Laboratory and Semipilot Bioreactor Feasibility Tests for

Laboratory and Semi-Pilot Bioreactor Feasibility Tests for. Desulphurization of Turkish Lignite using Leptospirillum ferriphilum. Srabani Mishra. 1, 2...
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Article Cite This: Energy Fuels 2018, 32, 2869−2877

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Laboratory and Semipilot Bioreactor Feasibility Tests for Desulphurization of Turkish Lignite using Leptospirillum ferriphilum Srabani Mishra,†,‡,§ Ata Akcil,*,† Sandeep Panda,† and Ismail Agcasulu† †

Mineral-Metal Recovery and Recycling (MMR&R) Research Group, Mineral Processing Division, Department of Mining Engineering, Suleyman Demirel University, TR32260 Isparta, Turkey ‡ Environment and Sustainability Department and §Academy of Scientific and Innovative Research (AcSIR), CSIR−Institute of Minerals and Materials Technology (CSIR−IMMT), Bhubaneswar 751013, Odisha, India ABSTRACT: The low rank coal lignite stands out to be a profuse source of energy in Turkey and accounts for nearly 43% of its total fossil fuel production. However, the high sulfur content associated with lignite averts its application in several sectors due to various health and environmental issues. In the present work, biodesulphurization of Turkish lignite was studied in 1 and 20 L aerated bioreactors using an iron oxidizing acidophile, Leptospirillum ferriphilum (L. ferriphilum). Additionally, the effect of Span 80 (S80) on biodesulphurization of the sample was studied, under the optimum concentration derived from our previous shake flask study, in order to notice its effect under scale-up aerated bioreactor conditions. Under lab scale (using 1 L bioreactors), in the absence of S80, L. ferriphilum could desulphurize a maximum of 65.6% sulfur from the lignite sample, while 56% desulphurization was achieved in the presence of 0.05% (v/v) S80. Further scale-up studies under semipilot conditions (in 20 L reactors) indicated a maximum desulphurization of 63% from the sample in absence of S80. The pH, oxidation−reduction potential (ORP), and Fe2+ iron and total iron concentrations were periodically monitored in all the reactors during the course of the experiments. XRD and FTIR characterization of the lignite samples pre and post biodesulphurization provided valuable information on the structural and phase changes due to the microbial action.

1. INTRODUCTION Coal has served as a proficient source of energy for centuries. Its applications extend to various sectors including cement, aluminum, steel manufacturing industries etc. However, the environmental issues associated with coal combustion, leading to the release of sulfur oxides and dioxides into the atmosphere, curbs its utilization in several areas. Therefore, several attempts are being made to reduce the sulfur content in coal through an effective and efficient method. Several factors such as type of coal, amount of sulfur in coal, distribution pattern of different forms of sulfur, etc. play a vital role in the process of coal desulphurization.1 The physicochemical techniques of sulfur removal either remain futile toward the reduction of organic sulfur or release high amount of SO2 into the atmosphere along with the use of high temperature and pressure.2,3 Therefore, in the present scenario, the biological approach is being focused and investigated more because of its several advantages over the physicochemical methods.3,4 It primarily offers the benefits of low operation cost, with concomitant production of value added products.5 Half of the world’s coal deposit comprises of low rank coals.6 Turkey is a country with vast reserves of lignite and nearly 43% of its fossil fuel production constitutes lignite.7 The high moisture, ash and sulfur content of low rank coals make it inapt for use as an energy source. Different microbial species including bacteria and fungus have been reported for the biodesulphurization of Turkish lignites at shake-flask level. In one of the studies, Rhodococcus rhodochrous was used for desulphurization of Mengen lignite, which led to a total sulfur removal of 30.2% from the sample.8 Apart from bacterial species, fungal species such as Phanerochaeta chrysosporium and © 2018 American Chemical Society

