Demineralization of Gondwana coal with Pseudomonas mendocina strain B6-1: a case study of coal from Gopinathpur top and bottom seams of Mugma mine, Dhanbad, Jharkhand (India)

In the present investigation an attempt has been made to demineralize the Gondwana coal of Gopinathpur top and bottom seams of Mugma mine, Raniganj coalfield, Dhanbad with the help of Pseudomonas mendocina strain B6-1. The change in the amount of ash yield and decrease in the concentration of selected minor elements like Na, K, Mn and Ca and environmentally sensitive selected trace elements such as Cd, Pb, Se, Ni, Mn, and Zn have been studied as a function of time of bacterial treatment as well as with variation in the bacterial biomass. After 28 days of bacterial treatment there was variable amount of decrease observed in ash yield as well as in the concentration of minor and trace elements. The removal of the elements was further enhanced with the increase in the bacterial biomass from 10 to 25 mg/mL. Due to over exploitation of superior grade coals in the country, the remaining coal resources, available for current use, are inferior in grade and contain high level of impurities and there is ample scope of bio-beneficiation of these coals using bacterial biomass.


Introduction
Coal is an important source of energy and is mainly used to generate electricity in the thermal power plants. Gondwana coals of India contribute more than 99 % of total coal resource of the country and are with medium to high ash yield. Due to over exploitation of superior grade coals, the remaining resources are inferior in grade having high level of impurities. Moreover, the Gondwana coals being of drift origin contain relatively high inorganic matter. These inorganic matters get associated with coal during various stages and have different modes of occurrences such as (a) dissolved salts and other inorganic substances in the pore water of coal, (b) inorganic elements which are incorporated within the organic compounds of macerals, and (c) crystalline or non-crystalline discrete inorganic particles representing actual mineral components. During mining activities the minerals derived from the non-coal bands from intra-seam further add to the impurities. Removal of those inorganic matters which are chemically bonded with the organic matter is difficult through physical cleaning and has become a matter of concern for the scientists. Besides, there are several elements which occur in association of organic and inorganic constituents as major ([1 wt%), minor (1-0.1 wt%) or trace (\0.1 wt%) elements (Vassilev and Vassileva 1997). The elements like As, Be, Cd, Cr, Cu, Pb, Mn, Hg, Mo, Ni, Sr, U, V, and Zn are considered to be environmentally sensitive (Pickhardt 1989). Significant contributions on the geochemistry of trace elements have been made by several scientists (Finkelman 1995;Ward et al. 2003;Ren et al. 2004;Seredin 2004;Eskenazy 2009;Zhao et al. 2014;Singh et al. 2015a, b). Geochemistry of trace elements of Indian coals has been studied by Singh et al. (1983Singh et al. ( , 2015a and Prachiti et al. (2011). Since studies on the demineralization of coal using bacterial biomass have not been carried out in details, there is much scope of such investigations. Contributions on limited numbers of coal samples have been made by Singh et al. (2012Singh et al. ( , 2014aSingh et al. ( , b, 2015c.
In the present investigation an attempt has been made to demineralize the Gondwana coal of Gopinathpur seam from Raniganj coalfield with the help of Pseudomonas mendocina strain B6-1. The change in the amount of the ash yield and change in the concentration of environmentally sensitive selected trace elements have been studied as a function of time of bacterial treatment as well as with variation in the bacterial biomass.

Methods of study
The coal samples for present investigation were collected from the Gopinathpur top and bottom seams of Mugma mine, Nirsa block. These Gondwana coals belong to Barakar Formation of Raniganj coalfield, Dhanbad district, Jharkhand. Two coal samples from Gopinathpur bottom seam (GB-1and GB-2) and one sample from Gopinathpur top seam (GT-1) have been subjected to bacterial treatment and various analyses.

Coal petrography
The maceral analysis has been carried out under Leitz Orthoplan Pol Microscope in Coal & Organic Petrology Lab, Banaras Hindu University. Oil recommended by Leitz (DIN 58889) has been used for the microscopy. The lineto-line and point-to-point spacings were maintained at 0.4 mm and more than 500 counts were taken on each sample. The methodology and recommendations given by Taylor et al. (1998) andICCP (2001) were followed.

Proximate analysis
The proximate analysis has been carried out as per ISI specifications (BIS 2003) to determine the contents of moisture, ash, volatile matter and fixed carbon. The analysis was carried out in Coal & Organic Petrology Lab, Banaras Hindu University.

