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Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Microbial treatment of sulfur-contaminated industrial wastes a
a
b
Marlenne Gómez-Ramírez , Karina Zarco-Tovar , Jorge Aburto , Roberto García de León b
& Norma. G. Rojas-Avelizapa
a
a
Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada del Instituto Politécnico Nacional , Querétaro , México b
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Instituto Mexicano del Petróleo , México D.F , México Published online: 30 Oct 2013.
To cite this article: Marlenne Gómez-Ramírez , Karina Zarco-Tovar , Jorge Aburto , Roberto García de León & Norma. G. Rojas-Avelizapa (2014) Microbial treatment of sulfur-contaminated industrial wastes, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:2, 228-232, DOI: 10.1080/10934529.2013.838926 To link to this article: http://dx.doi.org/10.1080/10934529.2013.838926
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Journal of Environmental Science and Health, Part A (2014) 49, 228–232 C Taylor & Francis Group, LLC Copyright ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2013.838926
Microbial treatment of sulfur-contaminated industrial wastes ´ MARLENNE GOMEZ-RAM I´REZ1, KARINA ZARCO-TOVAR1, JORGE ABURTO2, ´ ´ ROBERTO GARCIA DE LEON2 and NORMA. G. ROJAS-AVELIZAPA1 1
Centro de Investigaci´on en Ciencia Aplicada y Tecnolog´ıa Avanzada del Instituto Polit´ecnico Nacional, Quer´etaro, M´exico Instituto Mexicano del Petr´oleo, M´exico D.F, M´exico
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The present study evaluated the microbial removal of sulfur from a solid industrial waste in liquid culture under laboratory conditions. The study involved the use of two bacteria Acidithiobacillus ferrooxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCT-M1255 isolated from a Mexican soil. Experimentation for industrial waste biotreatment was done in liquid culture using 125-mL Erlenmeyer flasks containing 30 mL Starkey modified culture medium and incubated at 30◦ C during 7 days. The industrial waste was added at different pulp densities (8.25–100% w/v) corresponding to different sulfur contents from 0.7 to 8.63% (w/w). Sulfur-oxidizing activity of the strain AZCT-M125-5 produced 281 and 262 mg/g of sulfate and a sulfur removal of 60% and 45.7% when the pulp density was set at 8.25 and 16.5% (w/v), respectively. In comparison, the strain A. ferrooxidans ATCC 53987 showed a lower sulfur-oxidizing activity with a sulfate production of 25.6 and 12.7 mg/g and a sulfur removal of 6% and 2.5% at the same pulp densities, respectively. Microbial growth was limited by pulp densities higher than 25% (w/v) of industrial waste with minimal sulfur-oxidizing activity and sulfur removal. The rate of sulfur removal for Acidithiobacillus thioxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 was 0.185 and 0.0159 mg S g−1 h−1 with a pulp density of 16.5% (w/v), respectively. This study demonstrated that Acidithiobacillus thiooxidans AZCT-M125-5 possesses a high sulfur-oxidizing activity, even at high sulfur concentration, which allows the treatment of hazardous materials. Keywords: Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, solid industrial waste, sulfur-oxidizing activity, sulfur removal.
Introduction In the modern world, diverse types of activities, including agriculture, manufactories, and transportation, produce a large amount of wastes and new types of pollutants. Soil, air and water have traditionally been used as disposal sites of all these wastes,[1] and the problem has exponentially increased with population and industrial development.[2] Among the most important pollutants, sulfur compounds are of special interest because they are produced during petroleum refining, burned at fixed facilities to generate electricity as coal and coke and generated by vehicles.[3] The presence of elevated levels of sulfur in soils and sediments can create high levels of acidity in water sources and cause detrimental effects to the environment. Depending on the availability of oxygen in the sediments that creates anoxic conditions, sulfur can be transformed to hydrogen sulfide which is toxic to life and unpleasant at extremely low concentrations when volatilized. ´ Address correspondence to Marlenne Gomez-Ram´ ırez, Centro ´ en Ciencia Aplicada y Tecnolog´ıa Avanzada de Investigacion del IPN, Cerro Blanco 141, Colonia Colinas del Cimatario, Quer´etaro, 76090, Quer´etaro M´exico; E-mail: [email protected] Received April 26, 2013.
Additionally, the emission of reduced and/or oxidized sulfur compounds to the environment is associated with acid rain, corrosion phenomena, and can be toxic under certain conditions. Hydrogen sulfide, H2 S(g), constitutes the main pollutant in gaseous fuels such as liquefied petroleum gas (LPG), natural gas, biogas, and needs to be controlled due to its negative impact on health and the environment.[4] International environmental regulations strictly prohibit the use of this chemical during a war. However, sulfur mustard has been excessively produced, stored, and employed in various regions of the world, damaging human health and environment.[5] In Mexico, the high concentration of ozone and sulfur oxide has severely decreased air quality for many years.[6] Hence, strict pollution regulations have been implemented during the last 16 years.[7] One of them concerning sulfur pollution is the recovery of sulfur from LPG streams. Indeed, Mexican regulations [8] consider that all streams containing sulfur should be treated to recover it in order to reduce their emission to the atmosphere.[9] Nevertheless, sulfur-containing wastes are generated from gas processing and available treatments imply the use of chemical compounds that generate more toxic compounds.[10] Thus, it is important to develop economic and environmental friendly methods to treat huge amounts of polluted material with
Treatment of sulfur-contaminated industrial wastes sulfur compounds,[11] mainly those coming from crude oil and fuels processing. Based on previous data, the aim of this study was to evaluate the use of chemolithoautotrophic sulfur-oxidizing bacteria for the removal of sulfur contained in a solid industrial waste for future applications in petroleum industry.
