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Desulphurization of some low-rank Turkish lignites with crude laccase produced from Trametes versicolor ATCC 200801
Fuel Processing Technology 92 (2011) 71–76
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Fuel Processing Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f u p r o c
Desulphurization of some low-rank Turkish lignites with crude laccase produced from Trametes versicolor ATCC 200801 Pınar Aytar a, Serap Gedikli a, Mesut Şam b, Arzu Ünal c, Ahmet Çabuk d,⁎, Nazif Kolankaya e, Alp Yürüm f a
Graduate School of Natural and Applied Sciences, Eskişehir Osmangazi University, 26480, Eskişehir, Turkey Department of Biology, Faculty of Arts and Science, Aksaray University, Aksaray, Turkey Ministry of Agriculture and Rural Affairs, General Directorate of Agricultural Research, Ankara, Turkey d Department of Biology, Faculty of Arts and Science, Eskişehir Osmangazi University, 26480, Eskişehir, Turkey e Department of Biology, Division of Biotechnology, Faculty of Science, Hacettepe University, 06532, Ankara, Turkey f Grand Water Research Institute, Technion Israel Institute of Technology, 32000, Haifa, Israel b c
a r t i c l e
i n f o
Article history: Received 25 June 2009 Received in revised form 8 August 2010 Accepted 30 August 2010 Keywords: Desulphurization Laccase Turkish lignites
a b s t r a c t In this paper, data obtained during the oxidative desulphurization of some low-rank Turkish lignites with crude laccase enzyme produced from Trametes versicolor ATCC 200801 are presented. In order to optimize desulphurization conditions, effects of incubation time, pulp density, incubation temperature, medium pH, and also lignite source on the desulphurization have been examined. The values for incubation period, pulp density, temperature and pH in optimum incubation condition were found as 30 min, 5%, 35 °C, and pH 5.0, respectively. Under optimum conditions, treatment of coal samples with crude laccase has caused nearly 29% reduction in their total sulphur content. During the study, the rate of desulphurization of coal sample provided from Tunçbilek with crude laccase was found to be relatively higher than the other examined coal samples. Results of analytical assays have indicated that the treatment of coals with crude laccase has caused no change in their calorific values but reduced their sulphur emissions. 35%, 13%, and 25% reductions of pyritic sulphur, sulphate and organic sulphur in a period of 30 min were achieved, for a particle size of 200 μm under optimal conditions with enzymatic desulphurization. Also, statistical analyses such as Tukey Multiple Comparison tests and ANOVA were performed. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Demand for energy is increasing gradually due to developing industry and growing population. Fossil fuels constitute an important part of the natural energy sources. Coal, one of the fossil fuels, has a widespread usage at many industries. A number of researches have been conducted to increase the quality of coal, which made the usage of this fossil fuel relatively attractive. However, environmental implications, especially air polluting sulphur gasses, restrict the use of coal. To the best of our knowledge, generally, coals are reported to include 1–3 wt.% sulphur [1]. Emission of SO2 into the atmosphere is one of the most important pollution among other pollutants Discharge of SO2 from various sources, such as energy industries, can easily create air pollution and causes harmful effects on living things and environment. For instance, this causes acid rain affecting negatively plant and animals [2]. However this problem can be overcome by lowering high sulphur content of coal. Therefore, further research is needed in order to minimize the environmental impact of coal utilization.
⁎ Corresponding author. Tel.: +90 222 239 3750x2439; fax: +90 222 239 35 78. E-mail address: [email protected] (A. Çabuk). 0378-3820/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fuproc.2010.08.022
Some of the electric energy production in Turkey is provided by thermal power plants using fossil fuels. Lignite, the most abundant energy source in Turkey, is consumed in most of these plants, about 43%. Turkey has approximately 2% of the world's lignite reserves; however, the Turkish lignites have low calorific value and contain relatively higher amounts of ash and sulphur [3]. Generally, in the important lignite deposits of Turkey the total sulphur content varies between 0.50% and 6.51% [4]. Almost 80% of the total reserves have calorific values below 2500 kcal/kg [4]. In Turkey, lignite-fired thermal power plants are responsible for a considerable amount of air pollution. Thereby, it is important to equip the currently operating plants with newer technologies that will reduce amount of contaminants released into the air [5]. To solve the problem of emission of SO2 from coals, several microorganisms particularly bacteria have been reported for desulphurization of the coals [6–10]. While raw enzymes from bacteria isolated for desulphurization are generally well known, relatively little work has been carried out with fungi. Fungi, particularly lignin-degrading white rot fungi, are shown to be able to catalyse a wide range of reactions, including degradation of benzopyrens [11], dechlorination of chlorophenolic compounds [12], polycyclic aromatic hydrocarbons [13], dehalogenation of
P. Aytar et al. / Fuel Processing Technology 92 (2011) 71–76
hydroxybiphenyls [14], decolorization of textile dyes [15], desulphurization, and solubilization of coals [16–18] through the action of extracellular enzymes. Extracellular enzymes produced by white rot fungi have the potential to be useful biocatalysts due to their broad specificity, and ability to attack high-molecular weight substrates [19]. The lignin-degrading fungus Trametes versicolor utilizes a highly nonspecific oxidizing system to degrade lignin to produce CO2 as end product [20]. Peroxidase and laccase, enzymes of T. versicolor, due to having low substrate specificity can also degrade lignite structures [21]. For these reasons it has been proposed to utilize ligninolytic enzymes such as laccase in culture supernatant of T. versicolor. This study focuses on using the crude laccase enzyme for the biodesulphurization of low-rank lignites. In the first stage of present work, three lignites from various parts of Turkey with different sulphur content were biodesulphurizated using supernatant with high laccase activity provided from T. versicolor. The main intention was to determine the conditions of optimal enzymatic desulphurization, how it affect the distribution of sulphur proportions, and coal characteristics such as ash content, calorific value, and metal contents influencing the quality. The effects of various operating parameters, such as lignite type, incubation time, pulp density, incubation temperature and medium pH, were examined and each parameter studied was evaluated statistically. 2. Experimental 2.1. Coal samples and characteristics Lignite samples were provided from some coal fields of Turkey; Yatağan (Management of South Aegean Lignite), Soma (Management of Aegean Lignite) and Tunçbilek (Management of Garp Lignite). All of the coal samples used at reaction medium were ground at Retsch cross beater mill SK1 generating a grain of size 200 μm. Characterizations of the lignite used at the further stage of the experiment were carried out with LECO SC-144DR sulphur analyzer with infrared absorption detection procedure [22] for total sulphur analysis, LECO TGA701 for determining ash amount, and LECO AC-350 automatic calorimeter for determining heating value. Also, the sulphur forms were determined by following Standard Methods (ASTM D 2492) [23]. At the end of the incubation period of crude laccase treatment, the coal particles were separated by filter paper and washed with 10 ml of dilute (0.1 N) HCl in order to flush away the iron salts such as jarosite formed during the pyrite oxidation. They were then washed with sufficient amount of distilled water to ensure complete removal of the HCl solution. Thereafter, the coal samples were dried at 45 °C and analyzed for total sulphur content. The sulphur emission value (EV) which is grams of sulphur per megajoule (gS/MJ) was calculated by dividing the total sulphur content (wt.%) by calorific value (MJ) [24]. To determine metal contents, the coal samples were digested by extraction method of aqua regia (HCl/ HNO3 3:1) [25]. 5 g (±0.001 g) of the coal samples were weighed into ceramic crucible and burned at 600 °C. 30 ml of 1 M HCl and 10 ml of 1 M HNO3 were transferred to digest vessels. These solutions were kept overnight. After digestion, coal samples were filtered by quantitative filter paper Grade 201. The resultant solutions were transferred into 50 ml volumetric flasks and diluted to that amount using distilled and deionized water before metal analyses by ICP-AES model Perkin Elmer 3100. In inductively coupled plasma atomic emission spectrometry excited electrons emit energy at a given wavelength as they return to ground state. The elemental composition of the given samples was quantified relative to a reference standard.
