Laboratory and Semi-Pilot Bioreactor Feasibility Tests for

3 hours ago - Laboratory and Semi-Pilot Bioreactor Feasibility Tests for Desulphurization of Turkish Lignite using Leptospirillum ferriphilum. Srabani...
2 downloads 11 Views 955KB Size
Subscriber access provided by READING UNIV

Article

Laboratory and Semi-Pilot Bioreactor Feasibility Tests for Desulphurization of Turkish Lignite using Leptospirillum ferriphilum Srabani Mishra, Ata Akcil, Sandeep Panda, and Ismail Agcasulu Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b03069 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Energy & Fuels is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Laboratory and Semi-Pilot Bioreactor Feasibility Tests for Desulphurization of Turkish Lignite using Leptospirillum ferriphilum Srabani Mishra1, 2, 3, Ata Akcil1*, Sandeep Panda1, Ismail Agcasulu1 1

Mineral-Metal Recovery and Recycling (MMR&R) Research Group, Mineral Processing Division, Department of Mining Engineering, Suleyman Demirel University, TR32260 Isparta, Turkey 2

3

Environment and Sustainability Department, CSIR – Institute of Minerals and Materials Technology (CSIR – IMMT), Bhubaneswar-751013, Odisha, India

Academy of Scientific and Innovation 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 sulphur 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 1L and 20L 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 1L bioreactors), in the absence of S80, L. ferriphilum could desulphurize a maximum of 66.5% sulphur from the lignite sample, while 57.1% desulphurization was achieved in presence of 0.05% (v/v) S80. Further scale-up studies under semi-pilot conditions (in 20L reactors) indicated a maximum desulphurization of 63% from the sample in absence of S80. The pH, oxidation-reduction potential (ORP), 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. Keywords: Lignite; Biodesulphurization; Leptospirillum ferriphilum; Bioreactor; Span 80 *Corresponding Author Information: Ata Akcil, Email – [email protected]

1 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. Introduction Coal has served as a proficient source of energy since centuries. Its applications extend to various sectors including cement, aluminium, steel manufacturing industries etc. However, the environmental issues associated with coal combustion, leading to the release of sulphur oxides and dioxides into the atmosphere, curbs its utilization in several areas. Therefore, several attempts are being made to reduce the sulphur content in coal through an effective and efficient method. Several factors such as type of coal, amount of sulphur in coal, distribution pattern of different forms of sulphur etc. play a vital role in the process of coal desulphurization1. The physicochemical techniques of sulphur removal either remain futile towards the reduction of organic sulphur or release high amount of SO2 into the atmosphere along with the use of high temperature and pressure2,3. Therefore, in the present scenario, the biological approach is being focused and investigated more due to its several advantages over the physicochemical methods3,4. It primarily offers the benefits of low operation cost, with concomitant production of value added products5. Half of the world’s coal deposit comprises of low rank coals6. Turkey is a country with vast reserves of lignite and nearly 43% of its fossil fuel production constitutes lignite7. The high moisture, ash and sulphur 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 sulphur removal of 30.2% from the sample8. Apart from bacterial species, fungal species such as Phanerochaeta chrysosporium and Trametes versicolor have been used to carry out shake-flask experiments for sulphur removal from low rank coal9. In yet another study, Aytar et al10 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 sulphur 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 sulphur removal of 40% using Trametes versicolor. In addition to that, use of Aspergillus sp. by Aytar et al11 for the shake-flask biodesulphurization of coal obtained from the Mihaliccik open mine in Turkey resulted in a maximum organic sulphur removal of 38% and 2 ACS Paragon Plus Environment