Materials and methods
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Microorganisms and culture media Sulfur-oxidizing bacteria used throughout this study were Acidithiobacillus ferroxidans ATCC 53987 and a strain of Acidithiobacillus thiooxidans AZCT-M125-5, which was isolated from a Mexican soil with high sulfurcontent.[12,13] Culture medium used was a modified Starkey medium composed of (g/L): KH2 PO4 , 3; (NH4 )2 SO4 , 0.2; MgSO4 .7H2 O, 0.5; CaCl2 .2H2 O, 0.3; FeSO4. 7H2 O, 0.1; 30 ppb of molybdenum; elemental sulfur was added at a concentration of 1% (w/v). The medium was adjusted to pH 3 with sulfuric acid.[13] Industrial waste The industrial waste (IW) contaminated with sulfur is a cylindrical solid with 2–10 mm long and 3 mm in diameter and furnished by the Mexican Petroleum Institute (IMP). Its chemical analysis by X-ray energy dispersive spectroscopy (EDS) revealed the presence of 86.07% TiO2 , 12% CaSO4 and 1.92% Al2 O3 . In the experimental assays, the waste was gently ground using a porcelain mortar and pestle, dried and screened through a sieve of 595 µm. It was further washed with distilled water to remove impurities (mainly sulfate) using an IW/water ratio of 200 g/L and nine washings to remove sulfate. We used two IW lots containing with 7.2 and 8.63% sulfur, the first lot was used for the study of sulfur removal and the second for evaluation of pulp density, respectively. Sulfur analysis by HPLC A sample of 0.05 g of dry industrial waste (IW) was placed in a 30-mL screw capped vial, then 5 mL of tetrahydrofuran (THF) ACS grade were added. The mixture was stirred on an orbital rotating plate at 30 rpm for 2 h. After that time, THF was collected with the aid of a glass syringe and filtering through a Restek membrane filter. The filtrate was placed in a 1.5 mL glass tube. The resulting solution was analyzed by HPLC (Agilent, Waldbronn, Germany) fitted with a UV detector (Agilent) operating at 217 nm, an Econosphere 5-µm C18 reverse phase column (4.6 × 250 mm) was used at a flow rate of 1 mL/min using as eluent a mixture of methanol/water at 97/3.
229 Sulfur removal by Acidithiobacillus ferroxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCT-M125-5 at different IW pulp densities The inoculum was prepared in 250-mL flasks containing 50 mL of modified Starkey medium at pH 3, elemental sulfur at 1%, and incubated at 30ºC, 140 rpm during 6 days. Afterwards, this inoculum was used for studies of sulfur removal. Eight different experimental sets were prepared as follows: 125-mL Erlenmeyer flasks containing 20 mL of modified Starkey medium were inoculated with 2 mL (10%) of the inoculum (3 × 107 CFU/mL) and supplemented with the corresponding amount of IW. The IW pulp densities were fixed at 8.25, 16.5, 25, 33, 49.5, 66.2, 82.2, and 100% w/v corresponding to sulfur contents of 0.7, 1.4, 2.1, 2.8, 4.3, 5.7, 7.1, and 8.63% (w/w), respectively. Flasks were incubated at 30◦ C, 140 rpm, during 7 days. Controls without inoculum were also included to determine abiotic loss of sulfur. After the incubation period, the liquid phase was filtered using a cellulose acetate syringe filter (Alltech, Deerfield, IL, USA) and the filtrate was placed in a 40-mL glass tube. Sulfur oxidation by microorganisms was evaluated through sulfate turbidimetry determination according to a Mexican standard method.[14] Each determination was performed in duplicate. The production of sulfuric acid in the supernatant was also evaluated by pH measurement, using a digital potentiometer (Marchery-Nagel GmBH & Co., Dueren, Germany). Measurements of these parameters were carried out at the beginning and end of experimentation in duplicate. Industrial waste was dried at 40◦ C for 48 h and its residual sulfur content was determined by HPLC as already described.
Study of sulfur removal from industrial waste at 16.5% pulp density by Acidithiobacillus ferrooxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCT-M125-5 The inoculum for both Acidithiobacillus ferrooxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCTM125-5 was prepared as previously mentioned. In this case, an experimental set consisting of 125-mL Erlenmeyer flasks containing 20 mL of Starkey medium was prepared as follows per each microbial culture: the IW with 7.2% sulfur was added at 16.5% (w/v) pulp density and inoculated with 2 mL (10%) of a microbial solution (3 × 107 CFU/mL). Flasks were incubated at 30◦ C, 140 rpm and maintained for 216 h (9 days). The treatments were prepared in triplicate. We collected three flasks every 12 h, and the supernatant was analyzed for sulfate and pH as mentioned above. Residual sulfur content in the IW was evaluated as previously mentioned. We considered also control flasks with sulfurcontaining IW, modified Starkey medium but no inoculums in order to assess abiotic removal of sulfur.
230 Results and discussion
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Sulfur removal at different IW pulp densities For this study, we used two sulfur-oxidizing bacteria Acidithiobacillus ferrooxidans ATCC 53987 and the strain Acidithiobacillus thiooxidans AZCT-M125-5 previously isolated by our group. As aforementioned, the industrial waste was evaluated at different pulp densities (8.25, 16.5, 25, 33, 49.5, 66.2, 82.2, and 100% w/v) corresponding to sulfur contents of 0.7, 1.4, 2.1, 2.8, 4.3, 5.7, 7.1, and 8.63% (w/w), respectively. Previous studies showed that strains Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 were capable of chemolithoautotrophic growing using elemental sulfur as energy source up to 9% in liquid culture.[12] However, it is important to know whether Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 are able to assimilate sulfur imbibed in the industrial waste. We observed that strain Acidithiobacillus ferrooxidans AZCT-M125-5 showed the highest sulfur-oxidizing activity with a sulfate production of 281 and 262 mg/g of solid waste at 8.25 and 16.5% (w/v) IW pulp density, respectively (Fig. 1). Regarding strain Acidithiobacillus ferrooxidans ATCC 53987, the sulfur-oxidizing activity was one order of magnitude lower with a sulfate production of 25.6 and 12.7 mg of sulfate per g of solid waste corresponding to 8.25 and 16.5% (w/v) pulp density. Hence, the isolated strain Acidithiobacillus ferrooxidans AZCT-M125-5 was able to produce eleventimes more sulfate than Acidithiobacillus ferrooxidans ATCC 53987 under the same experimental conditions.
