620477

(v) KC620473+ KC620474+ KC620475

(vi) KC620473+ KC620476+ KC620474

(vii) KC620473+ KC620475+ KC620476

(viii) KC620473+ KC620477+ KC620475

(ix) KC620475+ KC620476+ KC620477

(x) KC620474+ KC620476+ KC700329

(xi) KC620475+ KC620478+ KC700330

(xii) KC620473+ KC620475+ KC620478

(xiii) KC620474+ KC700328+ KC700329

(xiv) KC700328+ KC700329+ KC700330

(XV) KC620476+ KC620477+ KC700330

(xvi) KC620474+ KC620476+ KC620477

(xvii) KC620478+ KC700328+ KC700329

The process of the invention is characterized thereby that no acid (e.g. nitric acid) pretreatment of the coal component before inoculation thereof with the bacterial component, is required or effected.

More particularly, the process can thus involve obtaining the bacterial strain(s) from a liquid or solid medium containing a hydrocarbon component or a coal component, and then using the bacterial strain(s) to effect the inoculation of the same, or another, coal component, as hereinbefore described. Typically, the bacterial strain(s) are obtained from (i) a solid medium in the form of soil, i.e. soil contaminated with a hydrocarbon component such as diesel and/or (ii) a coal slurry.

The process thus then involves using a novel bacterial strain obtained from a hydrocarbon contaminated source or a coal source or, preferably, a consortium of two or more such novel bacterial strains to treat a coal site, thereby to biodegrade the coal component at the coal site.

Thus, according to a second aspect of the invention, there is provided a biological process for treating a coal component, which process includes

obtaining at least one bacterial strain from a liquid or solid medium containing a hydrocarbon component or a coal component;

working up the bacterial strain into a bacterial component comprising said at least one bacterial strain;

inoculating the same, or another, coal component with the bacterial component; and

allowing the coal component to biodegrade and to transform into a particulate material, with the bacterial strain facilitating the biodegradation of the hydrocarbon component.

The obtaining of the bacterial strain from the liquid or solid medium containing the hydrocarbon or coal component may include isolating the bacterial strain from the liquid or solid medium. In particular, the bacterial strain may be isolated from a solid medium in the form of soil contaminated with the hydrocarbon component and/or a coal slurry.

The working up of the bacterial strain to produce the bacterial component may comprise combining or formulating at least two bacterial strains obtained as hereinbefore described, into a consortium, with the bacterial component thus comprising a consortium of two or more of the bacterial strains. The bacterial strains may be as listed or specified hereinbefore in Tables A and B. The bacterial component may, in particular, comprise a consortium of two or more of the bacterial strains listed in Table A. The coal component may be as hereinbefore described.

The bacterial component may include a minimal mineral salts medium in combination with the bacterial strain or the consortium of bacterial strains. The minimal mineral salts medium may comprise one or more of K₂HPO₄, KH₂PO₄, NHCI₄, MgCI₂ and CaCI₂, and may be an aqueous medium. The aqueous minimal mineral salts medium may then typically comprise (K₂HPO₄ 1 .71 g, KH₂PO₄ 1 .32g, NHCI₄ 1 .26g, MgCI₂ 6H₂O 0.01 1 g, CaCI₂ 0.02g)/L.

The minimal mineral salts medium may be enriched with a trace mineral solution comprising one or more of Na₃C6H₅O7, MnSO₄, CoSO₄, CoCI₂, ZnSO₄, CuSO₄, AIK(SO₄)₂, H₃BO₄, Na₂MoO₄, NiCI₂, Na₂SeO₃, V³⁺CI, and Na₂WO₄. The trace mineral solution may then comprise (Na₃C6H₅O₇-2H₂O 2.1 g, MnSO₄-2H₂O 0.5g, CoSO₄ or CoCI₂-6H₂O 0.1 g, ZnSO₄-7H₂O 0.1 g, CuSO₄-5H₂O 0.01 g, AIK(SO₄)₂ 0.01 g, H₃BO₄ 0.01 g, Na₂MoO₄-2H₂O 0.1 g, NiCI₂-6H₂O 0.025g, Na₂SeO₃ 0.2g, V(III)CI 0.01 g, Na₂WO₄-2H₂O 0.0033g)/100ml.