Page 2 of 26

Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

sulphate sulphur removal of 51%. A combination of gravity (MGS)/flotation technique along with biodesulphurization was used by Aksoy et al12 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 sulphur content reduced from 3.25% to 1.56% in the MGS sample following microbial treatment. Similarly, the sulphur 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 sulphur removal were observed to be 57.1% and 33% respectively13, 14. Although a lot of biodesulphurization studies have been reported till 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 used15. Apart from that, Cara et al16 have reported the packed column leaching of 5kg coal sample using mixed culture of acidophilic strains, where they achieved a maximum sulphur 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 sulphur removal of 20% was achieved in 14 days, out of which 45% comprised of pyritic sulphur17. Reports on higher scale biodesulphurization of US coal carried out by Pathak et al18 in a 8L bench scale stirred tank reactor using Acidithiobacillus ferrooxidans yielded a maximum pyritic sulphur removal of 67%. In addition to that, heap bioleaching experiments with semi-anthracite coal have been reported by Cara et al19. 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 sulphur. 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. Owing to 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 experiments20. It is very 3 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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. Materials and Methods 2.1. Coal Sample The sample used in the present study was collected from the TKI Can LinyitIsletme 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 sterilization4. 2.2. Microbial Growth The acidophilic strain, L. ferriphilum (DSM 14647) used in the present study was obtained from culture collection centre 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: 20g FeSO4.7H2O in 50 ml of 0.25N 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 112ºC for 30 mins 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.

4 ACS Paragon Plus Environment

Page 4 of 26

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

2.3. Biodesulphurization Experiments The efficiency of L. ferriphilum towards 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 studies20. The studies (experimental conditions discussed in subsections below) were initially performed in a laboratory scale reactor (1L working volume) followed by a semi-pilot scale reactor (20L 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 20L reactor system, having three impellers (two axial at the top & 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 1L 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 20L reactor were: internal height - 65 cm; internal diameter of the reaction vessel - 20 cm; outer diameter 30 cm with 5 openings. The experimental set-up of the reactor system is shown in Fig.1. 2.3.1. Lab and Semi-Pilot 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 32ºC through heated water circulation as discussed above. The reactors were continously 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 1L bioreactors tests, the experiments were further scaled-up on a semi-pilot level. In this experiment, similar operating conditions used for the lab 5 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

scale reactor were considered i.e. Pulp density – 5% (w/v), bacterial inoculum - 10% (v/v), temperature - 32ºC, time – 21 days and agitation speed – 150 rpm. The changes in pH, oxidoreductive potential, Fe2+ iron and total iron concentration in all the reactors (used for lab and semi-pilot 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 studies, in order 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, Oxido-Reductive 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 o-phenalthronine method21. A Thermo Scientific Orion DUAL STARTM 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. Sulphur Analysis and Proximate Analysis The change in total sulphur 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 pre and post microbial treated samples was determined through an X-ray powder diffractometer (Philips X’pert Pro, Panalytical) with CuKa (k = 1.54 A˚) radiation 6 ACS Paragon Plus Environment

Page 6 of 26

Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

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 (Perkin Elmer, FTIR- Spectrum GX model, 633 nm IR-HeNelaser) had a power less than 1mW and a scanning speed of 0.2 cm/sec. 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 surfactant20. 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 (1L) and semi-pilot (20L) scale under continous 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 sulphur 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 66.5%, while in BR2 it was observed to be 57.1%. The results obtained in the present study indicated that under continous stirring/agitation and aerated conditions, S80 in presence of L.ferriphilum did not have any effect on biodesulphurization i.e. sulphur removal was seen to be lowered nearly by 9%, which was also accompanied by slower iron oxidation (see the next section for further

7 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 26

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 sulphate (Fe2(SO4)3) through a series of steps. Since lignite samples are generally rich in pyritic sulphur content, the conversion of Fe2+ to Fe3+ facilitates the oxidative attack on the pyritic sulphur present in coal22. Thus, the microbial activity assists in the oxidation of Fe2+ to Fe3+, which further attacks coal leading to the oxidation and release of pyritic sulphur present in coal as shown in the following equations. FeS2 + 7/2O2 + H2O

FeSO4 + H2SO4

(Eq. 1)