Fig. 1. Effect of IW pulp density on sulfate production through sulfur oxidizing activity of Acidithiobacillus thiooxidans AZCTM125-5 and Acidithiobacillus ferrooxidans ATCC 53987 after 7 days of incubation at 30◦ C and 140 rpm (color figure available online).
G´omez-Ram´ırez et al. Pulp densities higher than 25% (w/v) highly affect sulfur-oxidizing activity of Acidithiobacillus ferrooxidans AZCT-M125-5, since sulfate production only reached 40 mg/g of industrial waste at this concentration. Regarding Acidithiobacillus ferrooxidans ATCC 53987, pulp densities higher than 33% (w/v) totally inhibited the sulfur-oxidizing activity. Such results may be attributed to different factors such as the lower water content in the medium that causes reduction of mass transport and accessibility of microorganisms to elemental sulfur; but also compromising their metabolic activities. It is well established that folded conformation of proteins in solution is determined largely by water properties. If cytoplasmic water exists in different hydrogen bonded states in different regions of the cell, both protein conformations and aggregation-disaggregation states are likely to be affected.[15] In addition, microbial cells must be in contact with hydrophobic sulfur particles and somehow oxidize it to hydrophilic sulfate. Thus, water activity is expected to have a significant influence on sulfur oxidation. Another reason might be the high osmotic pressure caused by elemental sulfur at high pulp density, which could affect the sulfur-oxidizing activity. Indeed, cells lose water when exposed to high osmotic solutions, and such condition might be deleterious to the maintenance of their normal structure, function, and metabolic control.[16] At higher pulp densities of the industrial waste (>25%), the mixture solid-culture medium becomes denser, making difficult mass transfer of oxygen and limiting sulfur oxidation or the availability of carbon dioxide as a sole carbon source.[17] We reestablished sulfur-oxidizing activity by using the same IW content but with a higher water volume, maintaining a IW/water ratio of 10:1 (data not shown). The lower sulfur-oxidizing activity of Acidithiobacillus ferrooxidans ATCC 53987 is explained in terms of its inability to efficiently take sulfur from the industrial waste, resulting in a lower sulfur-oxidizing activity. With regard to the pH, Acidithiobacillus thiooxidans AZCT-M125-5 caused the diminution from 1.9 to 1.25, while Acidithiobacillus ferrooxidans ATCC 53987 reached a slightly higher value since the pH value decreased from 1.9 to 1.6. Such change on pH value is due to the production of sulfuric acid from elemental sulfur and shows that the sulfur-oxidizing activity of strain AZCT-M125-5 is more important in regard to the strain Acidithiobacillus ferrooxidans ATCC 53987. We consider that the strain of Acidithiobacillus thiooxidans AZCTM125-5 can be used in the leaching of heavy metals through biohydrometallurgical approaches, aside from being useful in sulfur removal of industrial wastes as it was demonstrated in the present study. In regard to sulfur-oxidizing activity, it has been observed that the rate of sulfur oxidation increased for oxidizing bacteria at concentrations from 0.5 to 5[18] or 1 to 20 g/L of elemental sulfur as described elsewhere.[19] However, there is no report, to our knowledge, concerning the microbial
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Fig. 2. Residual sulfur concentration in IW by HPLC after treatment with Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 after 7 days of incubation, 140 rpm, and 30◦ C.
Fig. 3. Sulfur oxidation contained in IW by Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 at 30◦ C, 140 rpm; pH evolution during sulfur removal in IW.
treatment of an industrial waste containing sulfur using sulfur-oxidizing bacteria. In order to determine the removal of elemental sulfur from the IW due to the action of the isolate Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987, the treated industrial waste was further analyzed by HPLC. In the first case, we observed residual sulfur contents of 3.5 and 4.6% (w/w) at 8.25 and 16.5% (w/v) pulp densities and after 7 days of treatment, respectively (Fig. 2); which corresponds to, 60 and 46% sulfur removal from the IW. In the other side, Acidithiobacillus ferrooxidans ATCC 53987 reached a sulfur content of 8.3 and 8.6%, at the same pulp densities,, which corresponded to a sulfur removal of 6 and 2.5%, respectively. Nevertheless, the highest sulfur removal for Acidithiobacillus ferrooxidans ATCC 53987 was obtained at 33% (w/v) pulp density, which corresponds to 9% of sulfur removal.