Minor and trace element analysis
One gram (dry weight) of Gopinathpur seam coal sample was transferred in a digestion vessel and 10 mL of digestion mixture (10 part conc. HNO 3 and 1 part HClO 4 ) was added to it. The mixture was refluxed for 30 min and the step was repeated thrice so that no brown fumes were given off by the sample. It was filtered using Whatman (No. 41) filter paper. The digested samples were rinsed with 1 % Conc. HNO 3 and transferred in a separate test tube. The volume was made up to 20 mL. Concentrations of Cd, Pb, Se, Ni, Mn, and Zn elements were determined using Perkin-Elmer Inductively Coupled Plasma optical emission spectrometer (ICP-OES), model Optima 7000 DV, in the School of Life Sciences, Banaras Hindu University. Concentrations of other elements like Na, K and Ca were determined using Systronics Flame Photometer 128 in the Department of Botany, Banaras Hindu University.

Isolation and identification of bacteria
The bacteria used in the present investigation were isolated from Rajmahal coals and the detailed process has been discussed by Singh et al. (2014a, b). Identification of bacteria was carried out at Indian Institute of Vegetable Research (IIVR), Varanasi (Singh et al. 2014a, b). The bacterium was identified as P. mendocina strain B6-1.

Mass cultivation of Pseudomonas mendocina strain B6-1 bacteria
The isolated bacterial colonies have been grown in a growth medium and the growth of the colonies was measured individually at 660 nm with the help of spectrophotometer. The colony with maximum growth has been picked up, purified and transferred into liquid basal salt medium for mass cultivation. The medium had a composition of: glycerol 0.2 %, KH 2 PO 4 0.244 %, NaHPO 4 0.577 %, NH 4 Cl 0.2 % and MgCl 2 0.02 %.

Immobilization of Pseudomonas mendocina strain B6-1 bacteria
The exponential phase bacterial cells have been collected through centrifugation at 10000 rpm for 10 min and repeated washing. The final pellet was collected for further use. Mixture of 5 % sodium alginate solution was poured into broth of basal salt medium and was sterilized. Bacterial beads were prepared by adding 10 mg/mL of bacterial biomass into sodium alginate solution and this mixture was pumped drop wise into 0.2 M CaCl 2 solution in sterilized condition under laminar flow. The bacterial beads thus prepared were harvested and resuspended in 100 mL growth medium in elementary flask of 250 mL. The same method was adopted for the preparation of bacterial beads using 25 mg/mL biomass.

Method of treatment
Demineralization of coal samples of Gopinathpur seam was carried out with P. mendocina strian B6-1. These coal samples have been crushed to 70 mesh size. Two sets of coal samples (about 12 g) were taken (one for control and the other for treatment) in 50 mL basal salt medium and the pH was adjusted at five which was maintained with the help of 1 N HCl. The experiment was performed at room temperature in Erlenmeyer flasks of 100 mL, with volume of 50 mL basal salt medium. Each coal sample was inoculated with 100 number of bacterial beads of P. mendocina strian B6-1 and all the treated samples had a respective control (Blank), i.e. an assay with the same operation conditions but without inoculation. The experiments have been performed with coal samples of Gopinathpur seam of Mugma mine. The treated samples of every 2nd, 4th, 8th, 12th, 16th, 20th, 24th, and 28th day were taken out for the analysis. The pH and oxidation reduction potential (ORP) were monitored throughout the experiment. The solid fraction obtained after filtering it with Whatman filter paper was dried at room temperature and subjected to proximate analysis.

Results and discussion
3.1 Petro-chemical attributes   Nd not determined a World average (After Valković 1983) furnished in Table 1 while the graphical representation of the group maceral and mineral matter is provided in Fig. 1

Distribution of minor and trace elements
Gopinathpur seam coals have high concentrations of K (1189.5-5573 ppm; av 2650.7 ppm) and Na (4534-5618 ppm; av 5314.7 ppm) among the minor elements as compared to the world average in coals as given by Valkovic (1983). Similarly Se (6.8-9.0 ppm; 7.9 ppm) and Pb (1.8-13.2 ppm; av 9.3 ppm) are high in concentrations in these coals when compared with the Clarke values of the elements in Bituminous coals as given by Ketries and Yudovich (2009). Other elements analysed in these coals such as Ni (8.2-32.0 ppm; 19.6 ppm), Zn (9-31 ppm; av 17.8 ppm) and Mn (19.1-24 ppm; av 21.5 ppm) occur within the limits. The details of the concentrations of the analyzed minor and trace elements are furnished in Table 2.