The process may include adjusting the pH of the enriched minimal mineral salts medium to about 7, e.g. using an acid or base, as required..

The bacterial component may be in the form of a liquid suspension. The concentration of the bacterial consortium in the suspension may be in the range of 3.42-4.0 x 10⁹ cfu/ml, typically 3.62 x 10⁹ cfu/ml. The liquid suspension may be applied to the coal component at a rate of 400-500 L/Ha surface area of the coal component.

The bacterial component may be in solid form, and may be attached to or form part of inert carrier granules/pellets such as clay-based granules or pellets. The concentration of the bacterial consortium in these granules or pellets may be about 1 .5 mg/kg. The granules may be applied to the coal component at a rate of about 200-300 kg/Ha, preferably 250 kg/Ha surface area of the coal component. The coal component may comprise a coal-containing surface layer.

The coal component, when inoculated with the bacterial component, may be in combination with an inert particulate material. The inert particulate material may be soil, which is thus present in the surface layer together with coal or a coal derivative.

The coal-containing surface layer may comprise weathered coal, discard coal, roof coal or any other coal spoils. The process may include adding a neutralizing agent e.g. lime to the coal- containing surface layer, for pH control.

The process may include harvesting the particulate material, once sufficient degradation of the coal component has taken place.

The particulate soil-like material contains organic acids such as fulvic acid and/or organic matter that can readily be transformed into such organic acids such as humic, and the process may include processing the humic particulate material and recovering the organic acids and/or the organic matter.

The invention will now be described in more detail, with reference to the following non-limiting Example and the accompanying drawings.

In the drawings,

FIGURE 1 shows, for Example 1 , colour change of waste coal containing medium in the presence of bacterial consortia (top panel) and light micrographs indicating attachment of bacteria to coal particles (lower panel); FIGURE 2 shows, for Example 1 , FT-IR spectra of the freeze dried soluble fraction remaining after treatment of waste coal without (A) and with EBRU Culture 10 (B) or EBRU Culture 13 (C) bacterial consortia for 21 days;

FIGURE 3 shows, for Example 1 , FT-IR spectra of the freeze-dried insoluble fraction remaining after treatment of waste coal without (A) and with EBRU Culture 10 (B) or EBRU Culture 13 (C) bacterial consortia for 21 days;

FIGURE 4 shows, for Example 1 , the decline over time in mass (in grams) of substrate waste coal following exposure to selected bacterial consortia;

FIGURE 5 shows, for Example 1 , the decline over time in pH of waste coal media inoculated with selected bacterial consortia;

FIGURE 6 shows, for Example 1 , the kinetics of production of a humic- like substance from waste coal substrate by the action of selected bacterial consortia;

FIGURE 7 shows, for Example 1 , the kinetics of production of fulvic acid from humic acid by the action of selected bacterial consortia;

FIGURE 8 shows, for Example 3, growth and biomass accumulation by individual strains of the consortium ECCN 42b on discard coal;

FIGURE 9 shows, for Example 3, the decline in mass of substrate coal discard by individual strains and the consortium ECCN 42b comprising these strains;

FIGURE 10 shows, for Example 3, scaning electron microscopy illustrating colonization of coal discard by the individual strains (arrows: a=ECCN 19b; b=ECCN 21 b; c=ECCN 24b) of consortium ECCN 42b and by the consortium (rectangles: d); and

FIGURE 1 1 shows, for Example 3, by FT-IR the shift in and formation of new functional groups in residual coal discard material as a result of bacterial and bacterial consortia action for ECCN 42b. EXAMPLE

