MICROORGANISMS INVOLVED IN THE DESULPHURIZATION PROCESS OF COAL FROM TURCENI AND PAROSENI MINES
Carmen Mădălina Cişmaşiu, Gabriela Popescu, Lucia Dumitru, Corina Văcăroiu Roxana Cojoc and Simona Merciu
Romanian Academy, Institute of Biology, Center of Microbiology, Bucharest, Romania
Mona Monea, Adriana Botez, Cătălina Militaru
Research and Development National Institute for Metals and Radioactive Resources, Romania
ABSTRACT
Pollution from coal combustion is the largest problem in the current use of coal and the biggest constraint on its increased usage. When these fossil fuels are combusted, sulphur-bi-oxide is released into the atmosphere causing acid rains which destroyed buildings, kills forests and poisons aquatic organisms. The sulfur found in coal is either part of the molecular coal structure, is contained in minerals such as pyrite (FeS2) or occurs in minor quantities in the form of sulfate and elemental sulfur.
Biodesulphurization is a process that involves heterotrophic and chemolithotrophic microorganisms that would remove sulphur from fuels without degrading the fuel value of the product.
The results revealed that the higher number of heterotrophic neutrophilic bacteria was obtained from raw coal comparatively with non-magnetic and mixed samples. The acid production activity by heterotrophic bacteria increased with cellular growth, the lowest pH values being obtained after 96 hours of incubation at 280C.
The adapting of the P7 population to higher concentrations of metal ions determined a raised efficiency of biodesulphurization coal at values between 78.31-86.64%, compared to those (64.51-74.67%) obtained when using P9 population.
Raising the solid/liquid ratio from 5g/100 ml to 10g/100 ml determined the increasing of the bacterial oxidizing efficiency of the coal, which was evidenced through the desulphurization of lower percentages, which get to 58.34-78.31 for the pitcoal and 61.02-86.64% for lignite.
Key words: coal, biodesulfurization, Thiobacillus ferrooxidans
1. INTRODUCTION
Use of fossil fuels for electrical energy generation in the petrochemical industry is expected to increase in the next decade. The demand for low-sulfur fossil fuel has been intensified by increasingly regulatory standards for reduced levels of sulfur atmospheric emissions. The primary objection to the combustion of high-sulfur fuels is the generation of sulfur oxides which may play a major role in the formation of acid deposition with detrimental effects on medium. These considerations have led to renewed interest in the basic mechanisms of microbial sulfur transformations and the technology for microbial precombustion desulphurization of fossil fuels.
Most of the sulfur in coal occurs in two forms: inorganic and organic complexes. Significant elemental sulfur is rarely found. Inorganic sulfur occurs primarily as iron pyrite or marcasite, which have the chemical formula FeS2 but have different crystalline structures. Small amounts of lead, zinc, and copper sulfides are often observed, as well as some sulfate (mainly as gypsum (CaSO4.2H2O). When pyrite crystals are finely distributed within the coal matrix, mechanical cleaning can only remove part of the pyrite.
Generally, pyritic sulfur equals or exceeds organic sulfur in coal. Organic sulfur exists covalently bound in the complex coal matrix. Structures in the matrix include thiols, mercaptans, sulfide and disulfide linkages and complex thiophene moieties.
The role of bacteria in oxidation of insoluble iron sulfide (pyrite) and other metal sulfides to soluble ions is well established although the mechanism of pyrite solubilisation is still not completely resolved. Pyrite solubilisation has been most studied in the gram-negative chemolithotrophic Thiobacillus ferrooxidans, which utilizes either ferrous iron or reduced inorganic sulfur compounds as a sole energy source. The reactions involved in pyrite solubilization have been characterized as direct or indirect mechanisms for oxidation of the substrate by the bacteria. The direct mechanism may require the physical attachment of the bacteria to pyrite particles, resulting in the localized oxidation of pyretic sulfide to sulfate, and ferrous iron to ferric.
Alternately, solubilization of the substrate through dissociation may be required prior to oxidation by the microorganisms enzymatic systems. These reactions generate ferric iron, a strong oxidizing agent, which participates in the nonbiological (indirect) oxidation of pyrite, generating ferrous sulfate and elemental sulfur. Thus, in the indirect mechanism, the role of the bacteria is to deoxidize ferrous to ferric iron.
Thiobacillus species have been found tightly associated with pyrite crystals and appears that both the direct and indirect mechanisms operate in nature. Chang and Myerson (1982) have presented data supporting the idea of preferential attachment of bacteria on pyrite crystals to specific sites. Many factors, including the kinds and quantities of bacteria present and the nature of the pyrite in the coal, significantly influence the final reaction yields.
Successful desulfurization required attention to biological parameters (such as choice of microorganisms and nutrient supplements), control of the physical environment (pH, redox potential and temperature) and consideration of the coal condition and its source.