In general, the reaction leading Fe2+ to Fe3+ conversion is approximately 106 times higher in the presence of the microbes22. 2 FeSO4 + ½O2 + H2SO4

Fe2 (SO4)3 + H2O

(Eq. 2)

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

3Fe2+ + 2S0

(Eq. 3)

Following the above reaction, the elemental sulphur generated through the oxidation of pyrite can further be oxidized by the ferric iron as: S0 + 6Fe3+ + 4H20

6Fe2+ + SO42- + 8H+

(Eq. 4)

Changes in Fe2+ iron concentration were regularly monitored in the bioreactors in order to trace the bacterial activity. As observed from Fig 2a, the Fe2+ iron concentration in BR1, contaning no surfactant, gradually decreased following the 3rdday of incubation and became almost zero following the 6thday. 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 6th day of incubation and became nearly zero after the 9thday.

8 ACS Paragon Plus Environment

Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Surfactants have been reported of corrosion inhibition, where they prevent the oxidation of metals23. 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, while, 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 sulphur present in the coal. Earlier studies by Tuttle and Dugan24 related to the effect of organic compounds on the growth, Fe2+ and sulphur oxidation by Acidithiobacillus ferrooxidans had indicated that the presence of low molecular weight organic compounds inhibit the growth, iron and sulphur 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, non-specific 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 al25 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 continous 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 (Fig 2b). In case of BR1, the ORP was 9 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 3rd day of incubation (Fig 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 oxidation of Fe2+ iron in media and pyrite from the TL sample can be explained based on the reactions, Eq. 1, 2, 3 & 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 (Fig. 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 chanes 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. Semi-Pilot 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 presence of surfactant, S80. A possible explanation for this phenomenon has been given in section 3.1.1. Since, lower sulphur removal was achieved in presence of surfactant in 1L reactor, therefore, the biodesulphurization experiment in the 20L reactor was carried out in absence of surfactant in order to observe the desulphurization efficiency at semi-pilot scale. With 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 activites26.

10 ACS Paragon Plus Environment

Page 10 of 26

Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

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 Equations 1 & 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 sulphur removal achieved in the reactor was 63%. The initial sulphur 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 continous 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 sulphur distribution studies had indicated that the lignite sample contained 2.71% of pyritic sulphur, 1.57% sulphate sulphur and 2.56% organic sulphur20. 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 sulphur from coal resulting in structural and phase changes (discussed in section 3.3), which were responsible for the reduction in ash content and simultaneous increment in the fixed carbon content of the sample. 3.2.1. Changes in Fe2+, pH, ORP and total iron under semi-pilot conditions During the course of experiment in the 20L 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 Fig 3a, the system pH dropped from 1.8 to 1.44 that can be ascribed to the combined effect of Eq. 1 – 4 (see section 3.1.1). The slight increment in the system pH from 9th day till 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+ 11 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oxidation, along with the release of protons due to ferric iron precipitation leads to the development of an acidic pH in the medium27. The ORP of the system increased from 395.6 to 619 mV by the end of the experiment (Fig 3a). It gradually increased till 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 (Fig. 3b). The Fe2+ iron concentration in the system was almost nil by the 6th 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 till day 21. Similarly, the total iron concentration in the medium also increased from 4.04 to 14.11 g/L (Fig. 3b). It rose sharply till 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 Fig 4). The FTIR spectra not only provides information regarding the changes observed in sulphur containing functional groups following microbial treatment, but also reveals the changes undergone in the mineral matter content of coal due to microbial attack28. 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 12 ACS Paragon Plus Environment