Sulphur removal (%)
72
50 40 30 20 10 0 Tunçbilek
Yata an
Fig. 1. Desulphurization with crude laccase enzyme of different lignite coal (ınitial sulphur contents: 2.59% for Tunçbilek coal, 2.43% for Yatağan coal, 1.03% for Soma coal, and pH 6, 30 °C, 15 min, 5% of pulp density).
crude laccase production. Stock-cultures of the organism were maintained by growing the organism on malt-agar (Fluka Co.) slants. Liquid Vogel Medium, which was modified as described by Aktaş et al., was used as fermentation medium for crude laccase production from T. versicolor (ATCC 200801) [26]. In order to produce crude laccase, 1 ml of inoculant prepared by suspending the stock culture of organism in 10 ml of sterile saline solution was added into the 250 ml Erlenmeyer flask containing 100 ml of fermentation medium. Then flasks were allowed to incubation in rotating incubator-shaker (Edmund Bühler-Labortechnik-Materialtechnik Johanna Otto GmbH) adjusted to rotating speed of 150 rpm, at 30 °C for 10 days. At the end of the incubation period, liquid cultures of the organism were subjected to filtration to separate biomass from culture fluids. Culture filtrates including high laccase activity so obtained were used as desulphurization agent in further experiments. 2.3. Assay for laccase activity To assay the laccase enzyme activity in culture supernatant, 0.1 ml of enzyme source was added into test tube containing 4.9 ml of 0.1 M acetate buffer pH 4.6 and 1 mM guaiacol as substrate. The reaction mixture prepared was allowed to incubate at 37 °C for 5 min. As being different from tests, blanks containing inactive enzyme were prepared by boiling enzyme source. Enzyme activity in the tubes was measured by reading optical density in Jasco V530 UV/VIS. Spectrophotometer was adjusted to 465 nm wavelengths. One unit of activity was defined as enzyme activity that elicited an increase in A465 of 0.1 AU/min. 2.4. Experimental set-up and procedure for enzymatic coal desulphurization Enzymatic desulphurization experiments were performed by using the culture supernatant produced from T. versicolor. Lignite samples with different sulphur content, which were provided from Yatağan, Soma and Tunçbilek, were used as substrates in the first stage of experiments. The coal sample obtained from Tunçbilek was also used for further studies including the parameters: incubation time, pulp density, incubation temperature, and medium pH. Also, biodesulphurized lignite was evaluated in terms of ash, calorific value and metal analyses. Reactions were carried out in 250 ml conical Table 1 Homogenous subsets of coal type factor intent of desulphurization with performed Tukey Multiple Comparison tests. Coal type
N
Subset for alpha = .05 1
2.2. Production of crude laccase White rot fungal strain, T. versicolor (ATCC 200801) from basidiomycetes group was used as microorganismal source for
Soma
Lignite types
Soma Yatağan Tunçbilek Sig.
3 3 3
2
3
10.6387 13.1034 1.000
1.000
14.9104 1.000
50
Sulphur removal (%)
Sulphur removal (%)
P. Aytar et al. / Fuel Processing Technology 92 (2011) 71–76
40 30 20 10 0 15
30
45
60
90
120
240
73
50 40 30 20 10 0
360
1%
2%
3%
4%
Incubation time (minute)
5%
6%
7%
8%
9% 10% 15% 20%
Pulp density (w/v)
Fig. 2. Influence on incubation time of desulphurization with crude laccase (30 °C, pH 6 and 5% of pulp density).
Fig. 3. Influence on pulp density of coal of desulphurization with crude laccase (30 °C, pH 6 and 30 min).
flasks containing 100 ml buffer solution and appropriate volume of enzyme solution to give the final enzyme activity varying between 2.61 U/ml and 2.79 U/ml. Biodesulphurization influencing parameters, incubation time and temperature, were varied from 15 to 360 min, and from 20 to 60 °C, respectively. To determine appropriate pulp density (amount of substrate), final concentrations were varied between 1% and 20%. In order to investigate the effects of pH on the enzymatic desulphurization, Na-acetate buffer (0.1 M and pH: 3–5), KH2PO4 buffer (0.1 M and pH: 6–8) solutions were used in the reaction mixtures. Desulphurization reactions were conducted in shaking incubator adjusted to rotational speed of 125 rpm. Each experimental run was repeated at least twice and average values were presented. Experimental control tests were also carried out in the same conditions but using denatures of culture supernatant. In this way, it was possible to investigate the biodesulphurization process which might occur via chemical interactions and washing of coal samples. Thus, crude laccase containing culture supernatants which have the highest biodesulphurization capacity in batch conditions were determined.
content (Fig. 1). Similar results were reported by Shah et al (2002) [27]. According to Tukey Multiple Comparison tests, desulphurization process in all coal types is significantly different (Table 1). Since we have obtained higher yield of desulphurization with lignite sample provided from Tunçbilek (14.9104%) (p b 0.05), it was used as substrate in our further studies. The effect of incubation time on sulphur removal was tested by using the same batch system at the range of 15–360 min. For this experimental conditions, 30 min of incubation was found to be necessary to obtain the highest yield of desulphurization, and sulphur removal for this experiment duration was 28.5167% (p b 0.05) (Fig. 2). We conclude that removal of sulphur in different incubation times were significantly different (Table 2). Pulp density (or substrate concentration) was one of the conditional reaction parameters affecting the yield of enzymatic sulphur removal from coal samples. While a little variation in the yield of desulphurization was observed for the pulp densities up to 8%, sulphur removal process was reduced considerably after this pulp density. As seen in Fig. 3, pulp densities more than 8% resulted in decrease in the yield of sulphur removal probably as being associated with the decrease in enzyme and substrate ratio. Desulphurization process in examined values of pulp densities is significantly different. 5% of pulp density was chosen in respect of optimum sulphur removal as to both sulphur analyses and statistical analyses (Table 3). Also, percentage of desulphurization at this pulp density was found 28.5033% for coal provided from Tunçbilek (p b 0.05). Temperature of incubation was another incubational factor affecting the yield of crude laccase depending on desulphurization reactions. Tukey Multiple Comparison test related to temperature parameter was shown in Table 4. According to this, sulphur removal at 20 and 60 °C are the same homogenous group. Also, desulphurization at 30 and 35 °C are the same homogenous group. The other temperature levels were different in terms of desulphurization process. Statistical analyses illustrated that maximum desulphurization was observed at both 30 and 35 °C. As a result of the experiment performed to find out optimum reaction temperature for enzymatic desulphurization, 35 °C was determined as optimum temperature to
2.5. Statistical analyses One way analysis of variance (ANOVA) was used to investigate the significant differences between factors (coal type, pulp density, incubation temperature, incubation time, and medium pH) and Tukey test was also used for multiple comparisons. Significance level was taken α = 0.05 in all analysis. Analyses were performed in SPSS 13 software. 3. Results and discussion 3.1. Desulphurization optima of crude laccase treatment of the lignite coals As result of the experiment performed to search the effect of lignite source on yield of desulphurization, it was seen that percentage of sulphur removal was high for coal samples having more sulphur
Table 2 Homogenous subsets of incubation time factor intent of desulphurization with performed Tukey Multiple Comparison tests. Incubation time (min)
N
15.00 60.00 240.00 45.00 120.00 360.00 90.00 30.00 Sig.
3 3 3 3 3 3 3 3
Subset for alpha = .05 1
2
3
4
5
6
7
8
14.9133 15.4767 16.2967 16.8167 17.8400 18.0600 20.1333 1.000
1.000
1.000
1.000
1.000
1.000
1.000
28.5167 1.000
74
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Table 3 Homogenous subsets of pulp density factor intent of desulphurization with performed Tukey Multiple Comparison tests. N
20.00 10.00 9.00 15.00 6.00 7.00 1.00 8.00 3.00 4.00 2.00 5.00 Sig.
3 3 3 3 3 3 3 3 3 3 3 3
Subset for alpha = .05 1
2
3
4
5
6
11
12
17.7767 22.6967 22.9167 23.5833 24.0500 25.4800 26.0167 28.1933 1.000
1.000
1.000
1.000
In addition to the analyses related to the change of sulphur form, the immediate and ultimate analyses which are calorific value (gross calorific value also known as the higher calorific value), ash content and emission value for treated lignite (Tunçbilek) with crude laccase were carried out together for the determination of metal contents before and after enzymatic treatment for explaining changes of coal quality (Table 6). As it can be observed from Table 6, our experimental findings demonstrate that a crude laccase of T. versicolor has removed total sulphur and increased calorific value of lignite. A 35% reduction of pyritic sulphur, a 13% reduction of sulphate and a 25% reduction of organic sulphur in a period of 30 min were achieved, for a particle size of 200 μm under optimal conditions with enzymatic desulphurization. Furthermore metal contents and sulphur emission value of coal were reduced with crude laccase. High ash in the coal not only reduces the thermal value of coal but also leads to production of fly ash, which is a major environmental problem. Moreover, there was reduction in the ash that it proposed the presence of relationship between rate of biodesulphurization of lignite and ash eliminated. But it should be stressed that the reduction of ash content is usually because of chemical process rather than biological treatment [28]. The reduction in ash content may be due to dissolution of mineral matters [29]. The sulphur emission value (EV) in grams of sulphur per megajoule Table 4 Homogenous subsets of incubation temperature factor intent of desulphurization with performed Tukey Multiple Comparison tests.
