Environ. Sci. Technol. 2006, 40, 6782-6786
Control of Acidity Development on Solid Sulfur Due to Bacterial Action FRANCESCO CRESCENZI, ANTONELLA CRISARI, EDOARDO D’ANGELI, AND ALESSANDRO NARDELLA* EniTecnologie, Via E. Ramarini 32, Monterotondo (RM), Italy
The global production of sulfur, which is currently obtained almost exclusively as an involuntary byproduct of the oil and gas industry, is exceeding the market demand so that long term storage or even definitive disposal of elemental sulfur is often needed to handle production surplus. The storage of large quantities of elemental sulfur calls for solidifying liquid sulfur in huge blocks, hundred meters wide on each side and as high as 20 meters. Sulfur, in presence of water and air, can be oxidized to sulfuric acid by a ubiquitous microorganism: Thiobacillus. On large blocks, this natural phenomenon may lead to soil and water acidification. Research projects have addressed suppression of Thiobacilli activity to prevent acidification, but no industrial applications have been reported. This work describes the inhibition of sulfur biological oxidation attainable by exposing sulfur to a high ionic strength environment. The bacteriostatic action is produced by contacting sulfur with a solution of an inorganic salt, such as sodium chloride, having an ionic strength similar to sea water. Possible ways to exploit the inhibitory effect to prevent the generation of acidity from sulfur storage blocks are suggested.
Introduction Sulfur is one of the most important raw materials of the chemical industry, its worldwide production is about 40 million t per year (1). Today, more than 90% of world sulfur production is obtained as an involuntary byproduct of the oil and gas industry. The rigid production sometimes leads to accumulation of millions of tonnes of the commodity which, for various reasons, has no outlet in the market and possibly never will. Long-term storage of surplus sulfur is often the only viable option: sulfur is poured into large blocks, each weighing hundreds of thousand tonnes. Sulfur stockpiling consists of solidifying liquid sulfur coming from the recovery units, usually a Claus plant, into large blocks hundreds of meters wide on each side and from 10 to 20 meters high. Due to erosive phenomena, blocks tend to lose integrity, mainly at the sides exposed to rain and freezing conditions, sometimes leading to failure of the block’s walls. This phenomenon greatly increases the risk of sulfur dust spreading around the block’s surroundings. Although very poorly quantified, the release of sulfur dust in the area surrounding the storage site has been claimed to impact the environment. Sulfur entering the environment in disperse form undergoes incorporation into the biological cycle via oxidation to sulfuric acid. The phenomenon proceeds * Corresponding author e-mail:
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through the activity of Thiobacilli, extreme autotrophic bacteria that are able to gain energy by the oxidation of elemental sulfur (2-5). Thiobacillus is a widespread microorganism with a fundamental role in the oxidation of elemental sulfur, involving the contact of cells with sulfur particles, the oxidation of sulfur first to sulfite, and finally to sulfate. The biological oxidation of sulfur may be detrimental to the environment if the quantity of produced acid exceeds the buffering capacity of the local environment. If that occurs, pH tends to decrease, sometimes reaching dangerous values for the residential biota. This leads to the most important and yet unresolved environmental problem of present sulfur storage technology. Due to its low pH, runoff water around a sulfur block must be collected and neutralized before discharge. Besides being a cost, this constitutes a management problem and a long-term liability for the sulfur block’s owner. Recent estimates place yearly water management costs at values exceeding 1$/tonne of sulfur, which adds up to millions dollars per year for the actual or predicted storage capacity of many production sites. Aiming to develop more environmentally friendly solutions for sulfur storage, we identified a phenomenon that could be exploited to substantially decrease the risk of acid generation from a sulfur block. We have found that, while in general Thiobacilli can oxidize sulfur in the many different conditions present in natural habitats such as soil, marine environments, acidic pools in volcanic areas, etc., the bacterium seems to be affected by environments having both acidic pH and high ionic strength. A possible explanation for this phenomenon may be the fact that these conditions are seldom found in nature. In practical terms, in saline environments such as those that can be produced by wetting sulfur with salty water, Thiobacilli stop growing as soon as the pH begins to show acidic values. This inhibiting effect may be used to decrease the environmental impact of sulfur storage.