Methodology Bacterial strains

Bacteria were isolated from diesel contaminated soil in the Eastern Cape Province and from coal slurry in the Mpumalanga Province of South Africa. Pure colonies were isolated from bio prospected samples by inoculating coal slurry and diesel contaminated soils in nutrient broth followed by incubation for 48 h at 30 °C after which aliquots were inoculated onto nutrient agar and incubated for a further 24 h. Isolation of pure colonies was carried out after visible growth of the organisms. Ten bacterial strains were isolated and pure cultures of each, enriched with waste coal medium, were put in storage in 80% glycerol medium at -20^QC.

Preparation coal substrate

Low grade waste coal (also known as, and hence also herein referred to as, discard coal) material obtained from a waste coal dump in Mpumalanga Province, South Africa was oven dried at 50°C for 48 h and pulverized to a particle size of 0.2-0.5 mm in diameter. Sterilization of pulverized waste coal material was carried out by freeze-thawing (3 cycles) using liquid nitrogen.

Cultivation and incubation procedures

Mineral salt medium (MSM, containing K₂HPO₄ 1 .71 g/L, KH₂PO₄ 1 .32 g/L, NHCI₄ 1 .26 g/L, MgCI₂ 6H₂O 0.01 1 g/L, CaCI₂ 0.02 g /L) which was enriched with 4ml trace mineral solution (TMS containing Na3C6H₅O7-2H₂O 2.1 g, MnSO₄-2H₂O 0.5g, CoSO₄ or CoCI₂-6H₂O 0.1 g, ZnSO₄-7H₂O 0.1 g, CuSO₄-5H₂O 0.01 g, AIK(SO₄)₂ 0.01 g, H₃BO₄ 0.01 g, Na₂MoO₄-2H₂O 0.1 g, NiCI₂-6H₂O 0.025g, Na₂SeO₃ 0.2g, V(III)CI 0.01 g, Na₂WO₄-2H₂O 0.0033g)/100ml was prepared at pH 7.0 and 150 ml dispensed into 250 ml flasks and autoclaved at 121 °C for 15 min. After cooling to ambient, 1 g of sterilized pulverized waste coal was placed into each flask. 17 bacterial consortia were formulated from the 10 bacterial strains isolated. Each consortium was formed from centrifuged and thoroughly washed bacterial pellets by carefully resuspending each strain in an equal proportion in the ratio of either 1 :1 or 1 :1 :1 (v/v/v).

Each consortium was coded as EBRU Culture 1 , 2, 3, 4 to 17 and 1 ml of each consortium, in suspension, inoculated into MSM containing waste coal as the only carbon source. A set of un-inoculated samples served as negative controls. In addition, positive controls were made up of a consortium of the known strains Pseudomonas aeroginosa, Pseudomonas putida and Bacillus subtilis. All experiments were continuous and carried out at 30 °C for 21 days. Samples were abstracted on days 0, 2, 4, 7, 14, 19 and 21 for analysis of coal solubilization, FT-IR spectroscopy, pH, and determination of mass of residual coal. Each experiment was set up using a complete random block design and replicated three times. Analytical methods

Fourier transform infrared (FT-IR) spectroscopy

Fourier transform infrared spectroscopy (FT-IR) was used to monitor for modifications to the coal molecular structure after exposure of this material to microbial action. The biomass-bound coal and residual supernatant remaining after incubation were prepared and freeze dried and the dried material examined using a PerkinElmer universal ATR sampling accessory, spectrum 100, FT-IR spectrometer in the range of 4000-700 cm^-1. Measurement of waste coal solubilisation

The production of humic and fulvic acids from waste coal substrate was monitored by extracting using the well documented alkaline extraction method (Igbinigie et at, 2008; Novak et al., 2001 ; Velthorst et al., 1999). Typical solubility parameter curves were obtained when the soluble fraction was separated and the absorbance determined for both humic and fulvic acids at 450 and 370 nm respectively. pH analysis

A WTW 330 pH meter which was calibrated at pH 7 and 10 was used to determine pH. Mass determination of coal

Residual substrate of coal remaining after microbial action was purified by carefully centrifuging at 4000 x g for 20 min. Coal pellets were rinsed with sterile milliQ water and re-centrifuged at 1000 x g for 10 min. Resultant coal pellets were freeze dried and the mass of the dried coal recorded. This method was adopted for all experiments and at the conclusion of each experiment percentage decrease in weight of coal was calculated.