3 3 3 3 3 3 3
10
16.5467
3.2. Chemical evaluation of coal samples after crude laccase treatment
20.00 60.00 50.00 40.00 25.00 30.00 35.00 Sig.
9
15.4667
give maximum yield of desulphurization at ratio of 28.50% (p b 0.05) (Fig. 4). pH values of reaction medium were seemed to be effective on enzymatic yield of desulphurization of coal samples examined at experimental conditions. In the experiments to optimize the desulphurization conditions, pH 5.0 (p b 0.05) was found as optimum pH for the reactions giving maximum yield of desulphurization (28.8667%) (Fig. 5). As it can be seen from Table 5, desulphurization process in examined pH values is significantly different.
N
8
13.5500
1.000
Temperature (°C)
7
Subset for alpha = .05 1
2
3
4
5
19.7400 19.7500 21.3267 23.6367
1.000
1.000
1.000
1.000
28.4800 28.5000 1.000
1.000
1.000
1.000
1.000
28.5033 1.000
(g S/MJ) is the total sulphur content (wt.%) divided by calorific value (MJ). Increase of sulphur emission value causing negative environmental effect is one of the most significant indicators of air pollution. High value of EV indicates that there is a great environmental impact, while low values indicate a small impact [24]. 3.3. Proposed desulphurization mechanism From the results of our experiments performed to reveal the effect of crude laccase treatment on coal desulphurization and its optimization, it can be easily concluded that crude laccase obtained from T. versicolor is capable of sulphur removal from lignite coal samples examined. As for the removal of organosulphur compounds, which cause serious problems for petroleum industry, related products treated with the extracellular enzymes of some white rot fungi has been reported by some authors [19]. Schreiner et al. showed oxidation of thianthrene, one of sulphur compounds, by the ligninase of Phanerochaete chrysosporium which is white rot fungus [30]. Previously conducted research study has shown that lignin-degrading basidiomycete Coriolus versicolor (presently it is known as T. versicolor) exhibited metabolic response against sulphur-containing heterocyclic compounds [31]. Our desulphurization studies conducted with culture supernatant which has high laccase activity have clearly indicated that treatment of coal samples with crude laccase could result in reduction in their total sulphur content to some extent. There are some concerns about laccase that it may not be the only enzyme playing role in desulphurization process. Our previous study showed that T. versicolor and P. chrysosporium biomasses, which are white rot fungi, were potential organisms for biodesulphurization of coal at 40% efficiency [32]. Van Hamme et al. studied a model organic sulphur compound, dibenzyl sulfide (DBS) metabolism of white rot fungi and reported that they are able to metabolize a broad range of anthropogenic chemicals and hydrocarbons through intracellular cytochrome p-450 and extracellular enzymes [19]. They found 50 40 30 20 10 0 20
25.2467
1.000
1.000
Sulphur removal (%)
Pulp density (w/v)
25
30
35
40
50
60
Temperature (oC) Fig. 4. Influence on incubation temperature of desulphurization with crude laccase (pH 6, 5% of pulp density and 30 min).
Sulphur removal (%)
P. Aytar et al. / Fuel Processing Technology 92 (2011) 71–76
50
75
Table 6 Characteristics and initial and ultimate metal analyses of Tunçbilek lignite (before and after enzymatic treatment).
40 30 20 10 0 3,0
3,5
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
8,0
pH Fig. 5. Effect on medium pH of desulphurization with crude laccase (35 °C, 5% of pulp density and 30 min).
that the C–S bond in alkyl bridge of DBS is the target of oxidation by fungal cultures and the metabolism proceeds from DBS to dibenzyl sulphoxides followed by dibenzyl sulphone, prior to C–S bond cleavage. According to the research of Van Hamme et al., ring oxidation and opening occur only after this cleavage. The main factor involved in the metabolic conversion of dibenzyl sulphoxide to dibenzyl sulphone is shown to be enzyme consortium. Therefore, in this study, findings were lower than our previous study with same coal provided from Tunçbilek [32]. Extracellular enzyme source used for the biodesulphurization process interacted with organic fraction of coal samples. Probably, laccase and other extracellular enzymes take part on first oxidation step of desulphurization process. To the best of our knowledge, final degradation of the monomeric intermediates had been believed to be catalyzed by intracellular enzymes such as dioxygenases. Earlier studies on the application of laccase were promising. Dordick and co-workers studied the use of horseradish peroxidase and several other oxidative enzymes on coal-related compounds in organic media. They show that horseradish peroxidase, laccase, and chloroperoxidase are capable of oxidizing dibenzothiophene in 25% dimethylformamide [33]. In another study, enzymatic oxidation of DBT was evaluated by horseradish peroxidase, ligninolitic enzyme and also laccase. Reactions were carried out in monophasic organic media containing 25% (v/v) acetonitrile. Results show that 60% of DBT is converted into dibenzothiophene sulfoxide (12%) and dibenzothiophene sulfone (46%) [34]. A similar enzymatic desulphurization process using chloroperoxidase (CPO) from Caldariomyces fumago coupled with distillation at 325 °C oxidizes and removes preferentially the organosulphur compounds from a diesel at ambient temperature and atmospheric pressure [35,36]. Despite the elimination of up to 83% of organosulphur compounds, 2 mg of diesel was diluted in 1 ml of 20% acetonitrile-acetate buffer system which makes it inappropriate for industrial scale. Villasenor et al. studied DBT oxidation catalyzed by a laccase enzyme in the presence of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-
Total sulphur (%) Organic sulphur (%) Pyritic sulphur (%) Sulphate (%) Ash content (%) Calorific value (cal/g) Emission value (EV) Iron (mg/l) Zinc (mg/l) Manganese (mg/l) Copper (mg/l) Nickel (mg/l) Chromium (μg/l) Cobalt (μg/l) Cadmium (mg/l) Lead (μg/l) Magnesium (mg/l) Aluminum (mg/l)
Untreated coal
Treated with laccase
2.59 0.75 1.45 0.39 20.47 5182 0.119 5.4 ± 0.4 740 ± 2.4 30.5 ± 5.1 1.8 ± 0.2 27 ± 0.4 286 ± 53.4 661 ± 37.9 38.1 ± 0.1 822 ± 1.3 1520 ± 3.4 3.8 ± 0.4
1.84 0.56 0.94 0.34 16.97 5811 0.075 3 ± 0.1 639 ± 6.3 22.3 ± 0.8 1.1 ± 0.0 10 ± 0.3 150 ± 5.6 279 ± 4.2 4.8 ± 0.1 462 ± 3.3 886 ± 6.3 0.9 ± 0.0
sulfonic acid) (ABTS) [37]. However, the result found is a rather low rate process hindering the use of laccase system as a practical process for deep desulphurization. The limiting step of this process seems to be the oxidation of DBT by oxidized ABTS, rather than the oxidation of ABTS by laccase. Furthermore, enzyme catalysis by laccase would require the development of a much faster reaction scheme with a better mediator than ABTS. Ichinose et al. demonstrated that 4methyldibenzothiophene found in fossil fuel as a recalcitrant impurity was converted to water-extractable products such as hydroxymethylDBT-5-dioxide by developing an organic solvent-resistant fungal reaction system [38]. 4. Conclusions From the results it is clear that an enzymatic desulphurization method with crude laccase may be a welcomed alternative due to short time treatment compared to sulphur removal with biomass having the prolonged incubation time. Rather than refractory sulphur compounds, dealing with genuine fossil fuel like coal will open up new challenges to solve. Further study on solving a complete fungal desulphurization pathway will provide a novel strategy for biological sulphur removal. More efforts should be attempted in this direction. Acknowledgements We thank Coal Quality and Testing Laboratory from Garp Lignite Administrations and General Directorate of Mineral Research & Exploration for coal analyses. Also, we would like to express our
Table 5 Homogenous subsets of medium pH factor intent of desulphurization with performed Tukey Multiple Comparison tests. pH
N
Subset for alpha = .05 1
7.50 8.00 6.50 7.00 3.50 3.00 5.50 4.50 4.00 6.00 5.00 Sig.