Materials and Methods Biological Sulfur Oxidation. Water and soil acidification experiments were monitored using two different Thiobacilli microbial strains, one obtained from public cultivations and the other from natural environment. The synthetic Thiobacilli mixture was obtained from public cultivations of lyophilized strains of Thiobacillus thiooxidans DSM 504, Thiobacillus thioparus DSM 505, and Thiobacillus ferrooxidans DSM 583, each grown in its own specific media (6). A mixture of the pure cultures (0.5 mL), containing 108 cells/mL of the Thiobacilli strains, was added to 25 mL of Thiobacillus medium (TM). The medium TM was adjusted to pH 6 with 0.1 N HCl and 0.1 g of elemental sulfur was added (sulfur powder 98%-sieved UNI 0,05). The mixture was then incubated at 30 °C under gentle shaking (100 rpm) for 25 days. After reaching maximum cell concentration, bacterial mixtures were stored in 2 mL aliquots at -70 °C. In the following sections, this culture will be referred to as “mix”. Natural environment microbial mixture (consortium) was obtained by inoculating 25 mL of TM with 2 g of sulfur crumbs from a sulfur deposit in Canada, and incubating it at 30 °C under gentle stirring (100 rpm) for 25 days. During incubation, cell growth was monitored by measuring optical density (OD600.) increase and pH decrease. After reaching maximum 10.1021/es0610131 CCC: $33.50
2006 American Chemical Society Published on Web 10/04/2006
FIGURE 1. Experimental sulfur blocks cell concentration, bacterial mixtures were filtered to eliminate excess sulfur and then stored in 2 mL aliquots at -70 °C. The identification of the consortium as sulfur oxidizing bacteria was established observing the following characteristics, peculiar to Thiobacilli: (1) ability to reduce the pH of the growth medium below pH 2 (that classifies the microorganisms as acidophiles) (2) optimal growth at 30 °C (mesophiles) in the presence of CO2 as the sole carbon source (autotrophic) Thiobacilli Activity Inhibition. Biological sulfur oxidation experiments were conducted inoculating 25 mL of TM (adjusted to pH 6 with 0.1 N HCl) with 0.4% of the natural environment microbial mixture (consortium) and 0.1 g of elemental sulfur (sulfur powder 98%, sieved UNI 0,05). Effect of Temperature. The effect of temperature on microbial activity was investigated incubating the consortium at 5, 10, and 30 °C under gentle stirring (100 rpm). The Thiobacilli activity was monitored measuring the variations of pH. Effect of Ionic Strength. The ionic strength effect on microbial activity was investigated increasing the salinity of the growth medium with inorganic soluble salts. NaCl, Na2SO4, KCl, or KNO3 were used at concentrations up to 0.56 M. During incubation, carried out at 30 °C, Thiobacilli activity was monitored measuring pH decrease and sulfate production. Sulfate Analysis. Sulfate in water was determined via turbidimetry, according to standard methods (7). It was verified that chloride concentration up to 0.7 M did not interfere with the analysis. Sulfur Blocks Construction. Sulfur blocks used to test biological sulfur oxidation in outdoor conditions have been constructed. Pellets of 99% pure sulfur were melted at about 140 °C, and poured into stainless steel molds, in layers not exceeding 10 cm at a time, we waited for complete solidification before adding the following layer. Once the sulfur block was completed, the molds were disassembled.
Results and Discussion Inocula of Sulfur Oxidizing Bacteria. The two inocula of Thiobacilli were compared for their ability to oxidize sulfur. Identical time courses were found for growth and acidification curves of “mix“ and “consortium” cultures, thus we concluded that the two inocula were equivalent in their ability to oxidize sulfur and could be used alternatively. In most of the following experiments the natural consortium was used. Biological Oxidation of Sulfur in Outdoor Conditions. To observe the colonization of solid sulfur and the production of sulfuric acid in conditions simulating a real case, sulfur blocks about 0.5 m by side (Figure 1) were constructed outdoors.
FIGURE 2. Microphotograph of a colonized sulfur surface collected from the block surface. Blebs connecting sulfur and cells can be observed
FIGURE 3. Cumulative acid production per unit of sulfur surface, (S), in the runoff water coming in contact with the top (red), or with the lateral (blue) surfaces of the experimental block After 1 month, samples of sulfur were collected from the top surface of the blocks and observed using a scanning electronic microscope (SEM), Figure 2. Ample bacterial colonization of sulfur surface was observed. The bacteria released extensive amount of outer membrane vesicles, or blebs, that connect cells to sulfur and to other cells, showing that adhesion plays an important role in the colonization of sulfur surfaces in outdoor environments (8). Water acidification due to biological oxidation of sulfur was measured by collecting the water running off the block. Water from the top surface and from the lateral surfaces was collected separately. pH measurements showed that water from the top carried trace of evident acidification, while water coming from the side walls contained little or no acid (Figure 3). These results are probably due to the fact that acidification of rainwater is possible only if water stays in contact with sulfur for sufficient time for bacteria to grow. In our experimental block, this was possible only on the horizontal surface. Acid production on the top surface of the sulfur block had evident seasonal character. Figure 4 shows the strong difference observed between acidification in moist warm autumn weather and very little acid production in hot dry summer, when rapid evaporation quickly dried sulfur surfaces shortening the contact time between water and sulfur. That contact time between water and sulfur was an important parameter for acidification to occur is also VOL. 40, NO. 21, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Amount of acid present in the water running off the top of sulfur block measured in flooding events during late summer and early autumn.