from 1.95 to 1.4, which indicates that bacteria oxidized the elemental sulfur to sulfates. Hence, we observed a maximum sulfate production of 65 mg sulfate/g solid waste and a sulfur removal rate of 0.185 mg S g−1 h−1. In the case of Acidithiobacillus ATCC 53987, sulfate production remained practically unchanged throughout the incubation period, yielding a maximum sulfate production of 15 mg sulfate per g of solid waste after 216 h and a removal rate of 0.0159 mg S g−1 h−1. Moreover, the pH remained without changes during all the incubation period. These data show again that Acidithiobacillus thiooxidans AZCT-M125-5 is a better candidate to the biotechnological removal process of sulfur contained in solid wastes. There are reports concerning the use of sulfur-oxidizing microorganisms but to improve the copper leaching efficiency during chalcopyrite bioleaching. This is due to the microorganism’s ability to oxidize the sulfur layer formed on the mineral surface which produces sulfuric acid, which is a good bleaching agent.[20] The present study is, to the best of our knowledge, the first report in which sulfur-oxidizing activity is evaluated to sulfur removal of an industrial waste. The residual sulfur content in the solid waste at the end of the treatment using a pulp density of 16.5% by Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 was 5.2 and 6.84% (Fig. 4). The latter corresponds to a sulfur removal of 32 and 11.9%, respectively. Once again, Acidithiobacillus thiooxidans AZCT-M125-5 possesses a better ability to remove sulfur from the solid waste at the assayed conditions than Acidithiobacillus ferrooxidans ATCC 53987. The latter strain showed a good sulfur-oxidizing activity[12] and has been used in previous studies,[21–22] but Acidithiobacillus thiooxidans AZCT-M125-5 was more efficient in sulfur
Sulfur removal at 16.5% (w/v) pulp density of IW We was interested to determine the sulfur removal for both bacteria at the maximum IW concentration at which microorganisms exhibit a good sulfur-oxidizing activity. The study was followed during 216 h in batch culture using 16.5% (w/v) IW pulp density. We collected Erlenmeyer flasks every 12 h and both liquid and solid phases were analyzed for sulfate, pH in liquid phase and sulfur removal in solid phase. Acidithiobacillus thiooxidans AZCT-M125-5 has a growth lag phase of 96 h and indicating that microbial population needs a large period of adaptation to assimilate sulfur from the solid waste (Fig. 3). After that, the microorganism has an exponential growth phase that maintained at least 216 h. The pH value decreased during this log phase
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Fig. 4. Residual sulfur content in the IW after treatment with Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans HPLC at 140 rpm, 30◦ C, after 126 h.
removal from the target industrial waste. Future studies must include scale-up at lab column scale packed with the industrial waste in order to optimize and evaluate the effect of aeration rate, inoculum size, media renovation and incubation time on sulfur removal. This information might allow the estimation of the technical and economical feasibility in order to establish a bioleaching process at pilot scale.
Conclusions The present study demonstrates the applicability of sulfur-oxidizing bacterium of Acidithiobacillus thiooxidans AZCT-M125-5 for the removal of sulfur present in a contaminated industrial waste. The efficiency of this strain was higher than that shown by Acidithiobacillus ferroxidans ATCC 53987.
Acknowledgments This work was supported by Secretary of Energy and The National Council of Science and Technology of Mexico (Grant No. 136465) and Instituto Polit´ecnico Nacional.
References [1] Setyorini, D.; Prihatini, T.; Kurnia, U. Pollution of soil by agricultural and industrial waste. Jalan Ir. Juanda. 2002, 98, 1–11. ¨ Arisoy, M. A new approach for evaluating wastes. J. [2] Simsek, O.; Biol. Chem. 2007, 35, 17–24. [3] Guttikunda, S.K.; Carmichael, G.R.; Calori, G.; Eck, C.; JungHun, W. The contribution of megacities to regional sulfur pollution in Asia. Atmos. Environ. 2003, 37, 11–22.
G´omez-Ram´ırez et al. [4] Gonz´alez-S´anchez, A.; Revah, S. The effect of chemical oxidation on the biological sulfide oxidation by an alkaliphilic sulfoxidizing bacterial consortium. Enzyme Microb. Tech. 2007, 40, 292–298. [5] Hassan, Z.M.; Ebtekar, M.; Ghanei, M.; Taghikhani, M.; Daloii, M.R.N.; Ghazanfari, T. Immunobiological consequences of sulfur mustard contamination. Iran. J. Allergy Asthma Immunol. 2006, 5, 101–108. [6] Raga, G.B.; Raga, A.C. On the formation of an elevated ozone peak in Mexico city. Atmos. Environ. 2000, 34, 4097–4102. ´ [7] Hoyos, L.; Lara, P.; Ortiz, E.; Lopez, R.; Gonz´alez, J. Evaluation of air pollution control policies in Mexico city using finite markov chain observation model. Rev. Mat. 2009, 16, 255–266. [8] Secretar´ıa del Medio Ambiente y Recursos Naturales (SEMARNAT). Air pollution: recovery of sulfur from the oil refining process; ´ NOM-148-SEMARNAT-2006. Diario Oficial de la Federacion: Mexico, D. F., Mexico, 2007. ´ [9] Alc´antara-Gonz´alez, F.S.; Cruz-Gomez, M.J. An´alisis de la dis´ de azufre en productos, emisiones de SOx y la recutribucion ´ del mismo en el sistema nacional de refinacion. ´ Rev. Int. peracion Contam. Ambie. 2011, 27, 153–163. [10] Alberta Environment (AENV). Sulphur dioxide: environmental effects, fate and behavior; Alberta Environment: Edmonton, AB, March 2003; 6–9. [11] Janssen, A.J.H.; Ruitenberg, R.; Buisman, C.N.J. Industrial applications of new sulphur biotechnology. Water Sci. Technol. 