Thiobacillus ferrooxidans is the iron pyrite-oxidizing bacterial species most thoroughly studied in pure culture. T. ferrooxidans flourishes in low-pH environments and is chemoautotrophic, capable of synthesizing cellular material exclusively from inorganic substrates and obtaining energy via the oxidation of ferrous iron and sulfide. T. ferrooxidans cultures accumulate metabolic by-products that inhibit further growth and pyrite oxidation.
The best results of coal desulfurization have been obtained with mixed cultures. Oxidations using T. thiooxidans in conjunction with T. ferrooxidans have achieved remarkable rates of desulfurization. The presence of other species in the desulfurizing culture has proven useful in increasing the extent and rate of coal desulfurization. These species seem able to metabolize the inhibitory metabolites that accumulate during the growth T. ferrooxidans, while tolerating the hostile growth conditions. Some of these bacteria (including also heterotrophic bacteria) may also metabolize simple organic sulfur moieties, thus degrading a small percentage of the organic sulfur remaining in coal matrix. Generally, cell concentrations in the 1011-1013 cells/gram pyrite range have proven optimal for desulfurization, although higher concentrations may be beneficial in conjunction with increased CO2 levels.
2. MATERIAL AND METHODS
In our experiments were used six samples of lignite (from Halânga and Turceni) and three sample of pitcoal (from Paroseni) taken in 2008 year.
The total number of aerobic heterotrophic neutrophilic bacteria was estimated by decimal dilutions method of the samples followed by the dilutions inoculation in Petri dishes with selective medium (nutritive gelose) and incubation at 280C for 24 – 48 hours. After this period, the quantitative determination was performed by direct counting of the isolated colonies developed on selective medium. The isolated strains were characterized regarding the Gram staining, cellular morphology and biochemical features (oxidase and catalase activity, biosynthesis of organic acids).
The testing of organic acid biosynthesis was performed on selective solid medium containing peptone, glucose and a specific dye. The strains which determined the changing of medium colour from blue to green or yellow were counted as positive. These strains or mixed cultures were tested subsequently for organic acid production on the same selective liquid medium, in stirring conditions, at 280 C, for 96 hours. We determined both the cellular growth (spectrophotometrically at 660 nm) and acid production (by changing of pH value and medium colour), at the inoculation moment and at 24 hours intervals on all incubation period long.
In the desulphurization experiments of different coal samples by the heterotrophic neutrophilic bacteria was used lignite taken from Turceni and pitcoal from Paroşeni. In these experiments were used 6/1 and 8/1-2 mixed cultures isolated from the same coal samples. At the bacterial cultures was added 5 grams of coal and these were incubated at 280C, in stirring conditions (150 rpm), for 192 hours. At the end of the experimental period it was determined the diminished weight of the different coal samples under the action of heterotrophic neutrophilic cultures, which degraded organic sulphur from coal.
The chemolithotrophic iron-oxidizing bacteria were isolated from acid waters, mine sediments containing high concentrations of heavy metals from Baia (Tulcea county) and Valea Sesei (Alba county). In order to obtain populations of Thiobacillus ferrooxidans from these samples were used selective liquid culture medium 9 K (mineral medium with a pH of 2.5), in which the energetic substratum was represented by the ferrous sulphate in the optimum concentration of 43.22 g/l, which corresponds to 8.6 g/l Fe2+. To isolate strains it was used the medium 9K agared.
In a view to obtaining populations of acidophilic chemolithotrophic bacteria were got using isolated colonies on agarized selective culture media, following the dynamics of the physiological activity in inorganic media specific. Isolated colonies obtained are coloured brownish red. Using these technique 10 populations of Thiobacillus ferrooxidans were isolated.
In the experiments of bacterial desulphurization of different coal by the cultures of Thiobacillus ferrooxidans were used pitcoal from Paroşeni, lignite taken from two mines: Halânga and Turceni. In these experiments of biodesulfurization coal it was used the report solid/liquid of 5-10g/100ml in Leathen medium (Karavaiko, 1988), the granulometri of the coal in the experiment being of 0,75mm.
The experiments of testing the coal biodesulfurization was made in Erlenmeyer glasses (750 ml) with 90 ml specific medium and 10 ml inoculums aged 7 days. The bacterial cultures were incubated at 280C, on the rotational shaker, for 28 days at 150rpm.
In the experiments of coal desulphurization were used Thiobacillus ferrooxidans bacterial cultures (6 populations), selected on their resistance to higher concentrations of iron (18g/l Fe2+), Cu2+ and Zn2+ (3000 ppm).
Regarding the study of raising the efficiency of the coal biosulfurization process, the experiments were accompanied by chemical controls and biological controls (the P9 population – with a low resistance to metal ions).
At the end of the experimental period it was determined the diminished weight of the different coal under the action of Thiobacillus ferrooxidans cultures, which solubilized pyrite into soluble sulphate.