Results Bacterial identification and molecular characterization

Pure bacteria cultures were plated on agar medium and extraction of the DNAs was carried out which followed the method of Head et al., 1998. Molecular characterization of samples followed the method of Sambrook et a/., 1989. PGR analysis was carried out followed by cloning and sequencing (Muyzer et al., 1993 and Santegoeds et al, 1998). PCR products were sent to Inqaba Biotec for Sanger sequencing which were converted into text format using Chromas and then put into the NCBI BLAST database (Bond ei al., 2002). The sequences were submitted to GenBank using BankIT and assigned accession numbers which will be release in 2014.

Table 1 presents the accession number of each strain, the identity of the organism, and the source. Table 1. Identification of isolated bacteria and Genbank accession numbers

Genbank accession Closest Related Bacterial Source

numbers strain/species

KC620473 Bacillus massiliensis Diesel contaminated soil

KC620474 Seratia nematodiphila Diesel contaminated soil

KC620475 Proteus penneri Diesel contaminated soil

KC620476 Exiguobacterium Diesel contaminated soil aurantiacum

KC620477 Mycobacterium aurum Diesel contaminated soil

KC620478 Proteus mirabilis Diesel contaminated soil

KC758162 Bacillus flexus Diesel contaminated soil

KC700328 Citrobacter freundii Coal slurry

KC700329 Escherichia coli Coal slurry

KC700330 Bacillus subtilis Coal slurry

Table 2 shows the different bacterial consortia formulated and the codes assigned to each consortium.

Table 2. Bacterial consortia composition and the codes assigned to each consortium

Composition of Consortia Codes assigned Alternative to consortia Codes

(i) KC620473+ KC620475 EBRU Culture 1 ECCN 1 b

(ii) KC620473+ KC620474 EBPiU Culture 2 ECCN 2b

(Hi) KC620473+ KC620476 EBRU Culture 3 ECCN 3b

(iv) KC620473+ KC620477 EBRU Culture 4 ECCN 4b

(v) KC620473+ KC620474+ KC620475 EBRU Culture 5 ECCN 5b

(vi) KC620473+ KC620476+ KC620474 EBRU Culture 6 ECCN 6b

(vii) KC620473+ KC620475+ KC620476 EBRU Culture 7 ECCN 7b

(viii) KC620473+ KC620477+ KC620475 EBRU Culture 8 ECCN 8b

(ix) KC620475+ KC620476+ KC620477 EBRU Culture 9 ECCN 9b

(x) KC620474+ KC620476+ KC700329 EBRU Culture 10 ECCN 10b (xi) KC620475+ KC620478+ KC700330 EBRU Culture 11 ECCN 1 1 b

(xii) KC620473+ KC620475+ KC620478 EBRU Culture 12 ECCN 12b

(xiii) KC620474+ KC700328+ KC700329 EBRU Culture 13 ECCN 13b xiv) KC700328+ KC700329+ KC700330 EBRU Culture 14 ECCN 14b

(xv) KC620476+ KC620477+ KC700330 EBRU Culture 15 ECCN 15b

(xvi) KC620474+ KC620476+ KC620477 EBRU Culture 16 ECCN 16b

(xvii) KC620478+ KC700328+ KC700329 EBRU Culture 17 ECCN 17b

In the alternative codes, ECCN is derived from EBRU Culture Collection Number, and 'b' indicates that the culture is in bacterial form. When various bacterial consortia prepared as shown in Table 2 were provided with a substrate of waste coal, a colour change of the medium was typically observed. By way of example, EBRU Culture 2 and EBRU Culture 13 rendered the coal enriched medium nearly colourless within 14 d (see Figure 1 top panel). Also, microscopic analysis at ^χ 40 magnification revealed the close association of these microorganisms with the waste coal substrate particles indicative of attachment (see Figure 1 lower panel).