2001, 44, 85–90. ´ [12] Rojas-Avelizapa, N.G.; Gomez-Ram´ ırez, M.; Hern´andez-Gama, ´ R. IsoR.; Zarate-Garc´ıa, M.; Aburto-Anell, A.; Garc´ıa de Leon, lation of Sulfur-Oxidizing Bacterial with Potential Applications in Sulfur-Compounds Polluted Environments, Proceedings of the 3rd International Symposium on Environmental Management, Croatia, Oct. 26–28, 2011; Natalija, K., Hrvoje, K., Ana, L.B. Eds.; Faculty of Chemical Engineering and Technology, University of Zagreb: Zagreb, Croatia, 2011; 181–188. ´ [13] Rojas-Avelizapa, N. G.; Gomez-Ram´ ırez, M.; Hern´andez-Gama, ´ R. Isolation and selection of sulfurR.; Aburto, J.; Garc´ıa de Leon, oxidizing bacteria for the treatment of sulfur-containing hazardous wastes. Chem. Biochem. Eng. Q. 2013, 27, 109–117. [14] Secretar´ıa de Comercio y Fomento Industrial. Determination of ion sulfate in water samples for the feeding of boilers; NMX-K436-1977. Author: Mexico, D. F., Mexico, 1978. [15] Wiggins, P.M. Role of water in some biological processes. Microbiol. Rev. 1990, 54, 432–449. [16] Suzuki, I.; Lee, D.; Mackay, B.; Harahuc, L.; Oh, J.K. Effect of various ions, pH, and osmotic pressure on oxidation of elemental sulfur by Thiobacillus thiooxidans. Appl. Environ. Microbiol. 1999, 65, 5163–5168. ˇ [17] Kupka, D.; Skvarla, J.; Biroˇsov´a, E. Evaluation of growth rate of Acidithiobacillus ferrooxidans oxidizing elemental sulfur. Acta Metall. Slovaca. 2003, 9, 251–258. [18] Shen-Yi, C.; Jih-Gaw, L. Bioleaching of heavy metals from contaminated sediment by indigenous sulfur-oxidizing bacteria in an air-lift bioreactor: effects of sulfur concentration. Water Res. 2004, 38, 3205–3214. [19] Pokorna, B.; Mandl, M.; Borilova, S.; Ceskova, P.; Markova, R.; Janiczek, O. Kinetic constant variability in bacterial oxidation of elemental sulfur. Appl. Environ. Microbiol. 2007, 73, 3752–3754. [20] Zhou, Q.G.; Bo, F.; Bo, Z.H.; Xi, L.; Jian, G.; Fei, L.F.; Hua, C.X. Isolation of a strain of Acidithiobacillus caldus and its role in bioleaching of chalcopyrite. World J. Microbiol. Biotechnol. 2007, 23, 1217–1225. [21] Premuzic, E.T.; Lin, M.S. Biochemical Solubilization of Toxic Salts from Residual Geothermal Brines and Waste Waters. US Patent 5,366,891, November 22, 1994. [22] Premuzic, E.T.; Lin, M.S. Biochemical Upgrading of Oils. US Patent 5,858,766, January 12, 1999.
Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20
Microbial treatment of sulfur-contaminated industrial wastes a
a
b
Marlenne Gómez-Ramírez , Karina Zarco-Tovar , Jorge Aburto , Roberto García de León b
& Norma. G. Rojas-Avelizapa
a
a
Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada del Instituto Politécnico Nacional , Querétaro , México b
Click for updates
Instituto Mexicano del Petróleo , México D.F , México Published online: 30 Oct 2013.
To cite this article: Marlenne Gómez-Ramírez , Karina Zarco-Tovar , Jorge Aburto , Roberto García de León & Norma. G. Rojas-Avelizapa (2014) Microbial treatment of sulfur-contaminated industrial wastes, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 49:2, 228-232, DOI: 10.1080/10934529.2013.838926 To link to this article: http://dx.doi.org/10.1080/10934529.2013.838926
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Environmental Science and Health, Part A (2014) 49, 228–232 C Taylor & Francis Group, LLC Copyright ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2013.838926
Microbial treatment of sulfur-contaminated industrial wastes ´ MARLENNE GOMEZ-RAM I´REZ1, KARINA ZARCO-TOVAR1, JORGE ABURTO2, ´ ´ ROBERTO GARCIA DE LEON2 and NORMA. G. ROJAS-AVELIZAPA1 1
Centro de Investigaci´on en Ciencia Aplicada y Tecnolog´ıa Avanzada del Instituto Polit´ecnico Nacional, Quer´etaro, M´exico Instituto Mexicano del Petr´oleo, M´exico D.F, M´exico
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The present study evaluated the microbial removal of sulfur from a solid industrial waste in liquid culture under laboratory conditions. The study involved the use of two bacteria Acidithiobacillus ferrooxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCT-M1255 isolated from a Mexican soil. Experimentation for industrial waste biotreatment was done in liquid culture using 125-mL Erlenmeyer flasks containing 30 mL Starkey modified culture medium and incubated at 30◦ C during 7 days. The industrial waste was added at different pulp densities (8.25–100% w/v) corresponding to different sulfur contents from 0.7 to 8.63% (w/w). Sulfur-oxidizing activity of the strain AZCT-M125-5 produced 281 and 262 mg/g of sulfate and a sulfur removal of 60% and 45.7% when the pulp density was set at 8.25 and 16.5% (w/v), respectively. In comparison, the strain A. ferrooxidans ATCC 53987 showed a lower sulfur-oxidizing activity with a sulfate production of 25.6 and 12.7 mg/g and a sulfur removal of 6% and 2.5% at the same pulp densities, respectively. Microbial growth was limited by pulp densities higher than 25% (w/v) of industrial waste with minimal sulfur-oxidizing activity and sulfur removal. The rate of sulfur removal for Acidithiobacillus thioxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 was 0.185 and 0.0159 mg S g−1 h−1 with a pulp density of 16.5% (w/v), respectively. This study demonstrated that Acidithiobacillus thiooxidans AZCT-M125-5 possesses a high sulfur-oxidizing activity, even at high sulfur concentration, which allows the treatment of hazardous materials. Keywords: Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, solid industrial waste, sulfur-oxidizing activity, sulfur removal.