Trametes versicolor have been used to carry out shake-flask experiments for sulfur removal from low rank coal.9 In yet another study, Aytar et al.10 have carried out the oxidative desulphurization of Turkish lignite in shake-flasks using the enzymes derived from Trametes versicolor ATCC 200801, where they have achieved a total sulfur removal of 29%. They have also carried out the biodesulphurization of Tuncbilek lignite using Phanerochaeta chrysosporium ME446 and Trametes versicolor ATCC 200801, where they got a maximum sulfur removal of 40% using Trametes versicolor. In addition to that, use of Aspergillus sp. by Aytar et al.11 for the shake-flask biodesulphurization of coal obtained from the Mihaliccik open mine in Turkey resulted in a maximum organic sulfur removal of 38% and sulfate sulfur removal of 51%. A combination of gravity (MGS)/flotation technique along with biodesulphurization was used by Aksoy et al.12 for the desulphurization of lignite samples obtained from open and underground mines of Koyunagili, Mihaliccik Province, Eskisehir. In their investigation, the authors observed that the sulfur content reduced from 3.25 to 1.56% in the MGS sample following microbial treatment. Similarly, the sulfur content reduced from 3.55 to 1.81% in the sample treated with flotation technique followed by microbial treatment. Certain acidophilic strains such as Sulfolobus solfataricus and Acidithiobacillus ferrivorans have been also reported for the biodesulphurization of Turkish lignite, where the sulfur removal were observed to be 57.1 and 33%, respectively.13,14 Received: October 11, 2017 Revised: January 31, 2018 Published: January 31, 2018 2869

DOI: 10.1021/acs.energyfuels.7b03069 Energy Fuels 2018, 32, 2869−2877

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Energy & Fuels Although a lot of biodesulphurization studies have been reported to date, it is interesting to note that most of these studies have been performed at lab scale (in shake flaks) w.r.t. coal. Very few reports on scale-up coal biodesulphruization studies are available. For the scale-up studies, mostly stirred tank reactors and airlift reactors have been used.15 Apart from that, Cara et al.16 have reported the packed column leaching of a 5 kg coal sample using a mixed culture of acidophilic strains, where they achieved a maximum sulfur removal of 24% in 125 days. Besides, a pilot scale study (in a 525 L batch bioreactor) has been carried out with Tabas coal using Acidithiobacillus ferrooxidans, where a maximum sulfur removal of 20% was achieved in 14 days, out of which 45% comprised pyritic sulfur.17 Reports on higher scale biodesulphurization of US coal carried out by Pathak et al.18 in a 8L bench scale stirred tank reactor using Acidithiobacillus ferrooxidans yielded a maximum pyritic sulfur removal of 67%. In addition to that, heap bioleaching experiments with semianthracite coal have been reported by Cara et al.19 In the study, a pile was formed with 6 ton of gravity middlings coal sample having a grain size −12 + 0.5 mm. A maximum desulphrization of 23% was achieved, out of which 39% constituted of pyritic sulfur. Scale-up studies are highly essential to provide better insights into any developed process so that novel aspects can be proposed w.r.t. their industrial application in the future. The feasibility of experimental outcomes relies on its practicality at a higher scale. Because of the importance of such studies, the present work focuses on the scale-up biodesulphurization aspects of a Turkish Lignite sample using an iron oxiding acidophile, Leptospirlillum ferriphilum based on the outcomes/ optimum conditions obtained from our lab scale shake flask experiments.20 It is very important to note that scale-up aspects on biodesulpurization of Turkish Lignites has not been reported in literature and the use of Leptospirillum ferriphilum for Turkish coal desulphurization also stands out to be a novel aspect of research.

2.3. Biodesulphurization Experiments. The efficiency of L. ferriphilum toward biodesulphurization of Turkish Lignite (herein referred to as “TL” throughout the manuscript) was studied using double layered glass reactors of Rettberg, Germany. The optimized parameters obtained through our previous shake-flask studies were taken into consideration for the present studies.20 The studies (experimental conditions discussed in subsections below) were initially performed in a laboratory scale reactor (1 L working volume) followed by a semipilot scale reactor (20 L working volume). A water heating system was connected to each of the reactors, which circulated heated water into the double layers of the reactors for temperature maintenance. The 20 L reactor system, having three impellers (two axial at the top and one radial at the bottom of the reaction vessel), assisted in proper mixing of the sample. External aeration pipes were supplied to the reactors through its external opening on top of the lid in order to provide proper aeration in the reactors. The dimensions of the 1 L reactor were: internal height,16.5 cm; internal diameter of the reaction vessel, 10 cm; outer diameter of lid, 14.5 cm with 3 openings (for sample inlet and probes), 3 cm inner diameters each. Likewise, the dimensions of the 20 L reactor were: internal height, 65 cm; internal diameter of the reaction vessel, 20 cm; outer diameter, 30 cm with 5 openings. The experimental setup of the reactor system is shown in Figure 1.

