Indian Journal of Experimental Biology Vol. 53, June 2015, pp. 388-394
Bioleaching of nickel from spent petroleum catalyst using Acidithiobacillus thiooxidans DSM-11478 Mohita Sharma1,2, Varsha Bisht2, Bina Singh2, Pratiksha Jain1,2, Ajoy K Mandal2, Banwari Lal1,2 & Priyangshu M Sarma1,2* 1
Center for Bioresources and Biotechnology, TERI University, New Delhi-110 070, India Environmental and Industrial Biotechnology Division, The Energy and Resources Instititute (TERI), New Delhi-110 003, India
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Received 21 November 2014; revised 28 November 2014 The present work deals with optimization of culture conditions and process parameters for bioleaching of spent petroleum catalyst collected from a petroleum refinery. The efficacy of Ni bioleaching from spent petroleum catalyst was determined using pure culture of Acidithiobacillus thiooxidans DSM-11478. The culture conditions of pH, temperature and headspace volume to media volume ratio were optimized. EDX analysis was done to confirm the presence of Ni in the spent catalyst after roasting it to decoke its surface. The optimum temperature for A. thiooxidans DSM-11478 growth was found to be 32 °C. The enhanced recovery of nickel at very low pH was attributed to the higher acidic strength of sulfuric acid produced in the culture medium by the bacterium. During the bioleaching process, 89% of the Ni present in the catalyst waste could be successfully recovered in optimized conditions. This environment friendly bioleaching process proved efficient than the chemical method. Taking leads from the lab scale results, bioleaching in larger volumes (1, 5 and 10 L) was also performed to provide guidelines for taking up this technology for in situ industrial waste management. Keywords: Petroleum refinery, Proteobacteria, Thiobacillus concretivorus, Waste treatment
Catalysts used in the petroleum industry are made up of metals, metal oxides, and metal sulfides coated on various carriers such as alumina, silica, zeolites and carbon1. These catalysts aid in hydrocarbon transformation to purify and up-grade various petroleum streams and residues to produce a full range of transportation fuels, petrochemicals and plastics2. During processing, the catalysts are contaminated with impurities present in the crude oil feed and become deactivated and thus, lose their catalytic activity and selectivity over time3. The catalysts are pre-treated after a few cycles to regenerate and reactivate them. But after a certain stage, carbon filaments plug catalyst pores and voids, deeming it unfit for subsequent use. At this stage, catalysts are considered “spent”. The current generation rate of spent catalysts is estimated to be approximately 150000 to 170000 tons per annum worldwide4. Spent catalyst is a hazardous toxic waste with adverse biological effects as the metals present in the catalyst can leach to ground water aquifers after —————— *Correspondence: E-Mail:
[email protected] For suppl figs. please see respective IJEB page on line at NISCAIR repository, http://nopr.niscair.res.in
disposal and form toxic gases like hydrogen cyanide5. The spontaneous leaching of metals present in the spent catalyst due to natural weathering processes and uncontrolled bacterial activities can pose significant environment problems and become a potential threat to human health2,6. Moreover, increasingly stringent environmental regulations have led to meaningful treatment of waste catalysts enabling recovery of valuable metals through economically sustainable techniques7. Traditionally, pyrometallurgical and hydro metallurgical techniques are used for metal recovery from spent catalyst8. For nickel and aluminium recovery from the spent catalyst, aqua-regia, hydrochloric acid, sulphuric acid and sodium hydroxide are widely used9,10. These methods are expensive, less efficient, energy intensive and may produce secondary contaminants and more hazardous emissions like SO2 during contact process11. Bioleaching is an alternative to conventional methods, where microorganisms transform spent catalyst into soluble and extractable elements serving the dual purpose of detoxifying the spent catalyst with recovery of heavy metals in an economical and environment friendly manner 12,13. It is either by redox for proton formation or by
SHARMA et al: BIOLEACHING OF NICKEL FROM SPENT PETROLEUM CATALYST
excretion of complexing agents for ligand formation6. The predominant metal sulfide dissolving microbes, chemolithotrophs, belong to the genus Acidithiobacillus, class γ-proteobacteria of Gram-negative bacteria. They use inorganic |sulfur or ferrous iron as energy source and grow autotrophically by fixing CO212. The species of genera Acidimicrobium, Ferromicrobium, Sulfobacillus and archeal order Acidianus, Metallosphaera, Sulfolobales and Sulfurisphaera spp. are used for bioleaching of metals14-16. Chen et al. have reported the use of Acidithiobacillus strains for the treatment of sediment contaminated with heavy metals17. Beolchini et al.18, Bosio et al.3 and Mishra et al.19 have indicated the role of A. thiooxidans DSM-11478 in leaching of metals from spent refinery catalyst. Iron/sulphur oxidising bacteria (including Acidithiobacillus) was used by Beolchini et al. to study the dissolution kinetics of nickel, molybdenum and vanadium during bioleaching18. Treatment of spent material from the hydrogenation of vegetable oil containing a high-level of nickel was attempted by Bosio et al.3. Bioleaching of nickel, molybdenum and vanadium was studied by Mishra et al.19. In the present study, we report bioleaching of nickel from spent petroleum catalyst by A. thiooxidans DSM-11478. The culture conditions such as the growth medium, temperature and head-space volume to media volume ratio were optimized. Once the culture conditions were optimized, the process was progressively scaled-up from shake flask to fermenter (1, 5 and 10 L). Material and Methods Catalyst procurement, processing and analysis— The spent catalyst pellets were procured from Panipat refinery of Indian Oil Corporation Limited, Haryana, India. In order to process the spent catalyst into a coke-free powdered form, the catalyst pellets were roasted in the muffle furnace (Yorco® India) at 800 °C for 3 h. The chemical composition of the pre-processed and processed catalyst pellets was analysed by Energy Dispersive X- ray (EDX) spectroscopy (Rontec’s QuanTax 200). The pellets were later ground to particle size of 100 µm. The pre-processed and processed spent petroleum catalyst powder samples were analysed using Scanning Electron Microscopy (Zeiss EVO Series, model EVO50, Germany).
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Microorganism—The bacterial strain, Acidithiobacillus thiooxidans DSM-11478 used in the present study was procured from German Collection of Microorganisms and Cell Cultures, (DSMZ) Germany. Process parameter optimization
Media composition—The bacterial culture was initially revived on the DSMZ 271 medium followed by 2 other media, MSA and MSB. The composition of MSA per litre was: (NH4)2SO4 (3.00 g), KH2PO4 (0.50 g), MgSO4.7H2O (0.50 g), CaCl2.2H2O (0.01 g), Na2S2O3 (10.0 ml), trace element solution (2 ml) and vitamin solution (2 ml). MSB contained (NH4)2SO4 (2.00 g), K2HPO4 (0.50 g), MgSO4.7H2O (0.50 g), KCl (0.10 g), Ca(NO3)2 (0.01 g), S powder (10.00 g), trace element solution (2.00 ml) and vitamin solution (2.00 mL), per litre of distilled water. Thus, media were optimized for propagation and bioleaching experiments. Elemental sulfur powder was sterilized separately by keeping it at 100 °C for three consecutive days. This was aseptically layered on top of the media immediately after inoculation. Temperature—About 10 % of the active inoculum, grown in the MSA medium was inoculated in a 250 ml flask with 100 ml of the MSB medium. These were incubated at three different temperatures (32, 37 and 45 °C) at 150 rpm in the rotary shaker. Other parameters remained unchanged. The growth of the culture and drop in the pH values were observed by measuring the pH, OD, and protein estimation. Headspace volume: media volume ratio— Acidithiobacillus thiooxidans DSM-11478 is chemo-lithotroph and uses atmospheric CO2 as a sole carbon source. To optimize the CO2 required for propagation of A. thiooxidans DSM-11478, experiments were conducted maintaining different headspace:media volume ratios (2.5:1, 4.4:1, 5.0:1 and 6.6:1) at the optimized temperature. Other conditions were same as mentioned for termperature Bioleaching and chemical leaching experiments— Bioleaching of the spent petroleum catalyst was carried out in two-stages. In the first stage, bacterial growth experiments were conducted in 250 ml flasks, containing 100 ml of optimized medium (MSB) and 10 % (v/v) inoculum of the active culture grown in MSA medium. Commercial sulfur powder was used as the energy source. These flasks were incubated at 37 °C and 150 rpm in the rotary shaker. Control was set for each experiment with no inoculum. The
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consumption of the sulfur powder was observed with the change in the pH of the culture with time. After the stipulated drop in pH (20 days) when cultured in DSMZ 271 medium, whereas in medium MSB, it showed a significant increase in protein concentration and corresponding drop in pH just after 6 days of incubation (Suppl Fig. 1). This may be due to addition of trace element and vitamin in medium MSB. Though medium MSB served as an efficient method for conducting bioleaching experiments, a quick method for propagation of A. thiooxidans DSM-11478 was to incubate the culture in MSA medium. However, A. thiooxidans DSM-11478 growth in MSA medium failed to bring down the pH of the medium significantly as the pH did not drop below 2.9 from 4.5 even after a long period of incubation (40 days). For bioleaching experiments, lowering of pH below 1 is desirable. However, sodium thiosulfate has been reported to decompose at lower pH and at higher temperature26. This could possibly be a reason for failure of the medium MSA to perform bioleaching studies. Hence, MSA medium reduced the overall reaction time while transfer of inoculum to medium MSB produced sufficient acids to bring down the pH for leaching the nickel from the catalyst powder. Additionally, sulfur is a cheaper substrate than thiosulfate, making the whole process more cost effective.