Introduction In the modern world, diverse types of activities, including agriculture, manufactories, and transportation, produce a large amount of wastes and new types of pollutants. Soil, air and water have traditionally been used as disposal sites of all these wastes,[1] and the problem has exponentially increased with population and industrial development.[2] Among the most important pollutants, sulfur compounds are of special interest because they are produced during petroleum refining, burned at fixed facilities to generate electricity as coal and coke and generated by vehicles.[3] The presence of elevated levels of sulfur in soils and sediments can create high levels of acidity in water sources and cause detrimental effects to the environment. Depending on the availability of oxygen in the sediments that creates anoxic conditions, sulfur can be transformed to hydrogen sulfide which is toxic to life and unpleasant at extremely low concentrations when volatilized. ´ Address correspondence to Marlenne Gomez-Ram´ ırez, Centro ´ en Ciencia Aplicada y Tecnolog´ıa Avanzada de Investigacion del IPN, Cerro Blanco 141, Colonia Colinas del Cimatario, Quer´etaro, 76090, Quer´etaro M´exico; E-mail: [email protected] Received April 26, 2013.
Additionally, the emission of reduced and/or oxidized sulfur compounds to the environment is associated with acid rain, corrosion phenomena, and can be toxic under certain conditions. Hydrogen sulfide, H2 S(g), constitutes the main pollutant in gaseous fuels such as liquefied petroleum gas (LPG), natural gas, biogas, and needs to be controlled due to its negative impact on health and the environment.[4] International environmental regulations strictly prohibit the use of this chemical during a war. However, sulfur mustard has been excessively produced, stored, and employed in various regions of the world, damaging human health and environment.[5] In Mexico, the high concentration of ozone and sulfur oxide has severely decreased air quality for many years.[6] Hence, strict pollution regulations have been implemented during the last 16 years.[7] One of them concerning sulfur pollution is the recovery of sulfur from LPG streams. Indeed, Mexican regulations [8] consider that all streams containing sulfur should be treated to recover it in order to reduce their emission to the atmosphere.[9] Nevertheless, sulfur-containing wastes are generated from gas processing and available treatments imply the use of chemical compounds that generate more toxic compounds.[10] Thus, it is important to develop economic and environmental friendly methods to treat huge amounts of polluted material with
Treatment of sulfur-contaminated industrial wastes sulfur compounds,[11] mainly those coming from crude oil and fuels processing. Based on previous data, the aim of this study was to evaluate the use of chemolithoautotrophic sulfur-oxidizing bacteria for the removal of sulfur contained in a solid industrial waste for future applications in petroleum industry.
Materials and methods
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Microorganisms and culture media Sulfur-oxidizing bacteria used throughout this study were Acidithiobacillus ferroxidans ATCC 53987 and a strain of Acidithiobacillus thiooxidans AZCT-M125-5, which was isolated from a Mexican soil with high sulfurcontent.[12,13] Culture medium used was a modified Starkey medium composed of (g/L): KH2 PO4 , 3; (NH4 )2 SO4 , 0.2; MgSO4 .7H2 O, 0.5; CaCl2 .2H2 O, 0.3; FeSO4. 7H2 O, 0.1; 30 ppb of molybdenum; elemental sulfur was added at a concentration of 1% (w/v). The medium was adjusted to pH 3 with sulfuric acid.[13] Industrial waste The industrial waste (IW) contaminated with sulfur is a cylindrical solid with 2–10 mm long and 3 mm in diameter and furnished by the Mexican Petroleum Institute (IMP). Its chemical analysis by X-ray energy dispersive spectroscopy (EDS) revealed the presence of 86.07% TiO2 , 12% CaSO4 and 1.92% Al2 O3 . In the experimental assays, the waste was gently ground using a porcelain mortar and pestle, dried and screened through a sieve of 595 µm. It was further washed with distilled water to remove impurities (mainly sulfate) using an IW/water ratio of 200 g/L and nine washings to remove sulfate. We used two IW lots containing with 7.2 and 8.63% sulfur, the first lot was used for the study of sulfur removal and the second for evaluation of pulp density, respectively. Sulfur analysis by HPLC A sample of 0.05 g of dry industrial waste (IW) was placed in a 30-mL screw capped vial, then 5 mL of tetrahydrofuran (THF) ACS grade were added. The mixture was stirred on an orbital rotating plate at 30 rpm for 2 h. After that time, THF was collected with the aid of a glass syringe and filtering through a Restek membrane filter. The filtrate was placed in a 1.5 mL glass tube. The resulting solution was analyzed by HPLC (Agilent, Waldbronn, Germany) fitted with a UV detector (Agilent) operating at 217 nm, an Econosphere 5-µm C18 reverse phase column (4.6 × 250 mm) was used at a flow rate of 1 mL/min using as eluent a mixture of methanol/water at 97/3.