3 3 3 3 3 3 3 3 3 3 3
2
3
4
5
6
7
8
9
10
11
11.2667 13.2367 14.6667 16.1667 17.3667 20.0867 22.7900 25.4933 26.4700 28.4667 1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
28.8667 1.000
76
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gratitude to Chemistry Department at Balıkesir University for ICP measurements, Eskişehir Osmangazi University Natural and Applied Science Research Center and Dr. Coşkun Kuş from Selçuk University in Turkey for statistical analyses. References [1] S.S. Tripathy, R.N. Kar, S.K. Mishra, I. Twardowska, L.B. Sukla, Effect of chemical pretreatment on bacterial desulphurization of Assam coal, Fuel 77 (1998) 859–864. [2] F. Kargi, J. Robinson, Removal of sulphur compounds from coal by the thermophilic organism Sulfolobus acidocaldarius, Applied and Environmental Microbiology 44 (1985) 878–883. [3] M. Balat, Turkey's major lignite fields and significance of lignite for energy necessity, Energy Sources, Part B:Economics, Planning and Policy 3 (2008) 13–25. [4] N. İnaner, E. Nakoman, Properties of lignite deposits in western Turkey, 57th Annual Meeting of the International Committee for Coal and Organic PetrologyICCP, Patras, Greece 18–23 (September 2005) 11. [5] P.N. Say, Lignite-fired thermal power plants and SO2 pollution in Turkey, Energy Policy 34 (2006) 2690–2701. [6] M.D. Dahlberg, R.L. Rohrer, D.J. Fauth, R. Sprecher, G. Olson, Biodesulphurization of dibenzothiophene sulfone by Arthrobacter sp. and studies with oxidized Illinois No.6 coal, Fuel 72 (1993) 1645–1649. [7] G. Olsson, L. Larsson, O. Holst, H. Karlsson, Desulphurization of low-sulphur coal by Acidianus brierleyi: effects of microbial treatment on the properties of coal, Fuel Processing Technology 33 (1993) 83–93. [8] Y. Izumi, T. Ohshiro, H. Ogino, Y. Hine, M. Shimao, Selective desulphurization of dibenzothiophene by Rhodococcus erythropolis strain D-1, Applied and Environmental Microbiology 60 (1994) 223–226. [9] G. Mohebali, A.S. Ball, B. Rasekh, A. Kaytash, Biodesulphurization potential of a newly isolated bacterium, Gordonia alkanivorans RIPI90A, Enzyme and Microbial Technology 40 (2007) 578–584. [10] T.L. Peeples, R.M. Kelly, Bioenergetics of the metal/sulphur-oxidizing extreme thermoacidophile, Metallosphaera sedula, Fuel 72 (1993) 1619–1624. [11] A. Verdin, S.A. Lounès-Hadj, R. Durand, Degradation of benzo[a]pyrene by mitosporic fungi and extracellular oxidative enzymes, International Biodeterioration and Biodegradation 53 (2004) 65–70. [12] L. Roy-Arcand, F.S. Archibald, Direct dechlorination of chlorophenolic compounds by laccases from Trametes (Coriolus) versicolor, Enzyme and Microbial Technology 13 (1991) 194–203. [13] M.A. Pickard, R. Roman, R. Tinoco, R. Vazouez-Duhalt, Polycylic aromatic hydrocarbon metabolism by white rot fungi and oxidation by Coriolopsis gallica UAMH 8260 laccase, Applied and Environmental Microbiology 65 (1999) 3805–3809. [14] A. Schultz, U. Jonas, E. Hammer, F. Schauer, Dehalogenation of chlorinated hydroxybiphenyls by fungal laccase, Applied and Environmental Microbiology 67 (2001) 4377–4381. [15] I. Nilsson, A. Möller, B. Mattiason, M.S. Rubindamayugi, U. Welander, Decolorization of synthetic and real textile wastewater by the use of white-rot fungi, Enzyme and Microbial Technology 38 (2006) 94–100. [16] L. Gonsalvesh, S.P. Marinov, M. Stefanova, Y. Yürüm, A.G. Dumanli, G. DinlerDoganay, N. Kolankaya, et al., Biodesulphurized subbituminous coal by different fungi and bacteria studied by reductive pyrolysis. Part 1: ınitial coal, Fuel 87 (2008) 2533–2543. [17] G. Willmann, R. Fakoussa, Extracellular oxidative enzymes of coal-attacking fungi, Fuel Processing Technology 52 (1997) 27–41.
[18] C.F. Gökçay, N. Kolankaya, F.B. Dilek, Microbial solubilization of lignites, Fuel 80 (2001) 1421–1433. [19] J.D. Van Hamme, E.T. Wong, H. Dettman, M.R. Gray, M.A. Pickard, Dibenzyl sulfide metabolism by white rot fungi, Applied and Environmental Microbiology 69 (2003) 1320–1324. [20] A. Leonowicz, A. Matuszewska, J. Luterek, D. Ziegenhagen, M. Wasilewska, N. Cho, M. Hofrichter, J. Rogalski, Biodegradation of lignin by white rot fungi, Fungal Genetics and Biology 27 (1979) 175–185. [21] R. Hayatsu, R.G. Scott, R.E. Winans, L.P. Moore, R.L. McBeth, M.H. Studier, Ligninlike polymers in coals, Nature 278 (1979) 41–43. [22] American Society for Testing and Materials, Standart test methods for sulphur in the analysis sample of coal and coke using high-temperature tube furnace combustion methods, Annual Book of ASTM Standards, 2004. [23] ASTM D 2492, Standard Test Method for Forms of Sulphur in Coal, 2002. [24] A. Aller, O. Martinez, A. De Linaje, R. Mendez, A. Moran, Biodesulphurisation of coal by microorganisms isolated from the coal itself, Fuel Processing Technology 69 (2001) 45–57. [25] C. Watson, Analytical methods for the determination of trace metals and other elements, Official and Standardized Methods of Analysis, The Royal Society of Chemistry, Cambridge, 1994, pp. 457–458. [26] N. Aktaş, H. Çiçek, A.Ü. Taşpınar, G. Kibarer, N. Kolankaya, A. Tanyolaç, Reaction kinetics for laccase-catalyzed polymerization of 1-naphtol, Bioresource Technology 80 (2001) 29–36. [27] C.L. Shah, J.A. Abbott, N.J. Miles, X. Li, X. Jianping, Sulphur reduction evalution of selected high-sulphur Chinese coals, Fuel 81 (2002) 519–529. [28] M.J. Grossman, M.K. Lee, R.C. Prince, K.K. Garett, G.N. George, I.J. Pickering, Microbial desulphurization of a crude oil middle distillate fraction: analysis of the extent of sulphur removal and the effect of removal on remaining sulphur, Applied and Environmental Microbiology 65 (1999) 181–188. [29] C. Acharya, L.B. Sukla, V.N. Misra, Biological elimination of sulphur from high sulphur coal by Aspergillus-like fungi, Fuel 84 (2005) 1597–1600. [30] R. Schreiner, S. Stevens, M. Tien, Oxidation of thianthrene by the ligninase of Phanerochaete chrysosporium, Applied and Environmental Microbiology 54 (1988) 1858–1860. [31] H. Ichinose, M. Nakamizo, H. Wariishi, H. Tanaka, Metabolic response against sulphur-containing heterocyclic compounds by the lignin-degrading basidiomycete Coriolus versicolor, Applied Microbiology and Biotechnology 58 (2002) 517–526. [32] P. Aytar, M. Şam, A. Çabuk, Microbial desulphurization of Turkish lignites by white rot fungi, Energy and Fuels 22 (2008) 1196–1199. [33] J.S. Dordick, K. Ryu, J.P. McEldoon, Enzymatic catalysis on coal-related compounds in organic media: kinetics and potential commercial applications, Resource Recovery and Conservation 2–3 (1991) 195–209. [34] L.S. Madeira, V.S. Ferreira-Leitao, E.P. Silva Bon, Dibenzothiophene oxidation by horseradish peroxidase in organic media: effect of the DBT:H2O2 molar ratio and H2O2 addition mode, Chemosphere 71 (2008) 189–194. [35] M. Ayala, R. Tinoco, V. Hernández, P. Bremuntz, R. Vazquez-Duhalt, Biocatalytic oxidation of fuel as an alternative to biodesulphurization, Fuel Processing Technology 57 (1998) 101–111. [36] M. Ayala, N.R. Robledo, A. Lopez-Munguia, R. Vazquez-Duhalt, Substrate specificity and ionization potential in chloroperoxidasecatalyzed oxidation of diesel fuel, Environmental Science and Technology 34 (2000) 2804–2809. [37] F. Villasenor, O. Loera, A. Campero, G. Viniegra-Gonzalez, Oxidation of dibenzothiophene by laccase or hydrogen peroxide and deep desulphurization of diesel fuel by the later, Fuel Processing Technology 86 (2004) 49–59. [38] H. Ichinose, H. Wariishi, H. Tanaka, Bioconversion of recalcitrant 4-methyldibenzothiophene to water-extractable products using lignin-degrading basidiomycete Coriolus versicolor, Biotechnology Programme 15 (1999) 706–714.
