Biotechnol Lett (2011) 33:2141–2145 DOI 10.1007/s10529-011-0689-2

ORIGINAL RESEARCH PAPER

Bio-oxidation of H2S by Sulfolobus metallicus Marjorie Morales • Jonathan Arancibia Mariana Lemus • Javier Silva • Juan Carlos Gentina • German Aroca

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Received: 18 April 2011 / Accepted: 1 July 2011 / Published online: 10 July 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Sulfolobus metallicus is a hyperthermophilic and chemolithoautotrophic archaeon that uses elemental sulfur as an energy source. Its ability to oxidize H2S was measured either in the presence or absence of elemental sulphur, showing its ability for using both as an energy source. A biotrickling filter was set up and a biofilm of S. metallicus was established over the support. The maximum removal capacity of the biotrickling filter reached at 55°C was 40 g S/m3h for input loads higher than 70 g S/m3h. Thus, S. metallicus can be used in a biofiltration system for the treatment of waste gas emissions at high temperatures contaminated with H2S. Keywords Biofiltration
Biotrickling filter
H2S
Sulfolobus metallicus

Introduction The odour generated by industrial gaseous emissions is a significant environmental problem that arises when industrial installations are built near urban

M. Morales
J. Arancibia
M. Lemus
J. Silva
J. C. Gentina
G. Aroca (&) School of Biochemical Engineering, Pontificia Universidad Cato´lica de Valparaı´so, General Cruz 34, Valparaı´so, Chile e-mail: [email protected]

areas or where urban areas have grown around existing industrial areas. The odour is caused by the presence of volatile organic compounds (VOC), especially volatile organic sulfur compounds (VOSCs) such as H2S, methylmercaptane, dimethylsulfur and dimethyldisulfur. These compounds can be found in the gaseous emissions of several industrial operations including Kraft pulp mills, petroleum refineries, tanneries, waste water treatment plants, landfills, composting, solid waste treatment plants and some food industries, including fish canning and animal rendering operations (Ruokojarvi et al. 2001; Smet et al. 1998) In some cases, the VOSCs are emitted at low concentrations and high temperatures affecting large areas due to their low odor threshold. In 1920, the biological treatment of effluent gases was introduced as a competitive alternative to the conventional physicochemical treatment technologies (Kennes and Thallasso 1998). Mesophilic microorganisms have generally been used but the need to develop technologies for the biological treatment of gaseous emissions generated at high temperatures has recently arisen. The use of thermophilic microorganisms (Dhamwichukorn et al. 2001; Cox et al. 2001; Matteau and Ramsay 1999; Kong et al. 2001; Luvsanjamba et al. 2007; Takeuchi et al. 2000) to treat gaseous emissions avoids the need for an additional cost of decreasing the emission temperatures. Organisms belonging to the archaeal genus Sulfolobus are hyperthermophilic acidophiles, growing optimally between 50 and 80°C and pH 1.5–3.

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Several species can grow either as chemolithoautotrophs or as heterotrophs, using O2 as the terminal electron acceptor. They use sulfur compounds such as H2S, elemental sulfur (S8) and thiosulfate (S2O32-) as energy sources, acting as electron donors and generating sulfate as the final product. Sulfolobus metallicus is a hyperthermophilic aerobic archaeon that grows between 50 and 75° C and from pH 1–4.5. It is an obligate chemolithoautotroph that grows on elemental sulfur producing sulfuric acid (Huber and Stetter 1991). The aim of this study was to determine the capacity and ability of S. metallicus to oxidize H2S as an alternative energy source, even in the presence of S°, and to demonstrate the feasibility of using S. metallicus to develop a biofiltration system that can operate at high temperatures for treating waste gas emissions contaminated with H2S.