The major factors influencing the process include nutrients, oxygen, sulfur content, cell concentration, carbon dioxide, pH, temperature, particle size and exposed surface area of heavy metals and surfactants27. Shrihari et al. have previously reported that even though hydrodynamic and Van der Waal interactions are necessary for the attachment of cells in liquid medium to the solid sulfur particles, the cells can still grow without coming in direct contact with the solid by utilising an alternate substrate like thiosulfate generated by attached cells28. This is a plausible reason for successful propagation of A. thiooxidans DSM-11478 in thiosulfate based MSA medium. On further transfer to medium MSB, the effect of electric charges, surface irregularity, cell membrane characteristics of A. thiooxidans DSM-11478 cells facilitated the attachment to the surface of sulfur11. The process of bioleaching is highly dependent on the adsorption of bacteria onto the surface of elemental sulfur and its subsequent oxidation for sulfuric acid production29. When A. thiooxidans DSM-11478 is cultured in the sulfur medium, sulfate ions accumulate in the liquid medium that leads to sulfuric acid production required for bioleaching30. Hence, our study corroborates earlier findings that thiosulfate adapted cultures retain their ability to use sulfur as an energy source31,32.
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Effect of headspace volume: medium volume ratio—Bioleaching of Ni from spent catalyst powder can be mainly attributed to the acids produced by A. thiooxidans DSM-11478 that is further dependent on its metabolism and growth pattern. As the selected strain is a chemolithotroph, the effect of headspace volume on its growth and production of acids was studied. The resultant pH values obtained with respect to headspace volume and medium volume ratio of 2.5:1, 4.4:1, 5:1 and 6.6:1 were 0.9, 0.7, 0.4 and 0.1, respectively (Suppl Fig. 2). Since this chemolithotroph relies on atmospheric CO2 as a carbon source, a higher headspace volume was conducive for the growth of the bacterium as directly indicated by the corresponding reduction in pH (Suppl Fig. 2). These findings are in agreement with Wang et al., where the role of CO2 in the bioleaching performance by Acidithiobacillus species was essentially highlighted33. Effect of temperature—Effect of temperature on the growth of A. thiooxidans and the bioleaching process was monitored by conducting incubation and bioleaching experiments at 32, 37 and 45 °C, respectively. As seen from the results of growth and bioleaching performance, it can be concluded that the optimum temperature for the growth and propagation of A. thiooxidans was 37 °C (in medium MSA). However, for bioleaching based experiments, 32 °C was optimum (in medium MSB). It was also seen that 45 °C proved to be detrimental for the growth of A. thiooxidans. Analysis of sulfuric acid produced by A. thiooxidans— Titrimetric study was conducted to assess the strength of H2SO4 produced by A. thiooxidans in medium MSB that was to be subsequently used for bioleaching and chemical leaching experiments. The acid strength uniformly increased as the pH decreased (data not shown). The increase in normality of the acids
(biological and chemical) was 0.14 N when the pH decreased from 0.9 to 0.7. However, when the pH reduced from 0.7 to 0.4, strength of biologically produced acid (0.38 N) was more than chemical sample (0.34 N). During incubation of A. thiooxidans, the metabolic activity of the cells result in the production of H2SO4 in the medium that is mainly responsible for increase in acid strength of the samples to be used for bioleaching. Moreover, a decrease in culture volume was also observed due to evaporation of water from the media and this has been reported to be the major factor leading to H2SO4 accumulation and progressive reduction in the pH of culture34. Comparison of bioleaching with chemical leaching— Leaching experiments were conducted at pH 0.1, 0.4, 0.7 and 0.9. The leachate obtained after filtration of the leached samples by Whatman filter paper No. 3 were analysed for nickel content