229 Sulfur removal by Acidithiobacillus ferroxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCT-M125-5 at different IW pulp densities The inoculum was prepared in 250-mL flasks containing 50 mL of modified Starkey medium at pH 3, elemental sulfur at 1%, and incubated at 30ºC, 140 rpm during 6 days. Afterwards, this inoculum was used for studies of sulfur removal. Eight different experimental sets were prepared as follows: 125-mL Erlenmeyer flasks containing 20 mL of modified Starkey medium were inoculated with 2 mL (10%) of the inoculum (3 × 107 CFU/mL) and supplemented with the corresponding amount of IW. The IW pulp densities were fixed at 8.25, 16.5, 25, 33, 49.5, 66.2, 82.2, and 100% w/v corresponding to sulfur contents of 0.7, 1.4, 2.1, 2.8, 4.3, 5.7, 7.1, and 8.63% (w/w), respectively. Flasks were incubated at 30◦ C, 140 rpm, during 7 days. Controls without inoculum were also included to determine abiotic loss of sulfur. After the incubation period, the liquid phase was filtered using a cellulose acetate syringe filter (Alltech, Deerfield, IL, USA) and the filtrate was placed in a 40-mL glass tube. Sulfur oxidation by microorganisms was evaluated through sulfate turbidimetry determination according to a Mexican standard method.[14] Each determination was performed in duplicate. The production of sulfuric acid in the supernatant was also evaluated by pH measurement, using a digital potentiometer (Marchery-Nagel GmBH & Co., Dueren, Germany). Measurements of these parameters were carried out at the beginning and end of experimentation in duplicate. Industrial waste was dried at 40◦ C for 48 h and its residual sulfur content was determined by HPLC as already described.
Study of sulfur removal from industrial waste at 16.5% pulp density by Acidithiobacillus ferrooxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCT-M125-5 The inoculum for both Acidithiobacillus ferrooxidans ATCC 53987 and Acidithiobacillus thiooxidans AZCTM125-5 was prepared as previously mentioned. In this case, an experimental set consisting of 125-mL Erlenmeyer flasks containing 20 mL of Starkey medium was prepared as follows per each microbial culture: the IW with 7.2% sulfur was added at 16.5% (w/v) pulp density and inoculated with 2 mL (10%) of a microbial solution (3 × 107 CFU/mL). Flasks were incubated at 30◦ C, 140 rpm and maintained for 216 h (9 days). The treatments were prepared in triplicate. We collected three flasks every 12 h, and the supernatant was analyzed for sulfate and pH as mentioned above. Residual sulfur content in the IW was evaluated as previously mentioned. We considered also control flasks with sulfurcontaining IW, modified Starkey medium but no inoculums in order to assess abiotic removal of sulfur.
230 Results and discussion
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Sulfur removal at different IW pulp densities For this study, we used two sulfur-oxidizing bacteria Acidithiobacillus ferrooxidans ATCC 53987 and the strain Acidithiobacillus thiooxidans AZCT-M125-5 previously isolated by our group. As aforementioned, the industrial waste was evaluated at different pulp densities (8.25, 16.5, 25, 33, 49.5, 66.2, 82.2, and 100% w/v) corresponding to sulfur contents of 0.7, 1.4, 2.1, 2.8, 4.3, 5.7, 7.1, and 8.63% (w/w), respectively. Previous studies showed that strains Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 were capable of chemolithoautotrophic growing using elemental sulfur as energy source up to 9% in liquid culture.[12] However, it is important to know whether Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 are able to assimilate sulfur imbibed in the industrial waste. We observed that strain Acidithiobacillus ferrooxidans AZCT-M125-5 showed the highest sulfur-oxidizing activity with a sulfate production of 281 and 262 mg/g of solid waste at 8.25 and 16.5% (w/v) IW pulp density, respectively (Fig. 1). Regarding strain Acidithiobacillus ferrooxidans ATCC 53987, the sulfur-oxidizing activity was one order of magnitude lower with a sulfate production of 25.6 and 12.7 mg of sulfate per g of solid waste corresponding to 8.25 and 16.5% (w/v) pulp density. Hence, the isolated strain Acidithiobacillus ferrooxidans AZCT-M125-5 was able to produce eleventimes more sulfate than Acidithiobacillus ferrooxidans ATCC 53987 under the same experimental conditions.
Fig. 1. Effect of IW pulp density on sulfate production through sulfur oxidizing activity of Acidithiobacillus thiooxidans AZCTM125-5 and Acidithiobacillus ferrooxidans ATCC 53987 after 7 days of incubation at 30◦ C and 140 rpm (color figure available online).
G´omez-Ram´ırez et al. Pulp densities higher than 25% (w/v) highly affect sulfur-oxidizing activity of Acidithiobacillus ferrooxidans AZCT-M125-5, since sulfate production only reached 40 mg/g of industrial waste at this concentration. Regarding Acidithiobacillus ferrooxidans ATCC 53987, pulp densities higher than 33% (w/v) totally inhibited the sulfur-oxidizing activity. Such results may be attributed to different factors such as the lower water content in the medium that causes reduction of mass transport and accessibility of microorganisms to elemental sulfur; but also compromising their metabolic activities. It is well established that folded conformation of proteins in solution is determined largely by water properties. If cytoplasmic water exists in different hydrogen bonded states in different regions of the cell, both protein conformations and aggregation-disaggregation states are likely to be affected.[15] In addition, microbial cells must be in contact with hydrophobic sulfur particles and somehow oxidize it to hydrophilic sulfate. Thus, water activity is expected to have a significant influence on sulfur oxidation. Another reason might be the high osmotic pressure caused by elemental sulfur at high pulp density, which could affect the sulfur-oxidizing activity. Indeed, cells lose water when exposed to high osmotic solutions, and such condition might be deleterious to the maintenance of their normal structure, function, and metabolic control.[16] At higher pulp densities of the industrial waste (>25%), the mixture solid-culture medium becomes denser, making difficult mass transfer of oxygen and limiting sulfur oxidation or the availability of carbon dioxide as a sole carbon source.[17] We reestablished sulfur-oxidizing activity by using the same IW content but with a higher water volume, maintaining a IW/water ratio of 10:1 (data not shown). The lower sulfur-oxidizing activity of Acidithiobacillus ferrooxidans ATCC 53987 is explained in terms of its inability to efficiently take sulfur from the industrial waste, resulting in a lower sulfur-oxidizing activity. With regard to the pH, Acidithiobacillus thiooxidans AZCT-M125-5 caused the diminution from 1.9 to 1.25, while Acidithiobacillus ferrooxidans ATCC 53987 reached a slightly higher value since the pH value decreased from 1.9 to 1.6. Such change on pH value is due to the production of sulfuric acid from elemental sulfur and shows that the sulfur-oxidizing activity of strain AZCT-M125-5 is more important in regard to the strain Acidithiobacillus ferrooxidans ATCC 53987. We consider that the strain of Acidithiobacillus thiooxidans AZCTM125-5 can be used in the leaching of heavy metals through biohydrometallurgical approaches, aside from being useful in sulfur removal of industrial wastes as it was demonstrated in the present study. In regard to sulfur-oxidizing activity, it has been observed that the rate of sulfur oxidation increased for oxidizing bacteria at concentrations from 0.5 to 5[18] or 1 to 20 g/L of elemental sulfur as described elsewhere.[19] However, there is no report, to our knowledge, concerning the microbial
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Fig. 2. Residual sulfur concentration in IW by HPLC after treatment with Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 after 7 days of incubation, 140 rpm, and 30◦ C.