Figure 2 shows the shift in and formation of new functional groups in the soluble fraction of residual waste coal material as a result of bacterial consortia action. Decarboxylation which is the enzymatic and/or chemical removal of carboxyl groups and the release of carbon dioxide from the system had clearly occurred.

Figure 3 shows that hydroxylation and methylation were the major reactions that had occurred in the insoluble fraction of residual waste coal material as a result of bacterial consortia action. A total disappearance of ethers, esters, carboxylic acids and anhydrides was observed for waste coal treated with bacterial consortium EBRU Culture 10 (see Fig 3 B, region of 1400- 1200cm^"1).

Figure 4 shows the decline in mass of substrate waste coal over time due to the action of selected bacterial consortia. Greatest loss in mass of substrate waste coal was achieved with EBRU Culture 10 and EBRU Culture 13 and the bulk of the loss in weight occurred between days 7 and 14.

Figure 5 illustrates the decline in pH over time of a waste coal containing medium inoculated with selected bacterial consortia. Acidification is the buildup of hydrogen ions, also called protons, reducing the medium pH which occurs when a proton donor is added to the medium. The production of small organic acids and/or proton donors to the medium occurred by bacterial action on waste coal substrate as illustrated by FT-IR analysis of both the soluble and insoluble fractions remaining after inoculation and incubation (see Figures 2 and 3).

Humic-like substance is regarded as a major product of the biological breakdown of low ranked coals such as lignite (Sekhohola et al., 2013). Figure 6 shows the kinetics of accumulation of humic-like substance in the soluble fraction of waste coal containing medium inoculated with selected bacterial consortia. Humic-like substance was separated from fulvic acid using the alkaline extraction method (Igbinigie et at, 2008; Novak et al., 2001 ; Velthorst et al., 1999) and quantified (mg/L) spectrophotometrically at A=450 nm by interpolation from a standard curve prepared using an authentic humic acid standard.

Figure 7 shows the kinetics of accumulation of fulvic acid in the soluble fraction of a humic acid containing medium inoculated with selected bacterial consortia. Fulvic acid was separated from humic acid using the alkaline extraction method (Igbinigie et at, 2008; Novak et al., 2001 ; Velthorst et al., 1999) and quantified (mg/L) spectrophotometrically at A=370 nm by interpolation from a standard curve prepared using an authentic fulvic acid standard. Humic acid (HA) which is found in low grade coals such as lignite (Janos, 2003; del Rio et al., 1994: Lobartini et al., 1992), is the organic fraction which is soluble in alkaline media (i.e in 0.1 M NaOH/100ml) and insoluble in acidic media (at pH 1 -2) with average molecular weight of 2,000 to 3,000 Da (Zeng et al., 2002). HA was precipitated from liquid extracts in flasks by adjusting the pH of liquid extracts to <1 with 32% HCL. Flasks were left on a rotary shaker for 24hrs at ambient temperature which were later centrifuged at 4000 x g for 90min at 10 °C. Pellets were separated from supernatants with the resultant pellets taken as the humic acid fraction. 0.1 M NaOH was added to supernatants and allowed to stand for 1 hr before they were centrifuged at 4000 x g for 90min. Pellets were separated from supernatants and the supernatants were taken as the fulvic acid fraction.