Demineralization using Pseudomonas mendocina strain B6-1
Metal removal from coal by bacteria occurs through leaching, adsorption and accumulation. Leaching mechanisms of metals through bacteria takes place in various ways such as, (i) the formation of organic or inorganic acids (protons); (ii) oxidation and reduction reactions; and (iii) the excretion of complexing agents (Berthelin 1983). The chief inorganic acid in leaching environments is sulphuric acid which is formed by sulfur-oxidizing microorganisms. However, a series of organic acids are produced by bacterial metabolism which leads to organic acidolysis, complex and chelate formation and facilitates metal removal (Berthelin 1983). After liberation of metals from coal or organic matter through microbial leaching, the processes like adsorption and  Subsequently, the cations are removed. Metal accumulation, in bacteria, refers to translocation of the cations which is a metabolic dependent process and takes place across the wall and membrane into the cell. There is specific transport system for metal ions which involves channels and pores of cell membrane. Polyphosphate body in bacteria works as metal sink (Gadd 2009;Kornberg 1995).
In the present investigation three coal samples, two from Gopinathpur bottom seam (GB-1 and GB-2) and one sample from Gopinathpur top seam (GT-1), have been subjected to demineralization to see decrease in the content Table 4 Variation in the ash yield and ash reduction of coal samples of Gopinathpur top seam (GT-1) analyzed before and after bacteria treatment (with 10 and 25 mg biomass of bacteria)

Day
Ash yield (wt%) (GT-1) treated with 10 mg/mL biomass Ash reduction (%) (GT-1) by 10 mg/mL biomass   IC initial concentration of ash yield and the concentrations of minor/trace elements as a function of time (up to 28 days) and change in the concentration of bacterial biomass (10 and 25 mg/mL) comprising of P. mendocina strain B6-1.

Decrease in ash yield as a function of time
Coal samples (GB-1 and GB-2) from Gopinathpur bottom seam have been treated with 10 mg/mL bacterial biomass of P. mendocina B6-1 to see the decrease in the ash yield with time. In case of GB-1 sample there was a gradual decrease in the ash yield and after 28 days of bacterial treatment there was a reduction in the ash yield up to 3.12 %. However, in GB-2 coal sample there was relatively more reduction (up to 7.31 %) in the final ash yield. The details of the periodic reduction in the ash yield are furnished in Table 3 and are graphically shown in Fig. 3. 3.3.2 Decrease in ash yield as a function of change in concentration of bacterial biomass In this case one coal sample from Gopinathpur top seam (GT-1) was treated with 10 and 25 mg/mL of P. mendocina strain B6-1. In this sample a substantial decrease in the ash yield was noticed. After 28 days of bacterial treatment there was 33.99 % decrease in the ash when 10 mg/mL of biomass was used while 37.44 % reduction in the ash yield was recorded when 25 mg/mL of biomass was used. The details of the periodic ash reduction are furnished in Table 4 and are graphically shown in Fig. 4.

Removal of minor and trace elements as a function of time
In case of GB-1 coal sample variable removal (%) of minor elements has been recorded after 28 days of bacterial treatment with 10 mg/mL of biomass.  Tables 5 and 6, respectively. In case of GB-2 coal sample moderately high to very high removal of most of the minor and trace elements has been achieved after 28 days of bacterial treatment. Among the minor elements K, Ca, Mn and Na have given good response to bacterial treatment and removal was 14.27 %, 33.86 %, 53.38 % and 68.51 %, respectively while the trace elements like Pb, Cd, Zn, Ni and Se have been removed to a larger extent to the tune of 24.44 %, 33.33 %, 55.41 %, 62.08 % and 96.34 %, respectively. The details of decrease and percent removal are provided in Tables 7 and 8, respectively. The comparative removal of minor and trace elements in GB-1 and GB-2 coal samples of Gopinathpur bottom seam after 28 days of bacterial treatment using 10 mg/mL of biomass is shown in Fig. 5.

Removal of minor and trace elements as a function of change in the concentration of biomass
Coal sample (GT-1) from Gopinathpur top seam has been treated with P. mendocina B6-1 having 10 and 25 mg/mL Fig. 5 Comparative removal (%) of minor and trace elements in GB-1 and GB-2 coal samples, after 28 days of treatment with P. mendocina strain B6-1 using 10 mg/mL biomass    (4) Coal sample (GT-1) from Gopinathpur top seam was treated with 10 and 25 mg/mL of biomass (P. mendocina strain B6-1) which indicated a substantial decrease in the ash yield up to 33.99 % and 37.44 %, respectively after 28 days. Thus bacteria may be used as an important tool for the beneficiation of coals which are rich in impurities and environmentally sensitive elements. Nevertheless, there is need of more research work in this field to scale-up this experiment on Pilot and commercial levels.