2. MATERIALS AND METHODS

Figure 1. Experimental setup of (a) lab scale, 1 L reactor (b) semipilot, 20 L reactor.

2.1. Coal Sample. The sample used in the present study was collected from the TKI ̇ Ç an Linyit Iṡ ļ etmesi Md., Can, Canakkale, Turkey. The“as received” sample (+10 cm) was grounded and sieved to obtain a final size fraction of −75 + 45 μm. A representative portion of the grounded sample obtained through the conventional coning and quartering method was used for the biodesulphurization experiments after sterilization.4 2.2. Microbial Growth. The acidophilic strain, L. ferriphilum (DSM 14647) used in the present study was obtained from culture collection center of DSMZ, Germany. Prior to biodesulphurization experiments, the strain was activated in DSMZ recommended media having the following composition: • Solution A (mg/950 mL): (NH4)2SO4, 132; MgCl2·6H2O, 53; KH2PO4, 27; CaCl2·2H2O, 147; and trace element solution, 1 mL. The trace element solution had the following chemical composition (mg/L): MnCl2·2H2O, 62; ZnCl2, 68; CoCl2· 6H2O, 64; H3BO3, 31; Na2MoO4, 10; and CuCl2·2H2O, 67. • Solution B: 20 g of FeSO4·7H2O in 50 mL of 0.25 N H2SO4. The pH of Solution A was adjusted to 1.8 with H2SO4 and the pH of Solution B was maintained at 1.2, before keeping it for sterilization. Solution A and B were sterilized individually in an autoclave maintained at 112 °C for 30 min and later mixed after autoclaving. The growth medium had a final pH of 1.8. Following activation, the microbial strain was further adapted with Fe2+ iron and coal by repeatedly transferring the culture on recommended medium. The adapted strain was subsequently used in the biodesulphurization experiments.

2.3.1. Lab and Semipilot Scale Bioreactor Tests. For the 1L bioreactor, two different sets of experiments were carried out. In one of the bioreactors, 0.05% v/v of chemical surfactant, Span 80 (optimized concentration obtained through shake flask studies) was added to the DSMZ recommended growth medium with 10% (v/v) of bacterial inoculum. The other bioreactor contained only the growth medium and 10% (v/v) of inoculum without addition of chemical surfactant. The pulp density of TL sample was maintained as 5% (w/ v) in both the reactors. The experiment was carried out for a period of 21 days and the reactor temperature was maintained at 32 °C through heated water circulation as discussed above. The reactors were continuously aerated using an air diffuser system. Stirring speed of the impeller was set at 150 rpm in order to make a direct comparsion for the biodesulphurization efficiency under similar agitation. Taking into account the results derived from 1 L bioreactors tests, the experiments were further scaled-up on a semipilot level. In this experiment, similar operating conditions used for the lab scale reactor were considered, i.e., pulp density, 5% (w/v); bacterial inoculum, 10% (v/v); temperature, 32 °C, time, 21 days; and agitation speed, 150 rpm. The changes in pH, oxido-reductive potential and Fe2+ iron and total iron concentration in all the reactors (used for lab and semipilot scale studies) were regularly monitored at a time interval of every 3 days. Following biodesulphurization, the microbial-treated coal samples were washed with 0.01N dilute HCl prior to analytical 2870