Materials and methods Sulfolous metallicus was grown aerobically in a medium (Clark and Norris 1996) with the following composition: (NH4)2SO4 0.4 g/l; MgSO4.7H2O

Fig. 1 Laboratory-scale experimental biofilter (1), H2S generator (2), air heater (3), temperature controller (4), air compressor (5), humidifier (6), culture medium at 70°C (7), lead acetate solution (8)

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0.5 g/l; KH2PO4 0.2 g/l; KCl: 0.1 g/l and elemental sulfur as energy source at 5 g/l. The cultures were kept at 70°C, shaken at 150 rpm, and pH was adjusted to 2.5 with sulfuric acid. The inocula were at 10% (v/v) for successive cultures. Cell concentration was measured by a direct method using a Petroff Hauser cell counting chamber in a phase-contrast microscope. Sulfate was determined turbidimetrically (Clescerl et al. 1989). H2S in the bottles was determined by GC using a Supelpack S column and a flame photometric detector (400°C) with helium as carrier flow gas at 30 ml/min. The injector was at 60°C. The H2S in the biofilter was determined by a Dra¨ger model X-am 5000 sensor (Fig. 1). Determination of the oxidative ability To determine the ability of S. metallicus to oxidize H2S in the presence of S°, ten 250 ml bottles containing 45 ml culture medium without sulfur were prepared. Sulfur and/or inoculums (0.8 g protein/l) were added according to Table 1. The inoculua were taken from an actively growing culture. The bottles were sealed with a septum and 50 ml air containing H2S from 1000 to 1500 mg/l was added (Table 1).

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Table 1 Culture medium composition in bottles used to evaluate the H2S oxidative capacity of S. metallicus Bottle number

Inoculum (ml)

S° (g/l)

H2S (lg/ml)

1

5

5

1475

2

5

5

1208

3

5

5

–

4

5

5

–

5

5

–

1096

6

5

–

1208

7

–

5

1140

8

–

5

1015

9

–

–

1098

10

–

–

1194

The bottles were incubated at 68°C and shaken at 150 rpm for 24 h. Bottles 9 and 10 were incubated without cells or sulfur in order to measure the chemical oxidation of H2S. To provide a carbon source, 25 ml CO2 was also added to each bottle. H2S was measured in the gaseous phase and sulfate, protein and pH were measured in the liquid phase.

Sampling took place when the biotrickling filter reached a steady state (i.e.,\10% change), which was verified by the constant removal efficiency obtained at each loading rate. The loading rate was controlled by varying the air flow input, from 1 to 2 l/min, to obtain the residence time of gas of 120, 90 and 60 s.

Results S. metallicus oxidation ability of H2S All the H2S within the bottles, including those containing S° (number 1 and 2), was consumed (Fig. 2). Bottles 7–10 did not contain cells, so the reduction of H2S from the initial concentration is probably due to absorption in the liquid medium and chemical oxidation. Figure 3 shows the variation of cell concentrations in each bottle. The cellular concentration obtained in the bottles with both energy sources (bottles 1 and 2) was higher than in either of the bottles containing only sulfur (bottles 3 and 4) or only H2S (bottles 5 and 6), suggesting that both energy sources are used by the microorganism.

Biotrickling filter Set up of biotrickling filter The capacity of the biotrickling filter to eliminate H2S with increasing input loads is shown in Fig. 4. The maximum removal capacity of the biotrickling filter approached 40 g S/m3h at 55°C. For low input

1600 initial final

1400

H2S concentration (mg/l)

The biotrickling filter consisted of a glass column with an internal diameter of 0.06 m, a height of 1.2 m, and a packed volume of 2 l. The glass column had a jacket of insulating material. To maintain 55°C, hot Clark and Norris medium was recirculated. Thermocouples were installed inside the column to measure and control the temperature. The column had sampling ports every 12 cm. H2S was generated by mixing equimolar solutions of HCl and Na2S in a contacting column (Oyarzu´n et al. 2003). The generated gas was mixed with humidified hot air. The biofilm generation was carried out by keeping a continuous culture of S. metallicus through successive re-inoculations every 7 days obtaining a maximum of 2.6 9 106 cells/cm2 after 37 days. The pH was 2.6 at the start of the operation and declined sharply with each re-inoculation until 1.5. The resulting sulfate concentration was 1.3–3 g/l. To determine the efficiency and capacity of the biotrickling filter to remove H2S at 55°C, loading rates between 4 and 100 g S/m3h were fed to the bioreactor.