EXAMPLE 2

Similar tests to those conducted in Example 1 , were conducted on individual bacterial strains as listed in Table 3:

Table 3. Individual bacterial strains and the codes assigned to each

Similar results to those obtained with the bacterial consortia of Example 1 , were obtained, albeit that the individual strains of Table 3 were not as active as the consortia of Table 2.

EXAMPLE 3

Similar tests to those conducted in Example 1 , were conducted in respect of consortia as specified in Table 4: _Λ„

Table 4. Bacterial consortia composition and the codes assigned to each consortium

It was found that the consortia of Table 4 gave similar, and in most cases, improved results, particularly as regards the colonisation of the surface of coal discard, bacterial growth and biomass accumulation with coal discard as the sole carbon source, and with sustained biodegradation of the substrate and accumulation of humic-like material, as compared to the consortia of Table 2. By way of example, data obtained for ECCN 42b is given in Figures 8 to 1 1 .

In one embodiment of a practical implementation of the process of the invention, an overnight bacterial consortium, i.e. one of the bacterial strains/consortia listed in Tables 2, 3 and 4, is prepared in a medium made of simple sugars such as glucose. Cell biomass is harvested and pelletized in combination with nutrients which may be in the form of fertilizers containing nitrogen and phosphorus. Crushing of heavy coal particles and disking of the upper layer of the coal profile may be carried out for easy access of the bacteria and for oxidation process to take place. Neutralizing agents such as lime for pH control may be added to the coal layer i.e. to the upper 200mm of the coal containing layer. Neutralizing agents may be added at a rate of 20 tons/Ha to 40 tons/Ha, preferably 30 tons/Ha. Inoculation of pelletized bacteria in consortium at a rate of 200 kg/Ha to 300 kg/Ha, preferably 250 kg/Ha may be carried out on the disked coal profile within 100 cm to 200 cm deep, preferably 150 cm deep. Constant irrigation of the coal body may be carried out for speedy oxidation process. Once initial breakdown of coal has taken place due to bacterial conversion into humic-like substances, extraction of humic acids may be carried out on the oxidized coal. The coal extracted humic acid may be used as a soil conditioner to enhance the growth of plants.

In another embodiment of a practical implementation of the invention, one of the bacterial strains/consortia listed in Tables 2, 3 and 4, may be inoculated into a liquid medium containing coal in a batch reactor. Incubation time may be in the range of 30-40 days, preferably, 35 days. Once the initial breakdown cycle is completed, the coal medium containing bacteria consortium and minerals may be applied to a body of coal layer which has already been disked and neutralised by liming. Irrigation of the coal layer may be required to facilitate the speedy conversion/breakdown of coal to value added products such as humic-like substances. While the coal containing medium is used as an inoculum in treating waste coal dumps, it may also be applied in soils low in humus to act as an organic fertilizer for plant growth enhancement due to the high levels of humic-like substances contained in it.

The Applicant has surprisingly found that by means of the process of the invention, a pretreatment step in which the coal component is treated with an acid, such as nitric acid, is not required in order to obtain satisfactory biological remediation of the coal component; furthermore, it has not hitherto been known to use any of the bacterial strains listed in Tables A and B, or any of the consortia listed in Tables 2, 3 and 4, to degrade a coal component. It was also surprisingly found that coal degradation could be effected by bioprospecting organisms, particularly the bacterial strains of Tables A and B or the strains/consortia listed in Tables 2, 3 and 4, from a waste or discard coal site and forming consortia using organisms that degrade both coal and hydrocarbons. Also, it was found that at least some coal degradation can already be observed as soon as 3 days after inoculation of the coal component with the organism or consortia, as evidenced from loss of coal structure and associated colour change (due to depolymerization). References

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Claims

1 . A biological process for treating a coal component, the process including

TABLE A

and

2. The process according to Claim 1 , wherein the bacterial component comprises a consortium of two or more of the bacterial strains listed in Table A.