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Energy & Fuels studies, to remove any passivation and/or unwanted microbial products formed during the course of the experiments. 2.4. Analytical Studies. 2.4.1. Ferrous Iron Estimation, OxidoReductive Potential and pH Measurements. All the three reactors were regularly monitored for ferrous (Fe2+) iron, oxido-reductive potential (ORP) and pH. The concentration of Fe2+ iron in the biodesulphurization system was estimated by titration using the ophenalthronine method.21 A Thermo Scientific Orion DUAL STAR pH/ISE meter (reference electrode Ag/AgCl) was used to measure the changes in ORP and pH of the system. The system pH was maintained at 1.8 throughout the experiment by addition of dilute H2SO4. Analytical-grade chemical reagents were utilized in the present investigation. 2.4.2. Atomic Absorption Spectrophotometer (AAS). The total iron concentration in the reactors during the biodesulphurization experiment was monitored through atomic absorption spectrophotometer (AAS model Agilent AA240FS). The total working volume of the reactors was regularly maintained by the addition of required volume of pH 1.8 dH2O, which compensated for the volume loss due to evaporation or sampling. 2.4.3. Sulfur Analysis and Proximate Analysis. The change in total sulfur content following biodesulphuriation was determined using a LECO 932 CHNS Elemental Analyzer. The proximate analysis of the treated sample was carried out using a LECO TGA 701. 2.4.4. XRD and FTIR Analysis. The mineralogical changes in preand post-microbial- treated samples was determined through an X-ray powder diffractometer (Philips X’pert Pro, Panalytical) with CuKa (k = 1.54 Å) radiation and a programmable divergence slit. The voltage of X-ray powder diffractometer was 40Kv and its current was 20 mA. The spectral patterns of the original and treated samples was determined through Fourier transform infrared spectroscopy (FTIR). The FTIR instrument (PerkinElmer, FTIR- Spectrum GX model, 633 nm IRHeNelaser) had a power less than 1 mW and a scanning speed of 0.2 cm/sec.

Table 1. Biodesulphurization of TL Sample in 1 L Bioreactors reactor s

initial sulfur (%)

final sulfur (%)

% sulfur removal

BR1 BR2

7.63

2.62 3.36

65.6 56

L. ferriphilum did not have any effect on biodesulphurization, i.e., sulfur removal was seen to be lowered nearly by 9%, which was also accompanied by slower iron oxidation (see the next section for further discussion). Rather, a higher biodesulphurization was obtained under similar operation conditions, when only L. ferriphilum was present in the leaching media. 3.1.1. Changes in Fe2+, ORP, System pH, and Total Iron in Lab Scale Reactors. Fe2+ iron in the media solution acts as source of energy for the microbe’s (L. ferriphilum) growth and metabolism. The microbial oxidation of pyrite (FeS2) leads to the generation of ferric sulfate (Fe2(SO4)3) through a series of steps. Because lignite samples are generally rich in pyritic sulfur content, the conversion of Fe2+ to Fe3+ facilitates the oxidative attack on the pyritic sulfur present in coal.22 Thus, the microbial activity assists in the oxidation of Fe2+ to Fe3+, which further attacks coal leading to the oxidation and release of pyritic sulfur present in coal as shown in the following equations. FeS2 + 7/2O2 + H 2O → FeSO4 + H 2SO4 2+

(1)

3+

In general, the reaction leading Fe to Fe conversion is approximately 1 × 106 times higher in the presence of the microbes.22 2FeSO4 + 1/2O2 + H 2SO4 → Fe(SO4 )3 + H 2O

3. RESULTS AND DISCUSSION In our preliminary shake flask studies, Span 80 (S80) was found to have a marginal effect on biodesulphurization, which improved nearly by 5% in the presence of surfactant.20 0.05% v/v of S80 was found to be the optimum concentration and increasing surfactant concentration had a negative effect on the biodesulphurization efficiency of the microbe. Although the concentration of surfactant was lower and a slight improvement in biodesulphurization was observed, however it was necessary to carry out further scale-up studies that could possibly provide better insights into the overall process efficiency and the effect of surfactant in real agitated bioreactor conditions. Agitated bioreactors provide maintainace of all the necessary conditions along with homogeneous mixing of the sample. Therefore, in the present study biodesulphurization of TL sample was tested using stirred tank reactors at lab (1 L) and semipilot (20 L) scale under continuous stirring and aerated conditions. 3.1. Lab-Scale Bioreactor Tests. The lab-scale bioreactor experiments were aimed at testing the optimum conditions obtained through shake flask experiments in view of a scale-up application of the microbial process. The bioreactor containing L. ferriphilum without S80 addition was designated as BR1, while the bioreactor containing L. ferriphilum with 0.05% S80 was designated as BR2. The sulfur removal obtained in both the reactors is shown in Table 1. As can been seen in the table, the desulphurization efficiency of the microbe was seen to be less when S80 was present in the reactor. In BR1, the biodesulphurization efficiency was observed to be 65.6%, whereas in BR2 it was observed to be 56%. The results obtained in the present study indicated that under continuous stirring/agitation and aerated conditions, S80 in the presence of

(2)