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Fuel Processing Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f u p r o c
Desulphurization of some low-rank Turkish lignites with crude laccase produced from Trametes versicolor ATCC 200801 Pınar Aytar a, Serap Gedikli a, Mesut Şam b, Arzu Ünal c, Ahmet Çabuk d,⁎, Nazif Kolankaya e, Alp Yürüm f a
Graduate School of Natural and Applied Sciences, Eskişehir Osmangazi University, 26480, Eskişehir, Turkey Department of Biology, Faculty of Arts and Science, Aksaray University, Aksaray, Turkey Ministry of Agriculture and Rural Affairs, General Directorate of Agricultural Research, Ankara, Turkey d Department of Biology, Faculty of Arts and Science, Eskişehir Osmangazi University, 26480, Eskişehir, Turkey e Department of Biology, Division of Biotechnology, Faculty of Science, Hacettepe University, 06532, Ankara, Turkey f Grand Water Research Institute, Technion Israel Institute of Technology, 32000, Haifa, Israel b c
a r t i c l e
i n f o
Article history: Received 25 June 2009 Received in revised form 8 August 2010 Accepted 30 August 2010 Keywords: Desulphurization Laccase Turkish lignites
a b s t r a c t In this paper, data obtained during the oxidative desulphurization of some low-rank Turkish lignites with crude laccase enzyme produced from Trametes versicolor ATCC 200801 are presented. In order to optimize desulphurization conditions, effects of incubation time, pulp density, incubation temperature, medium pH, and also lignite source on the desulphurization have been examined. The values for incubation period, pulp density, temperature and pH in optimum incubation condition were found as 30 min, 5%, 35 °C, and pH 5.0, respectively. Under optimum conditions, treatment of coal samples with crude laccase has caused nearly 29% reduction in their total sulphur content. During the study, the rate of desulphurization of coal sample provided from Tunçbilek with crude laccase was found to be relatively higher than the other examined coal samples. Results of analytical assays have indicated that the treatment of coals with crude laccase has caused no change in their calorific values but reduced their sulphur emissions. 35%, 13%, and 25% reductions of pyritic sulphur, sulphate and organic sulphur in a period of 30 min were achieved, for a particle size of 200 μm under optimal conditions with enzymatic desulphurization. Also, statistical analyses such as Tukey Multiple Comparison tests and ANOVA were performed. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Demand for energy is increasing gradually due to developing industry and growing population. Fossil fuels constitute an important part of the natural energy sources. Coal, one of the fossil fuels, has a widespread usage at many industries. A number of researches have been conducted to increase the quality of coal, which made the usage of this fossil fuel relatively attractive. However, environmental implications, especially air polluting sulphur gasses, restrict the use of coal. To the best of our knowledge, generally, coals are reported to include 1–3 wt.% sulphur [1]. Emission of SO2 into the atmosphere is one of the most important pollution among other pollutants Discharge of SO2 from various sources, such as energy industries, can easily create air pollution and causes harmful effects on living things and environment. For instance, this causes acid rain affecting negatively plant and animals [2]. However this problem can be overcome by lowering high sulphur content of coal. Therefore, further research is needed in order to minimize the environmental impact of coal utilization.
⁎ Corresponding author. Tel.: +90 222 239 3750x2439; fax: +90 222 239 35 78. E-mail address: [email protected] (A. Çabuk). 0378-3820/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fuproc.2010.08.022
Some of the electric energy production in Turkey is provided by thermal power plants using fossil fuels. Lignite, the most abundant energy source in Turkey, is consumed in most of these plants, about 43%. Turkey has approximately 2% of the world's lignite reserves; however, the Turkish lignites have low calorific value and contain relatively higher amounts of ash and sulphur [3]. Generally, in the important lignite deposits of Turkey the total sulphur content varies between 0.50% and 6.51% [4]. Almost 80% of the total reserves have calorific values below 2500 kcal/kg [4]. In Turkey, lignite-fired thermal power plants are responsible for a considerable amount of air pollution. Thereby, it is important to equip the currently operating plants with newer technologies that will reduce amount of contaminants released into the air [5]. To solve the problem of emission of SO2 from coals, several microorganisms particularly bacteria have been reported for desulphurization of the coals [6–10]. While raw enzymes from bacteria isolated for desulphurization are generally well known, relatively little work has been carried out with fungi. Fungi, particularly lignin-degrading white rot fungi, are shown to be able to catalyse a wide range of reactions, including degradation of benzopyrens [11], dechlorination of chlorophenolic compounds [12], polycyclic aromatic hydrocarbons [13], dehalogenation of
P. Aytar et al. / Fuel Processing Technology 92 (2011) 71–76
hydroxybiphenyls [14], decolorization of textile dyes [15], desulphurization, and solubilization of coals [16–18] through the action of extracellular enzymes. Extracellular enzymes produced by white rot fungi have the potential to be useful biocatalysts due to their broad specificity, and ability to attack high-molecular weight substrates [19]. The lignin-degrading fungus Trametes versicolor utilizes a highly nonspecific oxidizing system to degrade lignin to produce CO2 as end product [20]. Peroxidase and laccase, enzymes of T. versicolor, due to having low substrate specificity can also degrade lignite structures [21]. For these reasons it has been proposed to utilize ligninolytic enzymes such as laccase in culture supernatant of T. versicolor. This study focuses on using the crude laccase enzyme for the biodesulphurization of low-rank lignites. In the first stage of present work, three lignites from various parts of Turkey with different sulphur content were biodesulphurizated using supernatant with high laccase activity provided from T. versicolor. The main intention was to determine the conditions of optimal enzymatic desulphurization, how it affect the distribution of sulphur proportions, and coal characteristics such as ash content, calorific value, and metal contents influencing the quality. The effects of various operating parameters, such as lignite type, incubation time, pulp density, incubation temperature and medium pH, were examined and each parameter studied was evaluated statistically. 2. Experimental 2.1. Coal samples and characteristics Lignite samples were provided from some coal fields of Turkey; Yatağan (Management of South Aegean Lignite), Soma (Management of Aegean Lignite) and Tunçbilek (Management of Garp Lignite). All of the coal samples used at reaction medium were ground at Retsch cross beater mill SK1 generating a grain of size 200 μm. Characterizations of the lignite used at the further stage of the experiment were carried out with LECO SC-144DR sulphur analyzer with infrared absorption detection procedure [22] for total sulphur analysis, LECO TGA701 for determining ash amount, and LECO AC-350 automatic calorimeter for determining heating value. Also, the sulphur forms were determined by following Standard Methods (ASTM D 2492) [23]. At the end of the incubation period of crude laccase treatment, the coal particles were separated by filter paper and washed with 10 ml of dilute (0.1 N) HCl in order to flush away the iron salts such as jarosite formed during the pyrite oxidation. They were then washed with sufficient amount of distilled water to ensure complete removal of the HCl solution. Thereafter, the coal samples were dried at 45 °C and analyzed for total sulphur content. The sulphur emission value (EV) which is grams of sulphur per megajoule (gS/MJ) was calculated by dividing the total sulphur content (wt.%) by calorific value (MJ) [24]. To determine metal contents, the coal samples were digested by extraction method of aqua regia (HCl/ HNO3 3:1) [25]. 5 g (±0.001 g) of the coal samples were weighed into ceramic crucible and burned at 600 °C. 30 ml of 1 M HCl and 10 ml of 1 M HNO3 were transferred to digest vessels. These solutions were kept overnight. After digestion, coal samples were filtered by quantitative filter paper Grade 201. The resultant solutions were transferred into 50 ml volumetric flasks and diluted to that amount using distilled and deionized water before metal analyses by ICP-AES model Perkin Elmer 3100. In inductively coupled plasma atomic emission spectrometry excited electrons emit energy at a given wavelength as they return to ground state. The elemental composition of the given samples was quantified relative to a reference standard.