Page 12 of 26

Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

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 position29. In the present study, the FTIR analysis of the treated sample was carried out following treatment in the 20L 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 H2O30, 31. From earlier reports32, 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 sulphate minerals present in coal. Similarly, marginal changes observed in the range of 1250-1500cm-1 corresponds to the –CH3 symmetric deformation, while the presence of –CH3 and –CH2 groups in cyclic structures may also partly contribute to this band31. 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 strain30. The reduced peak intensities observed in the range of 1000 – 750cm-1 can be attributed to aromatic structures and the presence of aluminosilicate phases in coal30,31,33. Likewise, reduced peak intensities observed in the range of 750 – 500 cm-1 can be attributed to Si – O bending vibrations30. Microbial attack might have resulted in the loss of silicate impurities from the sample leading to reduced peak intensity in this region. 3.3. XRD Analysis The mineralogical phase changes following microbial attack were studied through XRD analysis of the original and treated samples (shown in Fig 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, while new quratz 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 13 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

been previously reported by several researchers34,35. Origin of minerals in the coal is based on the source, mode of occurrence and prevalent environmental conditions36. 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 sulphur associated in the form of discrete physical entities with 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. 3.4. 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 sulphur oxidation or desulphurization, where an enhancement in the microbial activity has been observed in the presence of surfactant. Studies carried out by Cheng-guiet al37 have revealed the effect of two different surfactants on the sulphur oxidizing activities of Acidithiobacillus albertensis BY05. They have observed that the sulphur oxidizing activity of the microbial strain was enhanced only at specific surfactant concentrations. According to reports, the oxidation of sulphur particles is initiated with the attachment of the microbial cells to hydrophobic sulphur 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 cells37- 39. Studies carried out by He et al39 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 solution40, 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 continous aerated conditions as discussed in section 3.1.1). This in turn might be affecting the oxidation of Fe2+ to Fe3+ iron and subsequent 14 ACS Paragon Plus Environment

Page 14 of 26

Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

oxidation of pyritic sulphur present in coal. The mechanism of microbial action in both the reactors are shown in Fig 6 a & b. Conclusion Biodesulphurization of Turkish lignite was studied in the presence and absence of Span 80 in 1L bioreactor using Leptospirillum ferriphilum DSM 14647. A maximum desulphurization of 66.5% 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 sulphur 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 due to microbial action. The present study provided the optimum parameters to carry out coal biodesulphurization on a scale-up basis in stirred tank bioreactors. Acknowledgements SM and AA kindly acknowledges the Scientific and Technological Research Council of Turkey for the award of the TUBITAK – 2216 International Fellowship to SM in order to successfully carry out a part of her PhD research work at the MMR&R research group, SDU, Turkey. SM would also like to sincerely thank 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. SP kindly acknowledges the Scientific and Technological Research Council of Turkey for the award of TUBITAK – 2216 Post Doctoral Fellowship. References 1.

Liu, Q.; Hu, H.; Zhu, S.; Zhou, Q.; Li, W.; Wei, X.; Xie, K. Desulfurization of Coal by Pyrolysis and Hydropyrolysis with Addition of KOH/NaOH. Energy & Fuels. 2005, 19, 1673-1678

2.

Mukherjee, S.; Mahiuddin, S.; Borthakur P.C. Demineralization and Desulfurization of Subbituminous Coal with Hydrogen Peroxide. Energy & Fuels. 2001, 15, 1418-1424.

3.

Soleimani, M.; Bassi, A.; Margaritis, A. Biodesulfurization of refractory organic sulfurcompounds in fossil fuels. Biotechnol. Adv. 2007. 25, 570–596. 15 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4.

Page 16 of 26

Mishra, S.; Panda, P.P.; Pradhan, N.; Satapathy, D.; Subudhi, U.; Biswal, S.K.; Mishra, B.K. Effect of native bacteria Sinomonas flava 1C and Acidithiobacillus ferrooxidans on desulphurization of Meghalaya coal and its combustion properties. Fuel. 2014, 117, 415– 421.

5.

Agarwal, P.; Sharma, D.K. Comparative Studies on the Bio-desulfurization of Crude Oil with Other Desulfurization Techniques and Deep Desulfurization through Integrated Processes. Energy & Fuels. 2010, 24, 518–524.

6.