FIGURE 5. pH changes in water running off a sulfur block.
FIGURE 6. Acid production per unit sulfur surface (S) measured in early autumn after a flooding event on the sulfur block. The slope of the straight line is equal to 0.4 mmol(H+)/m2(S)d suggested by pH changes registered in the water after collection. Data in Figure 5 show that acidification continues after collection and that water leaving the block at neutral pH may undergo acidification thereafter. This is probably due to the fact that water running off a sulfur block inevitably carries with it small sulfur particles and some Thiobacilli. This fact was confirmed by chemical and microbiological analysis. The implication for field conditions is that acid production in runoff water is to be expected to continue even when water is no longer in contact with the sulfur block. The maximum biological sulfur oxidation rate taking place on sulfur surfaces in outdoor conditions was registered in early autumn, when optimal conditions of temperature and humidity were present. Specific acid production rate as high as 0.4 mmol (H+) per square meter of sulfur per day was recorded (Figure 6). Thiobacilli Activity Inhibition. To date, literature studies on Thiobacilli inhibition has been mostly focused on 6784
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FIGURE 7. pH change in a sample of water in contact with sulfur particles at different temperatures. Complete inhibition of microbial water acidification is observed at 5 °C. preventing corrosion of metallic structures by inhibiting biological production of sulfuric acid. Little attention has been given to avoiding sulfuric acid production from a sulfur stockpile. Mainly tested in corrosion prevention, biocides such as sodium lauryl sulfate (SLS) have been shown to be effective for inhibiting growth of Thiobacilli and biological oxidation of sulfur (9). For long-term storage, however, continuous inhibition of biological acidification is to be assured for the entire life of the storage site. Organic biocides exposed to outdoor conditions for a long time may be degraded, so that regular applications of biocides may be required, leading to high maintenance cost. Literature data show that there are other ways to affect biological oxidation of sulfur. Effective reduction of Thiobacilli activity has been obtained by keeping sulfur at low temperatures (10). It has also been reported that some strains of Thiobacilli are sensitive to the ionic strength and the osmotic pressure of the growth medium (11, 12). Inhibition in the latter cases has been associated to the accumulation of keto acids, toxic to Thiobacillus (13), and to the alteration of the cell membrane potential that, in turn, leads to temporary inhibition of respiratory processes (11). Not all Thiobacilli species are sensitive to ionic strength. Alophile strains that can survive in the presence of high concentration of salts have been isolated from seawater, geothermal fields and hypersaline lakes (14). Marine isolates need at least 10% seawater to grow. It appears that for these alophile strains, sodium chloride increases the efficiency of sulfur oxidation to sulfate (1516). To find low cost solutions to the problem of runoff water acidification from sulfur stockpiles, we have investigated the effects of temperature and ionic strength on the sulfur oxidation activity of Thiobacilli. Effect of Temperature on Thiobacilli Activity. Results reported in Figure 7 show that Thiobacillus induced acidogenesis is evident between 10 and 30 °C, whereas oxidation rate decreases considerably below 5 °C. Only few places in the world consistently show temperatures below 5 °C all year around, therefore, keeping sulfur outside the growth temperature range of Thiobacilli appears to be a viable way to prevent runoff water acidification only in very cold regions. Effects of High Ionic Strength on Thiobacilli Activity. Figure 8 shows the maximum bacterial population measured in a culture of Thiobacilli as a function of the ionic strength of the growth medium. Data indicate that there is a threshold
capability were completely recovered after reducing the ionic strength of the medium. Figure 11 shows sulfur oxidation
FIGURE 8. Ionic strength effect on Thiobacilli growth. Y axis shows the maximum attainable optical density of a Thiobacilli culture grown in media with different ionic strength (NaCl).
FIGURE 11. Recovery of microbial activity in a low salinity medium. Activity comparison between a formerly inhibited consortium (closed marks) and a never inhibited one (open marks), is measured as growth medium pH and optical density changes.