Ferric iron generated through microbial action is a strong oxidant, which acts on pyrite in the following manner: FeS2 + 2Fe3 + → 3Fe 2 + + 2S0

(3)

Following the above reaction, the elemental sulfur generated through the oxidation of pyrite can further be oxidized by the ferric iron as S0 + 6Fe3 + + 4H 20 → 6Fe 2 + + SO4 2 − + 8H+

(4)

2+

Changes in Fe iron concentration were regularly monitored in the bioreactors in order to trace the bacterial activity. As observed from Figure 2a, the Fe2+ iron concentration in BR1, contaning no surfactant, gradually decreased following the third day of incubation and became almost zero following the sixth day. However, in BR2, which contained 0.05% v/v surfactant, the rate of Fe2+ iron oxidation was slower in comparison to BR1. The decrease in Fe2+ iron concentration proceeded following the sixth day of incubation and became nearly zero after the ninth day. Surfactants have been reported of corrosion inhibition, where they prevent the oxidation of metals.23 Below the critical micelle concentration (CMC), the surfactant molecules tend to adsorb at the interface, thereby reducing the surface tension of the system. Above the CMC, the surfactant molecule form a monolayer, whereas the additional molecules come together to form micelles or multiple layers. Although shake-flask studies20 using L.ferriphilum had indicated nearly 5% enhancement in desulphurization, however, in the present case, proper agitation conditions along with aeration might have led to aggregation of surfactant molecules forming multiple layers on the coal surface and resulting in the slower oxidation of pyritic sulfur present in the coal. Earlier 2871

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Figure 2. Changes in (a) Fe2+, (b) ORP (mV), (c) pH, and (d) total iron concentration in lab scale (IL) reactors in presence and absence of S80.

Figure 3. Changes in (a) pH, ORP and (b) Fe2+, total iron concentration in 20 L bioreactor.

studies by Tuttle and Dugan24 related to the effect of organic compounds on the growth, Fe2+ and sulfur oxidation by Acidithiobacillus ferrooxidans had indicated that the presence of low molecular weight organic compounds inhibit the growth, iron and sulfur oxidation rates by the acidophile. Possible reasons behind it are the negative effects of organic compound on the iron-oxidizing enzyme system, abiological reaction of the organic compound with the Fe2+ iron, nonspecific disruption of the cell envelope or membrane by the organic compound etc. There is a possibility that the Fe2+ iron present in the medium might be reacting with Span-80, resulting in the inhibition or slowing down of the Fe2+ iron oxidation to ferric iron. Apart from that, studies carried out by An-an et al.25 have revealed the enhanced growth and metabolism of Acidithiobacillus ferrooxidans ATCC 23270 on S0 and CuFeS2 in the presence of nonionic surfactant, Tween 80. The change in composition of exopolymeric substances was stated as a possible reason behind the enhanced activity, which was also supported by the results of FTIR analysis. The down-regulation of extracellular proteins in the presence of surfactant, Tween 80, was also responsible for the increased efficiency of the acidophile. However, in the present study, a slow rate of Fe2+ iron oxidation was observed

in BR2 in the presence of surfactant. This indicated that presence of surfactant, S80, in the medium might be having a negative impact on the composition of exopolymeric substances secreted by L. ferriphilum (under the influence of continuous aeration and agitation), which could be another possible reason behind lowering of Fe2+ iron oxidation. With the gradual oxidation of Fe2+ in the media, ORP of the medium began to increase slowly along with the accumulation of ferric iron in the medium (Figure 2b). In case of BR1, the ORP was seen to steadily increase with time and the maximum value recorded was 607.6 mV. On the other hand, ORP was seen to suddenly decrease within the first 3 days of the experiment in BR2, which indicated that S80 probably hindered Fe2+ oxidation, however a slow increase in ORP was observed thereafter and 608.7 mV was recorded as maximum ORP by end of the experiment. The pH of the medium slowly began to drop following the third day of incubation (Figure 2c). The pH dropped from 1.8 to 1.6 in BR1 and 1.4 in BR2 by the end of the experiment. The drop in pH in BR2 was slower in comparison to BR1, which was concomitant with the rate of Fe2+ iron oxidation in both the reactors, respectively. The drop in pH and simulataneous 2872