Mills, S.J. Global perspective on the use of low quality coals.CCC 180 ISBN 978-92-9029500-6;

IEA

Clean

Coal

Centre,

2011;

pp

1-80

(Available

online

at:

https://www.usea.org/sites/default/files/012011_Global%20perspective%20on%20the%20us e%20of%20low%20quality%20coals_ccc180.pdf). 7.

Xia, W.; Xie, G.; Peng, Y. Recent advances in beneficiation for low rank coals. Powder Technol. 2015, 277, 206–221.

8.

Bozdemir, T.Ö.,Durusoy,T., Erincin, E.,Yürüm,Y., Biodesulfurization of Turkish lignites: 1. optimization of the growth parameters of Rhodococcus rhodochrous, a sulfur-removing bacterium, Fuel 75 (1996) 1596–1600.

9.

Gonsalvesh, L.; Marinov, S.P.; Stefanova, M.; Yürüm, Y.; Dumanli, A.G.; Doganay, G.D.; Kolankaya, N.; Sam, M.; Carleer, R.; Reggers, G. Biodesulphurized subbituminous coal by different fungi and bacteria studied by reductive pyrolysis. Part 1: Initial coal. Fuel. 2008, 87, 2533–2543.

10. Aytar, P.; Gedikli, S.; Şam, M.; Ünal, A.; Çabuk, A.; Kolankaya, A.; Yürüm, A. Desulphurization of some low-rank Turkish lignites with crude laccase produced from Trametes versicolor ATCC 200801. Fuel Process. Technol. 2011, 92, 71–76. 11. Aytar, P.; Aksoy, D.O.; Toptas, Y.; Çabuk, A.; Koca, S.; Koca, H. Isolation and characterization of native microorganism from Turkish lignite and usability at fungal desulphurization. Fuel. 2014, 116, 634–641. 12. Aksoy, D.O.; Aytar, P.; Toptaş, Y.; Çabuk, A.; Koca, S.; Koca, H. Physical and physicochemical cleaning of lignite and the effect of cleaning on biodesulfurization. Fuel. 2014, 132, 158–164. 13. Durusoy, T.; Ozbas, T.; Tanyolac, A.; Yurum, Y. Biodesulfurization of Some Turkish Lignites by Sulfolobus solfataricus. Energy & Fuels. 1992, 6, 804-808. 16 ACS Paragon Plus Environment

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

14. Aytar, P.; Kay, C.M.; Mutlu, M.B.; Çabuk, A. Coal Desulfurization with Acidithiobacillus ferrivorans, from Balya Acidic Mine Drainage. Energy & Fuels. 2013, 27, 3090–3098. 15. Mishra, S.; Pradhan, N.; Panda, S.; Akcil, A. Biodegradation of dibenzothiophene and its application in the production of clean coal. Fuel. Process. Technol. 2016. 152, 325-342. 16. Cara, J.; Vargas, M.; Moran, A.; Gomez, E.; Martınez, O.; Frutos, F.J.G. Biodesulphurization of a coal by packed-column leaching. Simultaneous thermogravimetric and mass spectrometric analyses. Fuel. 2006, 85, 1756–1762. 17. Farahnaz, E.; Reza, E.M. Biodesulfurization of Tabas coal in pilot plant scale, Iran. J.Chem. Chem. Eng. 2010, 29, 75–78. 18. Pathak, A.; Kim, D.J.; Srichandan, H.; Kim, B.G.Depyritization of US coal using iron oxidizing bacteria: batch stirred reactor study. Int. J. Chem. Mol. Nucl. Mat.Metallurg. Engineer. 2013, 7, 839–842. 19. Cara, J.; Carballo, M.T.; Moran, A.; Bonilla, D.; Escolano, O.; Frutos, F.J.G. Biodesulphurisation of high sulphur coal by heap leaching. Fuel. 2005, 84, 1905–1910. 20. Mishra, S.; Akcil, A.; Panda, S.; Erust, C. Biodesulphurization of Turkish Lignite by Leptospirillum ferriphilum: Effect of Ferrous iron, Span-80 and Ultrasonication. Hydrometallurgy. 2018, Accepted Manuscript, Article in Press. 21. Close, P.; Hornyak, E.J.; Baak, T.; Tillman, J.F.; Potentiometric titration of micro amounts of iron(II) with very dilute cerium (IV) sulfate, Microchemical J. 1996, 10, 334-339. 22. Blazquez, M.L.; Ballester, A.; Gonzalez, F.; Mier J.L. Coal biodesulphurization: A Review. Biorecovery. 1993, 2, 155-177. 23. Malik, M.A.; Hashim, M.A.; Nabi, F.; AL-Thabaiti, S.A.; Khan, Z. Anti-corrosion Ability of Surfactants: A Review. Int. J. Electrochem. Sci. 2011, 6, 1927 – 1948. 24. Tuttle, J.H.; Duga, P. Inhibition of growth, iron, and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds. Can. J. Microbiol. 1976, 22,719 – 30. 25. An-an, P.; Hong-chang, L.; Zhen-yuan, N.; Jin-lan, X. Effect of surfactant Tween-80 on sulfur oxidation and expression of sulfur metabolism relevant genes of Acidithiobacillus ferrooxidans. Transactions of Non-ferrous Met. Soc. China. 2012, 22, 3147−3155. 26. Panda, S.; Akcil, A.; Pradhan, N.; Deveci, H.; Current scenario of chalcopyrite bioleaching: A review on the recent advances to its heap leaching technology. Bioresource Technology. 2015, 196, 697-706. 17 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