Sulphur removal (%)
72
50 40 30 20 10 0 Tunçbilek
Yata an
Fig. 1. Desulphurization with crude laccase enzyme of different lignite coal (ınitial sulphur contents: 2.59% for Tunçbilek coal, 2.43% for Yatağan coal, 1.03% for Soma coal, and pH 6, 30 °C, 15 min, 5% of pulp density).
crude laccase production. Stock-cultures of the organism were maintained by growing the organism on malt-agar (Fluka Co.) slants. Liquid Vogel Medium, which was modified as described by Aktaş et al., was used as fermentation medium for crude laccase production from T. versicolor (ATCC 200801) [26]. In order to produce crude laccase, 1 ml of inoculant prepared by suspending the stock culture of organism in 10 ml of sterile saline solution was added into the 250 ml Erlenmeyer flask containing 100 ml of fermentation medium. Then flasks were allowed to incubation in rotating incubator-shaker (Edmund Bühler-Labortechnik-Materialtechnik Johanna Otto GmbH) adjusted to rotating speed of 150 rpm, at 30 °C for 10 days. At the end of the incubation period, liquid cultures of the organism were subjected to filtration to separate biomass from culture fluids. Culture filtrates including high laccase activity so obtained were used as desulphurization agent in further experiments. 2.3. Assay for laccase activity To assay the laccase enzyme activity in culture supernatant, 0.1 ml of enzyme source was added into test tube containing 4.9 ml of 0.1 M acetate buffer pH 4.6 and 1 mM guaiacol as substrate. The reaction mixture prepared was allowed to incubate at 37 °C for 5 min. As being different from tests, blanks containing inactive enzyme were prepared by boiling enzyme source. Enzyme activity in the tubes was measured by reading optical density in Jasco V530 UV/VIS. Spectrophotometer was adjusted to 465 nm wavelengths. One unit of activity was defined as enzyme activity that elicited an increase in A465 of 0.1 AU/min. 2.4. Experimental set-up and procedure for enzymatic coal desulphurization Enzymatic desulphurization experiments were performed by using the culture supernatant produced from T. versicolor. Lignite samples with different sulphur content, which were provided from Yatağan, Soma and Tunçbilek, were used as substrates in the first stage of experiments. The coal sample obtained from Tunçbilek was also used for further studies including the parameters: incubation time, pulp density, incubation temperature, and medium pH. Also, biodesulphurized lignite was evaluated in terms of ash, calorific value and metal analyses. Reactions were carried out in 250 ml conical Table 1 Homogenous subsets of coal type factor intent of desulphurization with performed Tukey Multiple Comparison tests. Coal type
N
Subset for alpha = .05 1
2.2. Production of crude laccase White rot fungal strain, T. versicolor (ATCC 200801) from basidiomycetes group was used as microorganismal source for
Soma
Lignite types
Soma Yatağan Tunçbilek Sig.
3 3 3
2
3
10.6387 13.1034 1.000
1.000
14.9104 1.000
50
Sulphur removal (%)
Sulphur removal (%)
P. Aytar et al. / Fuel Processing Technology 92 (2011) 71–76
40 30 20 10 0 15
30
45
60
90
120
240
73
50 40 30 20 10 0
360
1%
2%
3%
4%
Incubation time (minute)
5%
6%
7%
8%
9% 10% 15% 20%
Pulp density (w/v)
Fig. 2. Influence on incubation time of desulphurization with crude laccase (30 °C, pH 6 and 5% of pulp density).
Fig. 3. Influence on pulp density of coal of desulphurization with crude laccase (30 °C, pH 6 and 30 min).
flasks containing 100 ml buffer solution and appropriate volume of enzyme solution to give the final enzyme activity varying between 2.61 U/ml and 2.79 U/ml. Biodesulphurization influencing parameters, incubation time and temperature, were varied from 15 to 360 min, and from 20 to 60 °C, respectively. To determine appropriate pulp density (amount of substrate), final concentrations were varied between 1% and 20%. In order to investigate the effects of pH on the enzymatic desulphurization, Na-acetate buffer (0.1 M and pH: 3–5), KH2PO4 buffer (0.1 M and pH: 6–8) solutions were used in the reaction mixtures. Desulphurization reactions were conducted in shaking incubator adjusted to rotational speed of 125 rpm. Each experimental run was repeated at least twice and average values were presented. Experimental control tests were also carried out in the same conditions but using denatures of culture supernatant. In this way, it was possible to investigate the biodesulphurization process which might occur via chemical interactions and washing of coal samples. Thus, crude laccase containing culture supernatants which have the highest biodesulphurization capacity in batch conditions were determined.
content (Fig. 1). Similar results were reported by Shah et al (2002) [27]. According to Tukey Multiple Comparison tests, desulphurization process in all coal types is significantly different (Table 1). Since we have obtained higher yield of desulphurization with lignite sample provided from Tunçbilek (14.9104%) (p b 0.05), it was used as substrate in our further studies. The effect of incubation time on sulphur removal was tested by using the same batch system at the range of 15–360 min. For this experimental conditions, 30 min of incubation was found to be necessary to obtain the highest yield of desulphurization, and sulphur removal for this experiment duration was 28.5167% (p b 0.05) (Fig. 2). We conclude that removal of sulphur in different incubation times were significantly different (Table 2). Pulp density (or substrate concentration) was one of the conditional reaction parameters affecting the yield of enzymatic sulphur removal from coal samples. While a little variation in the yield of desulphurization was observed for the pulp densities up to 8%, sulphur removal process was reduced considerably after this pulp density. As seen in Fig. 3, pulp densities more than 8% resulted in decrease in the yield of sulphur removal probably as being associated with the decrease in enzyme and substrate ratio. Desulphurization process in examined values of pulp densities is significantly different. 5% of pulp density was chosen in respect of optimum sulphur removal as to both sulphur analyses and statistical analyses (Table 3). Also, percentage of desulphurization at this pulp density was found 28.5033% for coal provided from Tunçbilek (p b 0.05). Temperature of incubation was another incubational factor affecting the yield of crude laccase depending on desulphurization reactions. Tukey Multiple Comparison test related to temperature parameter was shown in Table 4. According to this, sulphur removal at 20 and 60 °C are the same homogenous group. Also, desulphurization at 30 and 35 °C are the same homogenous group. The other temperature levels were different in terms of desulphurization process. Statistical analyses illustrated that maximum desulphurization was observed at both 30 and 35 °C. As a result of the experiment performed to find out optimum reaction temperature for enzymatic desulphurization, 35 °C was determined as optimum temperature to
2.5. Statistical analyses One way analysis of variance (ANOVA) was used to investigate the significant differences between factors (coal type, pulp density, incubation temperature, incubation time, and medium pH) and Tukey test was also used for multiple comparisons. Significance level was taken α = 0.05 in all analysis. Analyses were performed in SPSS 13 software. 3. Results and discussion 3.1. Desulphurization optima of crude laccase treatment of the lignite coals As result of the experiment performed to search the effect of lignite source on yield of desulphurization, it was seen that percentage of sulphur removal was high for coal samples having more sulphur
Table 2 Homogenous subsets of incubation time factor intent of desulphurization with performed Tukey Multiple Comparison tests. Incubation time (min)
N
15.00 60.00 240.00 45.00 120.00 360.00 90.00 30.00 Sig.
3 3 3 3 3 3 3 3
Subset for alpha = .05 1
2
3
4
5
6
7
8
14.9133 15.4767 16.2967 16.8167 17.8400 18.0600 20.1333 1.000
1.000
1.000
1.000
1.000
1.000
1.000
28.5167 1.000
74
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Table 3 Homogenous subsets of pulp density factor intent of desulphurization with performed Tukey Multiple Comparison tests. N
20.00 10.00 9.00 15.00 6.00 7.00 1.00 8.00 3.00 4.00 2.00 5.00 Sig.
3 3 3 3 3 3 3 3 3 3 3 3
Subset for alpha = .05 1
2
3
4
5
6
11
12
17.7767 22.6967 22.9167 23.5833 24.0500 25.4800 26.0167 28.1933 1.000
1.000
1.000
1.000
In addition to the analyses related to the change of sulphur form, the immediate and ultimate analyses which are calorific value (gross calorific value also known as the higher calorific value), ash content and emission value for treated lignite (Tunçbilek) with crude laccase were carried out together for the determination of metal contents before and after enzymatic treatment for explaining changes of coal quality (Table 6). As it can be observed from Table 6, our experimental findings demonstrate that a crude laccase of T. versicolor has removed total sulphur and increased calorific value of lignite. A 35% reduction of pyritic sulphur, a 13% reduction of sulphate and a 25% reduction of organic sulphur in a period of 30 min were achieved, for a particle size of 200 μm under optimal conditions with enzymatic desulphurization. Furthermore metal contents and sulphur emission value of coal were reduced with crude laccase. High ash in the coal not only reduces the thermal value of coal but also leads to production of fly ash, which is a major environmental problem. Moreover, there was reduction in the ash that it proposed the presence of relationship between rate of biodesulphurization of lignite and ash eliminated. But it should be stressed that the reduction of ash content is usually because of chemical process rather than biological treatment [28]. The reduction in ash content may be due to dissolution of mineral matters [29]. The sulphur emission value (EV) in grams of sulphur per megajoule Table 4 Homogenous subsets of incubation temperature factor intent of desulphurization with performed Tukey Multiple Comparison tests.