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content and simultaneous increment in the fixed carbon content of the sample. 3.2.1. Changes in Fe2+, pH, ORP, and Total Iron under Semipilot Conditions. During the course of experiment in the 20 L reactor, the changes in the system pH, ORP, Fe2+ iron concentration and total iron concentration were also monitored periodically at an interval of every 3 days. As seen from Figure 3a, the system pH dropped from 1.8 to 1.44 that can be ascribed to the combined effect of eqd 1−4 (see section 3.1.1). The slight increment in the system pH from ninth day until the 15th day might be associated with the pyrite oxidation of coal, leading to the dissociation of pyrite particles and release of Fe2+ in the solution, which was rapidly consumed by the microbe. An equilibrium between the concurrent consumption of protons during Fe2+ oxidation, along with the release of protons due to ferric iron precipitation led to the development of an acidic pH in the medium.27 The ORP of the system increased from 395.6 to 619 mV by the end of the experiment (Figure 3a). It gradually increased until day 6 that indicated oxidation of Fe2+ in the media. A slight decrease in ORP was observed on day 9 that might be ascribed to pyrite oxidation (eq 1) releasing some Fe2+ into the media, that was further oxidized by the microbe which resulted in the increase of ORP thereafter. The Fe2+ content in the system was seen to decrease from 4.19 g/L to nearly 1.61 g/L by day 3 indicating microbial oxidation of the same (Figure 3b). The Fe2+ iron concentration in the system was almost nil by the sixth day of incubation, which was simultaneously accompanied by a decrease in the pH of the system. As discussed above, the DO was seen to reduce by day 3 and supply of oxygen in the reactor allowed L. ferriphilum to completely oxidize the Fe2+ iron in media. Thereafter, the Fe2+ content in the system was seen to slightly increase that might be ascribed to the pyrite oxidation from the sample. With time, L. ferriphilum oxidized the generated Fe2+ as a result of which its concentration was seen to vary until day 21. Similarly, the total iron concentration in the medium also increased from 4.04 to 14.11 g/L (Figure 3b). It rose sharply until day 12, after which its concentration slowy built up in the system. Increase in total iron concentration can be attributed to the release of iron (due to Fe2+ iron oxidation in media as well as oxidation of pyrite particles present in coal). 3.3. Analytical Characterization of TL Sample Pre- and Post-Biodesulphurization. 3.3.1. FTIR Analysis. The FTIR analysis of the original and L. ferriphilum treated samples was carried out in order to study any change in functional groups following treatment with the microbial isolate. Some changes in peak intensities were observed following treatment with the microbial isolate (shown in Figure 4). The FTIR spectra not only provides information regarding the changes observed in sulfur containing functional groups following microbial treatment, but also reveals the changes undergone in the mineral matter content of coal due to microbial attack.28 The depth from which the lignite sample is collected is one of the most significant factors, which reveals the amount of organic and inorganic matter that occurs in the lignite sample. FTIR spectroscopy is a practical approach, which determines the nature of organic matter in coal. The likely association of inorganic matter with the organic content of coal makes them a significant contributor to the FTIR spectrum. Crucial interactions between the organic and inorganic matters of coal hold a key role in the change of intensities for certain bands, along with major shifts in the band position.29 In the