27. Meruane, G.; Vargas, T. Bacterial oxidation of ferrous iron by Acidithiobacillus ferrooxidans in the pH range 2.5–7.0. Hydrometallurgy. 2003, 71, 149–158. 28. Elsamaka, G.G.; Oztas, N.A.; Yurum, Y. Chemical desulfurization of Turkish Cayirhan lignite with HI using microwave and thermal energy. Fuel. 2003, 82, 531–537. 29. Gezici, O.; Demir, I.; Demircan, A.; Ünlü, N.; Karaarslan, M. Subtractive-FTIR spectroscopy to characterize organic matter in lignite samples from different depths. Spectrochim. Acta A. Mol. Biomol. Spectrosc. 2012, 96, 63–69. 30. Baruaha, M.K.; Kotoky, P.; Borah, G.C. Distribution and nature of organic/mineral bound elements in Assam coals, India. Fuel. 2003, 82, 1783–1791. 31. Saikia, B.K.; Boruah, R.K.; Gogoi, P.K. FT-IR and XRD analysis of coal from Makumcoalfield of Assam. J. Earth Syst. Sci. 2007, 116, 575–579. 32. Saikia, B.J.; Parthasarathy, G. Fourier Transform Infrared SpectroscopicCharacterization of Kaolinite from Assam and Meghalaya, Northeastern India. J. Mod. Phys. 2010, 1, 206-210. 33. Qiu, Y.; Zhang, Q.; Tian, Y.; Zhang, J.; Cao, J.; Xiao, T. Composition and structure of luxing coal with different particle sizes. Petroleum & Coal. 2011, 53, 10-20. 34. Karayigit, I.; Onacak, T.; Gayer, R.A.; Goldsmith, S. Minerology and geochemistry of feed coals and their combustion residues from the Cayirhan power plant, Ankara, Turkey. Applied Geochemistry. 2001, 16, 911-919. 35. Sakintuna, B.; Yurum, Y.; Cetinkaya, S. Evolution of Carbon Microstructures during the Pyrolysis of Turkish Elbistan Lignite in the Temperature Range 700-1000 °C. Energy & Fuels. 2004, 18, 883-888. 36. Sharma, A.; Saikia, A.; Khare, P.; Baruah, B.P. J. Genesis of some tertiary Indiancoals from the chemical composition of ash e a statistical approach: Part 1. Earth Syst. Sci. 2014b, 123, 1705-1715. 37. Cheng-gui, Z.; Jin-lan, X.; Rui-yong, Z.; An-an, P.; Zhen-yuan, N.; Guan-zhou, Q. Comparative study on effects of Tween-80 and sodium isobutyl-xanthate on growth and sulfur-oxidizing activities of Acidithiobacillus albertensis BY-05. Trans. Nonferrous Met. Soc. China. 2008, 18, 1003-1007. 38. Vandevivere, P.; Kirchman, D.L. Attachment stimulates exopolysaccharide synthesis by a bacterium. Appl. Environ. Microbiol. 1993, 59, 3280-3286.