by atomic absorption spectroscopy (AAS) at appropriate dilutions. The corresponding graphs for both bioleaching and chemically leached samples were plotted for different catalyst concentrations (0.05 g, 0.075 g, 0.1 g, 0.15 g and 0.2 g) at different pH as shown in Fig. 2. Lowering of pH due to formation of acid during growth is responsible for dissolving metal oxides from spent catalysts with very high efficiency17,35. The bioleaching efficiency by microorganism was comparable to chemically leached samples. Furthermore, at pH 0.4 and 0.1 the bioleaching process superseded its chemical counterpart in terms of leaching efficiency at higher catalyst concentration (Fig. 2c and d). At these pH values, the yield of Ni was approximately 89 % with 0.2g of catalyst used. Lower pH, indicative of higher H2SO4 concentration has a pronounced effect on dissolution of nickel from the spent catalyst powder8.
Fig. 2—Comparison of bioleached vs. chemically leached samples at different catalyst concentrations and pH keeping sample volume constant at 20 ml. The leaching experiments were performed for a fixed time interval of 30 min at 100 °C. Graphs a, b, c and d show percentage of nickel leached in samples at pH 0.9, 0.7, 0.4 and 0.1, respectively.
SHARMA et al: BIOLEACHING OF NICKEL FROM SPENT PETROLEUM CATALYST
Fig. 3—Changes in pH value of the medium with time with respect to scale-up volumes.
Scale-up culture—As step towards future pilot scale studies, bioleaching was performed with larger volumes. Scale-up of the culture volume was conducted (0.1, 1, 5 and 10 L). These cultures were then evaluated by comparing the drop in pH with time as depicted in Fig. 3. The change in pH was similar for shake flask 0.1 L (0.96) and fermenter cultures till 1 L (0.98) and 5 L (0.98). Substantial drop in pH could be reached by 9th day of the experiment even when the medium volume was increased from 0.1 L to 5L. However, when the medium volume was increased up to 10 L, though drop in pH was achieved till 0.99 on day 12, further reduction in pH could not be achieved during this period. Hence, it can be concluded that a linear trend was maintained in reduction in pH with increasing incubation time till 5 L working volume but beyond 10 L, it was not the same. This could be due to alteration in headspace to medium volume requirements for the chemolithotrophic strain A. thiooxidans to effectively propagate and contribute towards bioleaching process for such higher working volumes.
A comparative study between bioleaching and chemical leaching has shown that bioleaching is more efficient than chemical leaching process in the mobilisation of nickel. The enhanced recovery of nickel at very low pH is attributable to higher acidic strength of the acid produced in the medium MSB. Progressive scale-up of the process was also done from 0.1 L to 10 L and substantial drop in the pH value with time was observed. However, critical parameters like pulp density, temperature, duration for conducting leaching experiments warrants further investigations for process optimization. Thus, bioleaching can be used as an alternative to reclamation of metals by chemical treatments at an industrial level. In addition, the product of leaching reaction NiSO4, which is commercially the most important nickel compound used for electroplating and it has demand in the ceramic industries and for laboratory use as well. Acknowledgement The authors acknowledge the Department of Biotechnology, Govt. of India, for financial support (Sanction no. BT/PR–11779/BCE/08/723/2009). References 1 2
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Conclusions In this study, the conditions for the bioleaching of nickel from spent petroleum catalyst with a chemolithotrophic strain A. thiooxidans were optimized. Roasting of spent catalyst before bioleaching experiments did not result in any loss of metallic constituents present in the catalyst. The optimum temperature for the growth of A. thiooxidans was 32 °C and media MSA and MSB were optimized for culture propagation and conducting bioleaching experiments respectively.
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