3. The process according to Claim 2, wherein the bacterial strains are enriched with a coal medium.

4. The process according to Claim 2 or Claim 3, wherein the bacterial strains are sourced from diesel contaminated soil or a coal slurry.

5. The process according to any one of Claims 2 to 4 inclusive, wherein the consortium comprises one of the following combinations of bacterial strains/specie:

(I) KC620473+ KC620475

(ii) KC620473+ KC620474

(iii) KC620473+ KC620476

(iv) KC620473+ KC620477

(v) KC620473+ KC620474+ KC620475

(vi) KC620473+ KC620476+ KC620474

(vii) KC620473+ KC620475+ KC620476

(viii) KC620473+ KC620477+ KC620475

(ix) KC620475+ KC620476+ KC620477

(x) KC620474+ KC620476+ KC700329

(xi) KC620475+ KC620478+ KC700330

(xii) KC620473+ KC620475+ KC620478

(xiii) KC620474+ KC700328+ KC700329

(xiv) KC700328+ KC700329+ KC700330

(xv) KC620476+ KC620477+ KC700330

(xvi) KC620474+ KC620476+ KC620477

(xvii) KC620478+ KC700328+ KC700329

6. The process according to any one of Claims 1 to 5 inclusive, which is characterized thereby that no acid pretreatment of the coal component before inoculation thereof with the bacterial component, is effected.

7. A biological process for treating a coal component, which process includes

8. The process according to Claim 7, wherein the obtaining of the bacterial strain from the liquid or solid medium containing the hydrocarbon or coal component includes isolating the bacterial strain from the liquid or solid medium.

9. The process according to Claim 8, wherein the bacterial strain is isolated from the solid medium, with the solid medium being in the form of soil contaminated with the hydrocarbon component and/or a coal slurry.

10. The process according to any one of Claims 7 to 9 inclusive, wherein the bacterial strain is selected from the group consisting of the following bacterial strains deposited with the Microbial Culture Collection (MCC), Maharashtra, India on 21 October 2013 (KC620476), 4 August 2014 (KC700328 and KC620473), 20 October 2015 (KC620474, KC620478, KC620475), 25 February 2015 (KC758162, KC700329, and KC620477) and 8 April 2015 (KC700330) under the accession numbers as listed in Table A: TABLE A

MCC Accession Number Genbank Accession Number

MCC 0034 KC620473

MCC 0021 KC620474

MCC 0027 KC620475 MCC 0016 KC620476

MCC 0042 KC620477

MCC 0022 KC620478

MCC 0039 KC758162

MCC 0033 KC700328

MCC 0041 KC700329

MCC 0062 KC700330

1 1 . The process according to Claim 10, wherein the bacterial component comprises a consortium of two or more of the bacterial stains listed in Table A.

12. The process according to any one of Claims 7 to 1 1 inclusive, wherein the bacterial component includes a minimal mineral salts medium in combination with the bacterial strain(s).

13. The process according to Claim 12, wherein the minimal mineral salts medium is enriched with a trace mineral solution comprising one or more of Na₃C₆H₅O7, MnSO₄, CoSO₄, CoCI₂, ZnSO₄, CuSO₄, AIK(SO₄)₂, H₃BO₄, Na₂MoO₄, NiCI₂, Na₂SeO₃, V³⁺CI, and Na₂WO₄.

14. The process according to any one of Claims 7 to 13 inclusive, wherein the bacterial component is in the form of a liquid suspension.

15. The process according to any one of Claims 7 to 13 inclusive, wherein the bacterial component is in solid granulated/pellet form, with the granules/pellets also comprising an inert carrier.

16. The process according to Claim 15, wherein the granules/pellets are applied to the coal component at a rate of about 200-300 kg/Ha.

17. The process according to any one of Claims 1 to 16 inclusive, wherein the coal component comprises a coal-containing surface layer.

18. The process according to any one of Claims 1 to 17, wherein the coal component, when inoculated with the bacterial component, is in combination with an inert particulate material.