The tests have been realized on the chemistry laboratory of the Research and Development National Institute for Metals and Radioactive Resources – Măgurele, by gravimetric determination in the BaSO4 form.
3. RESULTS
The results revealed that the higher number of heterotrophic neutrophilic bacteria was obtained both from raw lignite samples taken from Hălânga and Turceni (1.3 ´∙107 CFU/g and 5.4 ´∙106 CFU/g) and from raw pitcoal taken from Paroşeni (8.4∙´ 103 CFU/g). Comparatively, the lowest values of the heterotrophic bacteria number were determined in the case of non-magnetic samples taken from the same sites (6.3 ´∙103 CFU/g at Hălânga, 9.0 ´ 10 CFU/g at Turceni and 5.6∙´ 102 CFU/g at Paroşeni). Intermediary values were obtained for mixed samples of lignite (2.7∙´ 106 CFU/g at Hălânga and 3.8 ´ 106 CFU/g at Turceni, respectively) and for mixed pitcoal sample from Paroşeni (5.9 ´102 CFU/g) (table 1).
We isolated 123 strains of heterotrophic neutrophilic bacteria from nine lignite and pitcoal samples, which were subsequently cultivated on slants with the same selective solid medium.
The study of heterotrophic bacteria capacity to produce organic acids on selective solid medium have shown that from the 123 strains tested only 14 strains or mixed cultures can synthesized acids, which changing the pH value and the colour of the medium after 72 hours of incubation at 280C. Subsequently, the pure strains or mixed cultures selected as positive for organic acids production were grown on the same liquid medium and incubated in stirring conditions at 280C, for 96 hours. We determined both cellular growth and acid production at intervals of 24 hours, on all incubation period long.
The results showed that the majority of pure strains or mixed cultures tested was determined the decrease of pH values of culture media (from 6.3 – 6.8 at inoculation moment to 4.1 – 5.3 after 96 hours of incubation) owing to the organic acids biosynthesis capacity. Also, was observed that to majority of bacteria tested there are a correlation between the strains growth and organic acids production. The acid production activity by heterotrophic neutrophilic bacteria increased with cellular growth, the lowest pH values being obtained after 96 hours of incubation at 280C.
Table 1. The assessment of total number of heterotrophic neutrophilic bacteria from lignite and pitcoal samples.
Nr. / Coal sample / Total number of heterotrophic neutrophilic bacteria (CFU / g) / Isolated strains number / Strains or mixed cultures with organic acids biosynthesis activity1 / Raw lignite Halânga / 1.3 x 107 / 18 / -
2 / Mixed 2 lignite Halânga / 2.7 x 106 / 19 / -
3 / Non-magnetic Carpco Halânga (2) / 6.3 x 103 / 17 / 10
4 / Raw lignite Turceni / 5.4 x 106 / 15 / -
5 / Mixed 2 lignite Turceni / 3.8 x 106 / 11 / -
6 / Non-magnetic Carpco Turceni / 9.0 x 10 / 3 / 1
7 / Raw pitcoal Paroşeni / 8.4 x 103 / 15 / -
8 / Mixed pitcoal Paroşeni / 5.9 x 102 / 18 / 3
9 / Non-magnetic Carpco Paroşeni / 5.6 x 102 / 7 / -
Number of isolated heterotrophic neutrophilic bacterial strains / 123 / 14
In experiments with heterotrophic bacteria mixed cultures was obtained a diminution of coal mass, also. The results have showed that the loss of coal mass was more important in the lignite sample (13 %) comparatively with the mass reduction observed in pitcoal sample (7.6 %) (fig. 3.).
The Thiobacillus ferrooxidans cultures used in the experiments of biooxidizing the sulphur from the two coals tested were selected on the basis of their capacity to oxidize the sulphur in the presence of higher concentrations of metal ions. Thus, with a view to raising the efficiency of the bacterial desulphurization process of the coal, the P9 population of Thiobacillus ferrooxidans as a reference strain, due to its higher sensitivity to big concentrations of ferrous sulphate (10-18 g/l) in a culture medium.
The biooxidizing of sulphur from two coals of Paroseni and Halânga at different solid/liquid ratios (5-10 g/100 ml) in the presence of Thiobacillus ferrooxidans cultures in conditions of continuous stirring are presented in figures 4 – 6.
Fig. 1. The dynamic growth and organic acids production by 3/11-1 (a) and 3/15-2 (b) mixed cultures isolated from lignite sample taken from Halânga.
Fig. 2. The dynamic growth and organic acids production by 6/1 (c) and 8/1-2 (d) mixed cultures isolated from Turceni lignite sample and from Paroşeni pitcoal sample, respectively
After testing the biooxidizing of sulphur from the coal concentrate at the solid density of 5 g/100ml and 8 g/100ml, it was observed that the P7 population has determined a higher efficiency of coal desulphurization in the same time interval, a consequence of the higher tolerance to Fe2+, Cu2+ and Zn2+ in solution of these bacterial cultures.