18 ACS Paragon Plus Environment

Page 18 of 26

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

39. He, Z.G.; Yang, Y.P.; Zhou, S.; Hu, Y.H.; Zhong, H. Effect of pyrite, elemental sulfur and ferrous ions on EPS production by metal sulfide bioleaching microbes. Trans. Nonferrous Met. Soc. China. 2014, 24 1171−1178. 40. Rohwerder, T.; Gehrke, T.; Kinzler, K.; Sand W. Bioleaching review part A: Progress in bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation. Appl. Microbiol. Biotechnol. 2003, 63, 239-248. 41. Rawlings,

D.E.

Characteristics

and

adaptability

of

iron-

and

sulfur-oxidizing

microorganisms used for the recovery of metals from minerals and their concentrates Microb. Cell. Fact. 2005, 4:13.

19 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 26

List of Tables

Table 1.Biodesulphurizationof TL sample in 1L bioreactors Reactors

Initial Sulphur (%)

BR1 BR2

7.63

Final Sulphur (%)

% Sulphur Removal

2.62

66.5

3.36

57.1

20 ACS Paragon Plus Environment

Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

List of Figures

Fig.1 Experimental steup of (a) Lab scale, 1L reactor (b) Semi-pilot, 20L reactor

21 ACS Paragon Plus Environment

Energy & Fuels

6

a

No S-80 With S-80

5

3

a

mV

Fe2+ (g/L)

4

2 1 0 0

3

6

9

12 Days

15

18

21

650 b 600 550 500 450 400 350 300 250 200 150 0

b No S-80 With S-80

3

6

9 12 Days

15

18

21

3.0 c

c

2.0

1.5

14 d Total Iron (g/L)

No S-80 With S-80

2.5

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 26

12 10

d 8 6 No S-80 With S-80

4

1.0 0

3

6

9 12 Days

15

18

21

3

6

9

12 Days

15

18

21

Fig. 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.

22 ACS Paragon Plus Environment

Page 23 of 26

650

1.9

5

600

1.8

b

14

4

450

Fe 2+ (g/L)

500

1.6

ORP

a

550

3

b

2

8

1

6

0

4

1.5 400

10

1.4 0

3

6

9

12

15

18

21

0

3

Days

6

9

12 Days

15

18

21

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

23 ACS Paragon Plus Environment

Total Iron (g/L)

12

1.7 pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Energy & Fuels

100 80 % Transmittance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

60 40

Original CC L.f Semi-pilot

20 0 4000 3500 3000 2500 2000 1500 1000 500 Wavelength cm -1

Fig 4. FTIR spectra of original and microbially treated samples

24 ACS Paragon Plus Environment

Page 24 of 26

Page 25 of 26

2500 K

Counts

2000 1500

K

Q

K

Original K

P Q

P

P

P P P KK K K P

1000

P K

K

500 0 2000

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

K P Q KQ K

1500 K 1000

K K P P P

Semi-pilot

KQQ K Q

PK

K PP

K Q

Q

50

60

Q

500 10

20

30

40

70

80

Position 2-Theta Fig 5. XRD analysis of original and microbially treated samples (Note: P – Pyrite, K – Kaolinite, Q –Quartz).

25 ACS Paragon Plus Environment

Energy & Fuels 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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

26 ACS Paragon Plus Environment

Page 26 of 26