oxidation of Fe2+ iron in media and pyrite from the TL sample can be explained based on the reactions in eqs 1−4 as discussed above. With gradual progress in the experiment followed by subsequent reactions, the total iron concentration also increased from 4.19 to 13.1 g/L in BR1 and 14.09 g/L in BR2 (Figure 3d). The increment in the total iron concentration can be attributed to the oxidation of Fe2+ to Fe3+in the leaching media along with pyrite oxidation from coal that resulted in structural changes in the coal matrix (analytical characterization of the sample discussed in section 3.3) thereby enabling release of iron from the coal sample. 3.2. Semipilot Scale Bioreactor Tests. Following biodesulphurization experiments in 1L reactors, further studies were carried out in 20L reactor by considering the observations obtained from lab scale studies. In the lab scale bioreactor studies, it was observed that the microbe under the influence of aeration and continuous stirring did not respond well to desulphurization of the TL sample in the presence of surfactant, S80. A possible explanation for this phenomenon has been given in section 3.1.1. Because lower sulfur removal was achieved in the presence of surfactant in 1L reactor, therefore, the biodesulphurization experiment in the 20 L reactor was carried out in absence of surfactant in order to observe the desulphurization efficiency at semipilot scale. With the increase in depth/height of the reactor, the dissolved oxygen (DO) content in the reactor is expected to decrease due to the diffusion of the same as a result of simultaneous biochemical reactions proceeding with time. The initial DO on day 0, i.e., at the start of the experiment was seen to be 5.05 mg/L, which reduced to 0.21 mg/L by day 3. In such systems, typically 1−2 mg/L of DO is sufficient to drive the biochemical reactions and support microbial activities.26 Considering these aspects, oxygen supply was intermittently supplied for 1 day (on day 4), due to which the DO was seen to improve. By day 6, the DO recorded in the reactor was nearly 3 mg/L. Within first 6 days, the oxygen consuming steps such as Fe2+ and pyrite oxidation steps (see eqs 1 and 2) were observed to be complete (further discussions in the next section 3.2.1). Thereafter, the DO in the reactor system was observed to be maintained at nearly 3−4 mg/L and there was no requirement for further oxygen supply to the system. The experiments were run for 21 days and the maximum sulfur removal achieved in the reactor was 63%. The initial sulfur was seen to reduce from 7.63 to 2.82% following treatment with L. ferriphilum. It can thus be concluded that the results were almost comparable to the lab-scale bioreactor studies and continuous supply of oxygen was not an essential requirement to achieve higher desulphurization. Our previous studies on the original TL sample had revealed that it contained 24.7% ash, 39.13% volatile matter, 16.72% fixed carbon, and 19.45% moisture content. The sulfur distribution studies had indicated that the lignite sample contained 2.71% of pyritic sulfur, 1.57% sulfate sulfur, and 2.59% organic sulfur.20 In the present study, the proximate analysis of the biotreated TL sample (20L reactor) revealed that the ash content in the sample was 18.2% following microbial treatment, whereas, the volatile matter content was 40.05%. There was a notable increase in the fixed carbon content, which was observed to be 22.2% and the moisture content in the treated sample was found to be 19.54%. Microbial action on the coal matrix led to removal of sulfur from coal resulting in structural and phase changes (discussed in section 3.4), which were responsible for the reduction in ash 2873

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Figure 4. FTIR spectra of original and microbially treated samples.

present study, the FTIR analysis of the treated sample was carried out following treatment in the 20 L reactor. Reduced intensities in the bands were observed between 3750−3500 cm−1, which corresponds to the vibrations of hydroxyl groups present in aluminosilicates or due to the OH stretching vibrations of molecular H2O.30,31 From earlier reports,32 it has been observed that kaolin exhibits a typical spectrum at 3697, 3669, 3645, and 3620 cm−1. Therefore, the reduced peak intensities observed in this region might be associated with the kaolinite minerals, which were attacked by the microbial strain leading to lower peak intensity in the FTIR spectrum following treatment. Minor changes observed in the range of 1750−1500 cm−1 might be associated with the sulfate minerals present in coal. Similarly, marginal changes observed in the range of 1250−1500 cm−1 corresponds to the −CH3 symmetric deformation, whereas the presence of −CH3 and −CH2 groups in cyclic structures may also partly contribute to this band.31 Formation of small shoulder peak in the range of 1250−1000 cm−1 might correspond to the Si−O stretching vibrations. This region reveals the presence of silicate minerals in the coal sample, whose peak intensity was reduced following treatment with the acidophlic strain.30 The reduced peak intensities observed in the range of 1000−750 cm−1 can be attributed to aromatic structures and the presence of aluminosilicate phases in coal.30,31,33 Likewise, reduced peak intensities observed in the range of 750−500 cm−1 can be attributed to Si − O bending vibrations.30 Microbial attack might have resulted in the loss of silicate impurities from the sample leading to reduced peak intensity in this region. 3.4. XRD Analysis. The mineralogical phase changes following microbial attack were studied through XRD analysis of the original and treated samples (shown in Figure 5). Some major changes were observed following treatment of the coal sample with the acidophilic strain. Certain peaks had disappeared along with the formation of some new peaks following microbial treatment. Most of the pyrite peaks had disappeared following microbial attack, whereas new quartz peaks had surfaced. The peak intensities of kaolinite were also found to be reduced upon microbial treatment. The presence of clay minerals, quartz, pyrite, feldspar, gypsum, etc., in Turkish lignite samples has been previously reported by several researchers.34,35 Origin of minerals in the coal is based on the source, mode of occurrence and prevalent environmental conditions.36 A reduction in the peak intensities of the mineral phases, along with the formation of certain new peaks in the treated sample provided a clear evidence of the acute effect that the microbial strain had on the lignite sample. Disruption of the coal matrix by microbial attack led to the removal of pyritic sulfur associated in the form of discrete physical entities with