3 3 3 3 3 3 3
10
16.5467
3.2. Chemical evaluation of coal samples after crude laccase treatment
20.00 60.00 50.00 40.00 25.00 30.00 35.00 Sig.
9
15.4667
give maximum yield of desulphurization at ratio of 28.50% (p b 0.05) (Fig. 4). pH values of reaction medium were seemed to be effective on enzymatic yield of desulphurization of coal samples examined at experimental conditions. In the experiments to optimize the desulphurization conditions, pH 5.0 (p b 0.05) was found as optimum pH for the reactions giving maximum yield of desulphurization (28.8667%) (Fig. 5). As it can be seen from Table 5, desulphurization process in examined pH values is significantly different.
N
8
13.5500
1.000
Temperature (°C)
7
Subset for alpha = .05 1
2
3
4
5
19.7400 19.7500 21.3267 23.6367
1.000
1.000
1.000
1.000
28.4800 28.5000 1.000
1.000
1.000
1.000
1.000
28.5033 1.000
(g S/MJ) is the total sulphur content (wt.%) divided by calorific value (MJ). Increase of sulphur emission value causing negative environmental effect is one of the most significant indicators of air pollution. High value of EV indicates that there is a great environmental impact, while low values indicate a small impact [24]. 3.3. Proposed desulphurization mechanism From the results of our experiments performed to reveal the effect of crude laccase treatment on coal desulphurization and its optimization, it can be easily concluded that crude laccase obtained from T. versicolor is capable of sulphur removal from lignite coal samples examined. As for the removal of organosulphur compounds, which cause serious problems for petroleum industry, related products treated with the extracellular enzymes of some white rot fungi has been reported by some authors [19]. Schreiner et al. showed oxidation of thianthrene, one of sulphur compounds, by the ligninase of Phanerochaete chrysosporium which is white rot fungus [30]. Previously conducted research study has shown that lignin-degrading basidiomycete Coriolus versicolor (presently it is known as T. versicolor) exhibited metabolic response against sulphur-containing heterocyclic compounds [31]. Our desulphurization studies conducted with culture supernatant which has high laccase activity have clearly indicated that treatment of coal samples with crude laccase could result in reduction in their total sulphur content to some extent. There are some concerns about laccase that it may not be the only enzyme playing role in desulphurization process. Our previous study showed that T. versicolor and P. chrysosporium biomasses, which are white rot fungi, were potential organisms for biodesulphurization of coal at 40% efficiency [32]. Van Hamme et al. studied a model organic sulphur compound, dibenzyl sulfide (DBS) metabolism of white rot fungi and reported that they are able to metabolize a broad range of anthropogenic chemicals and hydrocarbons through intracellular cytochrome p-450 and extracellular enzymes [19]. They found 50 40 30 20 10 0 20
25.2467
1.000
1.000
Sulphur removal (%)
Pulp density (w/v)
25
30
35
40
50
60
Temperature (oC) Fig. 4. Influence on incubation temperature of desulphurization with crude laccase (pH 6, 5% of pulp density and 30 min).
Sulphur removal (%)
P. Aytar et al. / Fuel Processing Technology 92 (2011) 71–76
50
75
Table 6 Characteristics and initial and ultimate metal analyses of Tunçbilek lignite (before and after enzymatic treatment).
40 30 20 10 0 3,0
3,5
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
8,0
pH Fig. 5. Effect on medium pH of desulphurization with crude laccase (35 °C, 5% of pulp density and 30 min).
that the C–S bond in alkyl bridge of DBS is the target of oxidation by fungal cultures and the metabolism proceeds from DBS to dibenzyl sulphoxides followed by dibenzyl sulphone, prior to C–S bond cleavage. According to the research of Van Hamme et al., ring oxidation and opening occur only after this cleavage. The main factor involved in the metabolic conversion of dibenzyl sulphoxide to dibenzyl sulphone is shown to be enzyme consortium. Therefore, in this study, findings were lower than our previous study with same coal provided from Tunçbilek [32]. Extracellular enzyme source used for the biodesulphurization process interacted with organic fraction of coal samples. Probably, laccase and other extracellular enzymes take part on first oxidation step of desulphurization process. To the best of our knowledge, final degradation of the monomeric intermediates had been believed to be catalyzed by intracellular enzymes such as dioxygenases. Earlier studies on the application of laccase were promising. Dordick and co-workers studied the use of horseradish peroxidase and several other oxidative enzymes on coal-related compounds in organic media. They show that horseradish peroxidase, laccase, and chloroperoxidase are capable of oxidizing dibenzothiophene in 25% dimethylformamide [33]. In another study, enzymatic oxidation of DBT was evaluated by horseradish peroxidase, ligninolitic enzyme and also laccase. Reactions were carried out in monophasic organic media containing 25% (v/v) acetonitrile. Results show that 60% of DBT is converted into dibenzothiophene sulfoxide (12%) and dibenzothiophene sulfone (46%) [34]. A similar enzymatic desulphurization process using chloroperoxidase (CPO) from Caldariomyces fumago coupled with distillation at 325 °C oxidizes and removes preferentially the organosulphur compounds from a diesel at ambient temperature and atmospheric pressure [35,36]. Despite the elimination of up to 83% of organosulphur compounds, 2 mg of diesel was diluted in 1 ml of 20% acetonitrile-acetate buffer system which makes it inappropriate for industrial scale. Villasenor et al. studied DBT oxidation catalyzed by a laccase enzyme in the presence of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-
Total sulphur (%) Organic sulphur (%) Pyritic sulphur (%) Sulphate (%) Ash content (%) Calorific value (cal/g) Emission value (EV) Iron (mg/l) Zinc (mg/l) Manganese (mg/l) Copper (mg/l) Nickel (mg/l) Chromium (μg/l) Cobalt (μg/l) Cadmium (mg/l) Lead (μg/l) Magnesium (mg/l) Aluminum (mg/l)
Untreated coal
Treated with laccase
2.59 0.75 1.45 0.39 20.47 5182 0.119 5.4 ± 0.4 740 ± 2.4 30.5 ± 5.1 1.8 ± 0.2 27 ± 0.4 286 ± 53.4 661 ± 37.9 38.1 ± 0.1 822 ± 1.3 1520 ± 3.4 3.8 ± 0.4
1.84 0.56 0.94 0.34 16.97 5811 0.075 3 ± 0.1 639 ± 6.3 22.3 ± 0.8 1.1 ± 0.0 10 ± 0.3 150 ± 5.6 279 ± 4.2 4.8 ± 0.1 462 ± 3.3 886 ± 6.3 0.9 ± 0.0
sulfonic acid) (ABTS) [37]. However, the result found is a rather low rate process hindering the use of laccase system as a practical process for deep desulphurization. The limiting step of this process seems to be the oxidation of DBT by oxidized ABTS, rather than the oxidation of ABTS by laccase. Furthermore, enzyme catalysis by laccase would require the development of a much faster reaction scheme with a better mediator than ABTS. Ichinose et al. demonstrated that 4methyldibenzothiophene found in fossil fuel as a recalcitrant impurity was converted to water-extractable products such as hydroxymethylDBT-5-dioxide by developing an organic solvent-resistant fungal reaction system [38]. 4. Conclusions From the results it is clear that an enzymatic desulphurization method with crude laccase may be a welcomed alternative due to short time treatment compared to sulphur removal with biomass having the prolonged incubation time. Rather than refractory sulphur compounds, dealing with genuine fossil fuel like coal will open up new challenges to solve. Further study on solving a complete fungal desulphurization pathway will provide a novel strategy for biological sulphur removal. More efforts should be attempted in this direction. Acknowledgements We thank Coal Quality and Testing Laboratory from Garp Lignite Administrations and General Directorate of Mineral Research & Exploration for coal analyses. Also, we would like to express our
Table 5 Homogenous subsets of medium pH factor intent of desulphurization with performed Tukey Multiple Comparison tests. pH
N
Subset for alpha = .05 1
7.50 8.00 6.50 7.00 3.50 3.00 5.50 4.50 4.00 6.00 5.00 Sig.