Fig. 3. Sulfur oxidation contained in IW by Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 at 30◦ C, 140 rpm; pH evolution during sulfur removal in IW.
treatment of an industrial waste containing sulfur using sulfur-oxidizing bacteria. In order to determine the removal of elemental sulfur from the IW due to the action of the isolate Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987, the treated industrial waste was further analyzed by HPLC. In the first case, we observed residual sulfur contents of 3.5 and 4.6% (w/w) at 8.25 and 16.5% (w/v) pulp densities and after 7 days of treatment, respectively (Fig. 2); which corresponds to, 60 and 46% sulfur removal from the IW. In the other side, Acidithiobacillus ferrooxidans ATCC 53987 reached a sulfur content of 8.3 and 8.6%, at the same pulp densities,, which corresponded to a sulfur removal of 6 and 2.5%, respectively. Nevertheless, the highest sulfur removal for Acidithiobacillus ferrooxidans ATCC 53987 was obtained at 33% (w/v) pulp density, which corresponds to 9% of sulfur removal.
from 1.95 to 1.4, which indicates that bacteria oxidized the elemental sulfur to sulfates. Hence, we observed a maximum sulfate production of 65 mg sulfate/g solid waste and a sulfur removal rate of 0.185 mg S g−1 h−1. In the case of Acidithiobacillus ATCC 53987, sulfate production remained practically unchanged throughout the incubation period, yielding a maximum sulfate production of 15 mg sulfate per g of solid waste after 216 h and a removal rate of 0.0159 mg S g−1 h−1. Moreover, the pH remained without changes during all the incubation period. These data show again that Acidithiobacillus thiooxidans AZCT-M125-5 is a better candidate to the biotechnological removal process of sulfur contained in solid wastes. There are reports concerning the use of sulfur-oxidizing microorganisms but to improve the copper leaching efficiency during chalcopyrite bioleaching. This is due to the microorganism’s ability to oxidize the sulfur layer formed on the mineral surface which produces sulfuric acid, which is a good bleaching agent.[20] The present study is, to the best of our knowledge, the first report in which sulfur-oxidizing activity is evaluated to sulfur removal of an industrial waste. The residual sulfur content in the solid waste at the end of the treatment using a pulp density of 16.5% by Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans ATCC 53987 was 5.2 and 6.84% (Fig. 4). The latter corresponds to a sulfur removal of 32 and 11.9%, respectively. Once again, Acidithiobacillus thiooxidans AZCT-M125-5 possesses a better ability to remove sulfur from the solid waste at the assayed conditions than Acidithiobacillus ferrooxidans ATCC 53987. The latter strain showed a good sulfur-oxidizing activity[12] and has been used in previous studies,[21–22] but Acidithiobacillus thiooxidans AZCT-M125-5 was more efficient in sulfur
Sulfur removal at 16.5% (w/v) pulp density of IW We was interested to determine the sulfur removal for both bacteria at the maximum IW concentration at which microorganisms exhibit a good sulfur-oxidizing activity. The study was followed during 216 h in batch culture using 16.5% (w/v) IW pulp density. We collected Erlenmeyer flasks every 12 h and both liquid and solid phases were analyzed for sulfate, pH in liquid phase and sulfur removal in solid phase. Acidithiobacillus thiooxidans AZCT-M125-5 has a growth lag phase of 96 h and indicating that microbial population needs a large period of adaptation to assimilate sulfur from the solid waste (Fig. 3). After that, the microorganism has an exponential growth phase that maintained at least 216 h. The pH value decreased during this log phase
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Fig. 4. Residual sulfur content in the IW after treatment with Acidithiobacillus thiooxidans AZCT-M125-5 and Acidithiobacillus ferrooxidans HPLC at 140 rpm, 30◦ C, after 126 h.
removal from the target industrial waste. Future studies must include scale-up at lab column scale packed with the industrial waste in order to optimize and evaluate the effect of aeration rate, inoculum size, media renovation and incubation time on sulfur removal. This information might allow the estimation of the technical and economical feasibility in order to establish a bioleaching process at pilot scale.
Conclusions The present study demonstrates the applicability of sulfur-oxidizing bacterium of Acidithiobacillus thiooxidans AZCT-M125-5 for the removal of sulfur present in a contaminated industrial waste. The efficiency of this strain was higher than that shown by Acidithiobacillus ferroxidans ATCC 53987.
Acknowledgments This work was supported by Secretary of Energy and The National Council of Science and Technology of Mexico (Grant No. 136465) and Instituto Polit´ecnico Nacional.
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