Figure 5. XRD analysis of original and microbially treated samples (P, pyrite; K, kaolinite; Q, quartz).

the coal. Microbial action also led to the reduction in peak intensities of the kaolinite phases, ultimately resulting in overall mineralogical phase changes undergone by the treated sample. 3.5. Probable Mechanism and Mode of Action of L. ferriphilum for TL Biodesulphurization under Scale-up Conditions. In recent years, some reports have been made regarding the effect of surfactant towards sulfur oxidation or desulphurization, where an enhancement in the microbial activity has been observed in the presence of surfactant. Studies carried out by Cheng-gui et al.37 have revealed the effect of two different surfactants on the sulfur oxidizing activities of Acidithiobacillus albertensis BY-05. They have observed that the sulfur oxidizing activity of the microbial strain was enhanced only at specific surfactant concentrations. According to reports, the oxidation of sulfur particles is initiated with the attachment of the microbial cells to hydrophobic sulfur particles, which is originally inhibited due to the hydrophilic extracellular layer of bacterial cells. Extracellular polymeric substances (EPS) are secreted by the bacterial cells to facilitate efficient attachment with the substrate and the amount and composition of the EPS secreted depends on the growth substrate of the bacterial cells.37−39 Studies carried out by He et al.39 have indicated that the ferric iron ions present in EPS play a crucial role in the attachment of the bacterial cells to the mineral surfaces. During attachment to pyrite surfaces, the positively charged EPS plus ferric iron complex undergoes an electrochemical interaction with the negatively charged pyrite surface, further leading to the dissolution of sulfide via oxidation. Thus, the Fe3+ ion in EPS stands out to be one of the most imperative aspects, which determines the bioleaching efficacy of the microbes. The EPS layer aids as the reaction space for the oxidation of Fe2+ iron to ferric iron. This biooxidation preferably takes place in the EPS layer instead of the bulk solution.40,41 In the present study, the presence of surfactant in the medium might be interfering with the EPS secretion by the microbial strain (under agitation and continuous aerated conditions as discussed in section 3.1.1). This in turn might be affecting the oxidation of Fe2+ to Fe3+ iron and subsequent oxidation of pyritic sulfur present in coal. The mechanism of microbial action in both the reactors are shown in Figure 6 a, b. 2874

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Figure 6. Schematic representation of the microbial action in (a) 1 L and (b) 20 L bioreactor.

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CONCLUSION

AUTHOR INFORMATION

Corresponding Author

Biodesulphurization of Turkish lignite was studied in the presence and absence of Span 80 in a 1 L bioreactor using Leptospirillum ferriphilum DSM 14647. A maximum desulphurization of 65.6% was achieved in the 1L bioreactor in the absence of Span 80. Optimum conditions obtained in 1L bioreactor were used to carry out biodesulphurization in 20L bioreactor, where a maximum sulfur removal of 63% could be achieved. XRD results of the original and treated samples showed reduced peak intensities of pyrite and kaolinite peaks, while some new quartz peaks were also observed following biodesulphurization. FTIR analysis of the samples revealed some structural changes that occurred because of microbial action. The present study provided the optimum parameters to carry out coal biodesulphurization on a scale-up basis in stirred tank bioreactors.

*E-mail: [email protected]. ORCID

Ata Akcil: 0000-0002-9991-0543 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.M. and A.A. kindly acknowledge the Scientific and Technological Research Council of Turkey for the award of the TUBITAK 2216 International Fellowship to S.M. in order to successfully carry out a part of her PhD research work at the MMR&R research group, SDU, Turkey. S.M. also sincerely thanks the Council of Scientific and Industrial Research (CSIR), India, and the Director, CSIR-IMMT, for their kind permission to visit SDU, Turkey, for the aforesaid purpose under the TUBITAK Fellowship program. S.P. kindly acknowledges the Scientific and Technological Research Council of 2875

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Turkey for the award of TUBITAK 2216 Post Doctoral Fellowship.

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