3 3 3 3 3 3 3 3 3 3 3
2
3
4
5
6
7
8
9
10
11
11.2667 13.2367 14.6667 16.1667 17.3667 20.0867 22.7900 25.4933 26.4700 28.4667 1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
28.8667 1.000
76
P. Aytar et al. / Fuel Processing Technology 92 (2011) 71–76
gratitude to Chemistry Department at Balıkesir University for ICP measurements, Eskişehir Osmangazi University Natural and Applied Science Research Center and Dr. Coşkun Kuş from Selçuk University in Turkey for statistical analyses. References [1] S.S. Tripathy, R.N. Kar, S.K. Mishra, I. Twardowska, L.B. Sukla, Effect of chemical pretreatment on bacterial desulphurization of Assam coal, Fuel 77 (1998) 859–864. [2] F. Kargi, J. Robinson, Removal of sulphur compounds from coal by the thermophilic organism Sulfolobus acidocaldarius, Applied and Environmental Microbiology 44 (1985) 878–883. [3] M. Balat, Turkey's major lignite fields and significance of lignite for energy necessity, Energy Sources, Part B:Economics, Planning and Policy 3 (2008) 13–25. [4] N. İnaner, E. Nakoman, Properties of lignite deposits in western Turkey, 57th Annual Meeting of the International Committee for Coal and Organic PetrologyICCP, Patras, Greece 18–23 (September 2005) 11. [5] P.N. Say, Lignite-fired thermal power plants and SO2 pollution in Turkey, Energy Policy 34 (2006) 2690–2701. [6] M.D. Dahlberg, R.L. Rohrer, D.J. Fauth, R. Sprecher, G. Olson, Biodesulphurization of dibenzothiophene sulfone by Arthrobacter sp. and studies with oxidized Illinois No.6 coal, Fuel 72 (1993) 1645–1649. [7] G. Olsson, L. Larsson, O. Holst, H. Karlsson, Desulphurization of low-sulphur coal by Acidianus brierleyi: effects of microbial treatment on the properties of coal, Fuel Processing Technology 33 (1993) 83–93. [8] Y. Izumi, T. Ohshiro, H. Ogino, Y. Hine, M. Shimao, Selective desulphurization of dibenzothiophene by Rhodococcus erythropolis strain D-1, Applied and Environmental Microbiology 60 (1994) 223–226. [9] G. Mohebali, A.S. Ball, B. Rasekh, A. Kaytash, Biodesulphurization potential of a newly isolated bacterium, Gordonia alkanivorans RIPI90A, Enzyme and Microbial Technology 40 (2007) 578–584. [10] T.L. Peeples, R.M. Kelly, Bioenergetics of the metal/sulphur-oxidizing extreme thermoacidophile, Metallosphaera sedula, Fuel 72 (1993) 1619–1624. [11] A. Verdin, S.A. Lounès-Hadj, R. Durand, Degradation of benzo[a]pyrene by mitosporic fungi and extracellular oxidative enzymes, International Biodeterioration and Biodegradation 53 (2004) 65–70. [12] L. Roy-Arcand, F.S. Archibald, Direct dechlorination of chlorophenolic compounds by laccases from Trametes (Coriolus) versicolor, Enzyme and Microbial Technology 13 (1991) 194–203. [13] M.A. Pickard, R. Roman, R. Tinoco, R. Vazouez-Duhalt, Polycylic aromatic hydrocarbon metabolism by white rot fungi and oxidation by Coriolopsis gallica UAMH 8260 laccase, Applied and Environmental Microbiology 65 (1999) 3805–3809. [14] A. Schultz, U. Jonas, E. Hammer, F. Schauer, Dehalogenation of chlorinated hydroxybiphenyls by fungal laccase, Applied and Environmental Microbiology 67 (2001) 4377–4381. [15] I. Nilsson, A. Möller, B. Mattiason, M.S. Rubindamayugi, U. Welander, Decolorization of synthetic and real textile wastewater by the use of white-rot fungi, Enzyme and Microbial Technology 38 (2006) 94–100. [16] L. Gonsalvesh, S.P. Marinov, M. Stefanova, Y. Yürüm, A.G. Dumanli, G. DinlerDoganay, N. Kolankaya, et al., Biodesulphurized subbituminous coal by different fungi and bacteria studied by reductive pyrolysis. Part 1: ınitial coal, Fuel 87 (2008) 2533–2543. [17] G. Willmann, R. Fakoussa, Extracellular oxidative enzymes of coal-attacking fungi, Fuel Processing Technology 52 (1997) 27–41.
[18] C.F. Gökçay, N. Kolankaya, F.B. Dilek, Microbial solubilization of lignites, Fuel 80 (2001) 1421–1433. [19] J.D. Van Hamme, E.T. Wong, H. Dettman, M.R. Gray, M.A. Pickard, Dibenzyl sulfide metabolism by white rot fungi, Applied and Environmental Microbiology 69 (2003) 1320–1324. [20] A. Leonowicz, A. Matuszewska, J. Luterek, D. Ziegenhagen, M. Wasilewska, N. Cho, M. Hofrichter, J. Rogalski, Biodegradation of lignin by white rot fungi, Fungal Genetics and Biology 27 (1979) 175–185. [21] R. Hayatsu, R.G. Scott, R.E. Winans, L.P. Moore, R.L. McBeth, M.H. Studier, Ligninlike polymers in coals, Nature 278 (1979) 41–43. [22] American Society for Testing and Materials, Standart test methods for sulphur in the analysis sample of coal and coke using high-temperature tube furnace combustion methods, Annual Book of ASTM Standards, 2004. [23] ASTM D 2492, Standard Test Method for Forms of Sulphur in Coal, 2002. [24] A. Aller, O. Martinez, A. De Linaje, R. Mendez, A. Moran, Biodesulphurisation of coal by microorganisms isolated from the coal itself, Fuel Processing Technology 69 (2001) 45–57. [25] C. Watson, Analytical methods for the determination of trace metals and other elements, Official and Standardized Methods of Analysis, The Royal Society of Chemistry, Cambridge, 1994, pp. 457–458. [26] N. Aktaş, H. Çiçek, A.Ü. Taşpınar, G. Kibarer, N. Kolankaya, A. Tanyolaç, Reaction kinetics for laccase-catalyzed polymerization of 1-naphtol, Bioresource Technology 80 (2001) 29–36. [27] C.L. Shah, J.A. Abbott, N.J. Miles, X. Li, X. Jianping, Sulphur reduction evalution of selected high-sulphur Chinese coals, Fuel 81 (2002) 519–529. [28] M.J. Grossman, M.K. Lee, R.C. Prince, K.K. Garett, G.N. George, I.J. Pickering, Microbial desulphurization of a crude oil middle distillate fraction: analysis of the extent of sulphur removal and the effect of removal on remaining sulphur, Applied and Environmental Microbiology 65 (1999) 181–188. [29] C. Acharya, L.B. Sukla, V.N. Misra, Biological elimination of sulphur from high sulphur coal by Aspergillus-like fungi, Fuel 84 (2005) 1597–1600. [30] R. Schreiner, S. Stevens, M. Tien, Oxidation of thianthrene by the ligninase of Phanerochaete chrysosporium, Applied and Environmental Microbiology 54 (1988) 1858–1860. [31] H. Ichinose, M. Nakamizo, H. Wariishi, H. Tanaka, Metabolic response against sulphur-containing heterocyclic compounds by the lignin-degrading basidiomycete Coriolus versicolor, Applied Microbiology and Biotechnology 58 (2002) 517–526. [32] P. Aytar, M. Şam, A. Çabuk, Microbial desulphurization of Turkish lignites by white rot fungi, Energy and Fuels 22 (2008) 1196–1199. [33] J.S. Dordick, K. Ryu, J.P. McEldoon, Enzymatic catalysis on coal-related compounds in organic media: kinetics and potential commercial applications, Resource Recovery and Conservation 2–3 (1991) 195–209. [34] L.S. Madeira, V.S. Ferreira-Leitao, E.P. Silva Bon, Dibenzothiophene oxidation by horseradish peroxidase in organic media: effect of the DBT:H2O2 molar ratio and H2O2 addition mode, Chemosphere 71 (2008) 189–194. [35] M. Ayala, R. Tinoco, V. Hernández, P. Bremuntz, R. Vazquez-Duhalt, Biocatalytic oxidation of fuel as an alternative to biodesulphurization, Fuel Processing Technology 57 (1998) 101–111. [36] M. Ayala, N.R. Robledo, A. Lopez-Munguia, R. Vazquez-Duhalt, Substrate specificity and ionization potential in chloroperoxidasecatalyzed oxidation of diesel fuel, Environmental Science and Technology 34 (2000) 2804–2809. [37] F. Villasenor, O. Loera, A. Campero, G. Viniegra-Gonzalez, Oxidation of dibenzothiophene by laccase or hydrogen peroxide and deep desulphurization of diesel fuel by the later, Fuel Processing Technology 86 (2004) 49–59. [38] H. Ichinose, H. Wariishi, H. Tanaka, Bioconversion of recalcitrant 4-methyldibenzothiophene to water-extractable products using lignin-degrading basidiomycete Coriolus versicolor, Biotechnology Programme 15 (1999) 706–714.