FIGURE 9. Inhibitory effect on acidogenesis obtained using different salts at 0.56 mol/L. On the Y axis, the pH change registered in Thiobacilli culture media added with different inorganic salts is shown. Sulfate production accompanying pH decrease is shown for the NaCl added medium. value for the ionic strength above which Thiobacilli stop growing. Tests carried out using 0.56 M solutions of NaCl, Na2SO4, KCl, and KNO3 demonstrated that the inhibitory effect on Thiobacilli growth does not depend on the specific salt used. These results are illustrated in Figure 9, where it is seen that acid production, regardless the salt used, stops as soon as pH reaches acidic values. Sulfate analysis showed that microbial sulfur oxidation started rapidly, continued at a decreasing pace while pH was turning acidic, and stopped completely just below pH 6. The effect of ionic strength on Thiobacilli growth can also be seen observing with a microscope the sulfur surfaces. In Figure 10 the scant colonization in the high ionic strength medium is compared with the coverage of the sulfur surfaces with a mat of Thiobacilli observed in the unmodified environment. The effect of NaCl on Thiobacilli growth appears to be bacteriostatic. In fact, microbial growth and sulfur oxidation
carried by a Thiobacilli consortium collected from an inhibited high ionic strength medium compared with the oxidation activity expressed by bacteria that had never been inhibited. The two growth curves have similar slopes indicating that duplication time is the same for the two cultures. Inhibitory effects due to high ionic strength appear to be persistent. Neither induced resistance nor colonization by alophile strains were observed in a sample of sulfur pellets treated with sodium chloride and kept outdoors for nearly 400 days. Pellets were regularly flooded with water and left to dry. As expected, pH of an untreated control sample rapidly decreased below 2 and remained acidic thereafter, while sulfur pellets treated with NaCl never developed acidity. The bacteriostatic effect inhibiting sulfur oxidation exerted by high ionic strength solutions could be exploited to prevent the acidification of runoff water coming from sites where large quantities of sulfur are stockpiled in outdoor conditions. Many practical ways can be envisioned to keep sulfur in contact with a salty solution. The easiest way would be to pour sulfur blocks in a salt cavern or below a salty water table, such as those found in saline marshes. A more general approach would call for periodically sprinkling sulfur surfaces with a salty solution to be replenished as soon as dilution from precipitation would lead ionic strength below the minimum effective value for bacteriostatic action. The inhibitory effect could be extended to longer times by protecting salt treated surfaces from water flooding with a layer of soil. In regions with low annual rainfall, management of the soil cover could lead to a negative balance between
FIGURE 10. Microphotograph of colonized sulfur surface, in presence (left) or in absence of NaCl (right). VOL. 40, NO. 21, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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precipitation and evapotranspiration, preventing any loss of salt from sulfur treated surfaces.
Acknowledgments This work has been supported by ENI. We acknowledge Mr. A. Molino and Mr. P. Sacceddu for technical assistance.
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(9) Hyne, J. B.; Laishley, E. J.; Bryant, R. D. Sodium Lauryl Sulfate a biocide for controlling acidity development in bulk commercially formed sulfur. Alberta Sulfur Res. Ltd. Q. Bull. Vol. XXXIII 1996, (1), 29-41. (10) Fawzi Abed, M. A. Rate of elemental sulfur oxidation in some soils of Egypt as affected by the salinity level, moisture content, texture, temperature and inoculation. Beitr. Trop. Landwirtsch. Veterina¨rmed. 1976, 14(2), 179-85. (11) 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(11), 5163-5168. (12) Keller, P. The effect of some salts on Thiobacillus thioparus. Can. J. Microbiol. 1969, 15(3), 314-318. (13) Borichewski, R. M. Keto acids as growth limiting factors in autotrophic growth of Thiobacillus thiooxidans. J. Bacteriol. 1967, 93(2), 597-599. (14) Wood, A. P.; Kelly, D. P. Isolation and characterization of Thiobacillus halophilus sp. nov., a sulfur-oxidising autotrophic eubacterium from a western Australian hypersaline lake. Arch. Microbiol. 1991, 156(4), 277-280. (15) Tilton, R. C.; Cobet, A. B.; Jones, G. E. Marine Thiobacilli. I. Isolation and distribution. Can. J. Microbiol. 1967, 13(11), 15211528. (16) Devendran, K.; Chandramohan, D.; Natarajan, R. Studies on marine Thiobacilli. Bull. Dept. Mar. Sci. Univ. Cochin. 1975, VII(1), 91-102 .
Received for review April 28, 2006. Revised manuscript received July 31, 2006. Accepted August 25, 2006. ES0610131