1200 1000 800 600 400 200 0

0

1

2

3

4

5

6

7

8

9

10

Bottle number

Fig. 2 Initial and final H2S concentration in bottles after 24 h, at initial pH 2.5, shaking at 150 rpm and 70°C

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1,8e+7 initial final

1,6e+7

Biomass (cells/ml)

1,4e+7 1,2e+7 1,0e+7 8,0e+6 6,0e+6 4,0e+6 2,0e+6 0,0

0

1

2

3

4

5

6

7

8

9

10

Bottle number

3

Removal Capacity H2S gS/m3h h

Fig. 3 Initial and final biomass concentration in bottles after 24 h, at initial pH 2.5, shaking at 150 rpm and 70°C

40

30

20

10

0 0

20

40

60

80

100

Input Load H2S gS/m3h

Fig. 4 Removal capacity for different input loads of H2S from 4 to 80 gS/m3h at 55°C and different residence times: 120 s (dark circle), 80 s (open circle), 60 s (open square)

loads of H2S (between 4 and 8 g S/m3h) the removal efficiency of pollutants was close to 100%. For medium loads (from 26 to 50 g S/m3h) the removal efficiency was in the range of 68–72%. Maximum input loads of over 70 g S/m3h had the lowest removal efficiency in the range of 47–54%.

Discussion H2S depletion in the sealed bottles showed that S. metallicus used it as an energy source. H2S was also consumed in the presence of S°. This behavior may be due to the fact that S° is an intermediate compound in the oxidation of H2S to sulfate (Lomans et al. 2002). H2S is

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probably consumed before S° because of the low solubility of S° in water where it is dispersed in a colloidal form, meanwhile H2S and its dissociated species are in a soluble form in aqueous medium, depending on the gas phase concentration and pH of the solution, being more available to the microorganism. Samples drawn from non-inoculated bottles had a final H2S concentration in the gas phase that was decreased by around 200 mg/l, compared to initial values. The reduction could be due to absorption of H2S in the liquid phase, chemical reactions or nonhomogeneous sampling caused by the higher density of H2S compared to air. The final cell concentration was higher in the bottles containing both energy sources than bottles containing either S° or H2S; although, the cell concentration in the bottles containing only S° was greater than that obtained in the bottles containing only H2S. This is probably because S. metallicus for growth using S° as the energy insted of H2S, for this reason the sulfur oxidising microorganisms prefer the consumption of S° (with sulfate formation) over the consumption of H2S (yield more S°), because in the first case the energy available is higher (Janssen et al. 1995). The measurements of the H2S removal from a gas stream using a biotrickling filter shows that the elimination capacity obtained, which was 39 g S/m3h for H2S inputs higher than 70 g S/m3h, were similar to other results (Indrani et al. 2007) using a microbial community obtained from a hot spring, where the maximum removal capacity of the biotrickling filters was around 40 g S/m3h at 70°C for H2S inputs higher than 60 g S/m3h. The removal efficiency of the BTF studied decreased when the input load of H2S increased, probably because the biofilm was not able to consume all of the H2S due either to the short residence time of the gas or because of mass transfer limitations. This work clearly shows that it is possible to use S. metallicus for the biological removal of H2S from a contaminated gas stream at high temperatures. Acknowledgments This research was financed by CONICYT, Project FONDECYT 1080422, and the Pontificia Universidad Cato´lica de Valparaı´so Project DII 203.777.

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