Electronic Journal of Biotechnology ISSN: 0717-3458   © 2003 by Universidad Católica de Valparaíso -- Chile Vol. 6 No. 3, Issue of December 15, 2003

There is a great diversity of microorganisms that produce hydrogen sulfide (Aiking et al. 1982; Kim and Olson, 1989; Barton and Tomei, 1995; Cooney et al. 1996; Levine et al. 1998; Wang et al. 2000), from organic (i.e. sulfur-containing amino acids) and inorganic sources (i.e. sulfates). Although hydrogen sulfide is a toxic pollutant gas generally occurring in wastewater, it has been used to precipitate metals in wastewater treatment reactors and has been proposed for stabilization of metals in soils and for formation of metal sulfide “quantum” particles for microelectronics applications (Holmes et al. 1997).

When a novel hydrogen sulfide producer bacterial strain is isolated, it is necessary the physiological and biochemical characterization of such strain, in order to optimize the hydrogen sulfide production for its further application (Fortin et al. 1994; Peyton et al. 1995; White and Gadd, 1996; White and Gadd, 1998; Smith, 2000).

In this paper a minibioreactor-gas collector system designed for characterization and study of hydrogen sulfide production from bacteria, is described. In addition, a simple analytical method, based on H₂S indirect conductimetric determination as a modification of the method of lead acetate for detection of hydrogen sulfide (Hunter and Crecelius, 1938), was developed.

Materials and Methods

Development of the analytical method

For the quantitative determination of hydrogen sulfide produced in the minibioreactor, a conductimetric method was developed as a modification of the classical method for determination of hydrogen sulfide in bacterial cultures (Hunter and Crecelius, 1938). The method has two stages: first, the hydrogen sulfide released during a bacterial culture, is collected in a 0.090 mol/L lead acetate solution, via the reaction:

Pb(CH₃COO)_{2 (aq.)} + H₂S _(g)                  PbS _(s) + 2CH₃COOH _(aq.)

In the second stage, excess Pb²⁺ is determined conductimetrically by titration with a 4 mmol/L sodium hydrogenphosphate solution, via the reaction:

3Pb²⁺ _(aq.) + 2HPO₄^2- _(aq.)                 Pb₃(PO₄)_{2 (s)} + 2H⁺ _(aq.) 

In this way, the hydrogen sulfide formed in the fermentation process is indirectly quantified.

Minibioreactor-gas collector design

The minibioreactor with a rubber cap (Figure 1), was made from a culturing tube (50 mL) functioning as the vessel of the bioreactor (A). Air input was through a 2 mm glass tube (B), with one of its ends coupled to the air filter (C) for sterile air input (Midisart, 0.2 µm). The fermentation gases pass through the exhaustion pipe (D) and were absorbed in the gas collector (E). The samples of culture were taken through the sample valve (F).

Gas collector was a two-way burette modified, containing 10 mL of lead acetate 90 mM. The gases from culture pass through the lead acetate solution in one position of stopcock (G); in the other position of stopcock, the samples were taken, as the way to evaluate Pb2+ ion excess.

Results and Discussion

In relation to the developed system, its novelty is in the whole as a hydrogen sulfide determination tool. The device has the advantage that it is possible to study the hydrogen sulfide production either by aerobic or anaerobic microorganisms. One of the main advantages of the designed system is the possibility of working at small scale, making it more attractive economically, because it decreases the volumes of necessary reagents in order to prepare the corresponding culture medium. Another advantage is that the agitation takes place without use of impeller, which reduces the energy expenses; achieving, with the flow of the pressurizing gas, an appropriate agitation for the growth of the studied microorganism.

In spite of the simplicity of the design, it is possible to maintain the constant temperature if an external thermostat is used. Several minibioreactors have been designed with diverse purposes (Man Bock et al. 1999; Jiho Min et al. 2000; Man Bock et al. 2001). However, until date, there are not reports in relation to the use of this type of device for the study of bacteria-produced hydrogen sulfide.

In order to make a validation of the developed method for determination of hydrogen sulfide, it was compared to classical iodometric method (Ayres, 1968). It was found that both methods do not differ in accuracy and precision.

The developed system was applied in detection and quantification of hydrogen sulfide levels produced by Tsukamurella paurometabola DSM 20162, grown in a culture medium containing cysteine 1.5 mM as a sole carbon source. The kinetic behaviour showed a hydrogen sulfide production growth-associated, which indicates that T. paurometabola uses cysteine as a carbon source for growing; and due to catabolism produces hydrogen sulfide, which has been reported for other microorganisms (Kim and Olson, 1989; Acree et al. 1971; Chu et al. 1997).

Concluding Remarks

A minibioreactor-gas collector system was developed for studying the biological production of hydrogen sulfide in microorganisms. This device promises to be useful in studies at small scale, being simple and economical. In addition, an analytical method for indirect determination of hydrogen sulfide chemically absorbed in the gas collector was developed, as a modification of the method of Hunter and Crecelius (1938); detecting hydrogen sulfide levels higher than 0.5 µmol, which allows to determine the levels of hydrogen sulfide produced by such strains that produce small amounts of this gas.

References

ACREE, T.E.; SONOFF, E. P. and SPLITTSTOESSER, D.F. Determination of hydrogen sulfide in fermentation broths containing sulfur dioxide. Applied Microbiology, 1971, vol. 22, no. 1, p.110-112.

AIKING, H.; KOK, K.; VAN HEERIKHUIZEN, H. and VAN´T RIET, J. Adaptation to cadmium by Klebsiella aerogenes growing in continuous culture proceeds mainly via formation of cadmium sulfide. Applied and Environmental Microbiology, 1982, vol. 44, p. 938-944.

AYRES, G.H. Quantitative Chemical Analysis. 2^nd ed. New York, Harper & Row Publishers, 1968, 710 p.

BARTON, L.L. and TOMEI, F. Characteristics and activities of sulfate-reducing bacteria. In: BARTON, L.L. ed. Sulfate-reducing bacteria. New York, Plenum Press, New York, 1995, p. 1-32. ISBN 0-306-44857-2.

COONEY, M.; ROSCHI, E.; MARISON, I.; COMMINELLIS, C. and VON STOCKAR, U. Physiologic studies with the sulfate-reducing bacterium Desulfovibrio desulfuricans: evaluation for use in a biofuel cell. Enzyme and Microbial Technology, 1996, vol. 18, no. 5, p. 358-365.

CHU, L.; EBERSOLE, J.L.; KURZBAN, G.P. and HOLT, S.C. Cystalysin, a 46-Kilodalton Cysteine Desulfhydrase from Treponema denticola, with Hemolytic and Hemoxidative Activities. Infection and Immunity, August 1997, vol. 65, no. 8, p. 3231-3238.

FORTIN, D.; SOUTHAM, G. and BEVERIDGE, T.J. Nickel sulfide, iron-nickel sulfide and iron sulfide precipitation by a newly isolated Desulfotomaculum species and its relation to nickel resistance. FEMS Microbiology and Ecology, 1994, vol. 14, no. 2, p. 121-132.

HOLMES, J.D.; RICHARDSON, D.J.; SAED, S.; EVANS-GOWING, R.; RUSELL, D.A. and SODEAU, J.R. Cadmium-specific formation of metal sulfide `Quantum particles´ by Klebsiella pneumoniae. Microbiology, 1997, vol. 143, no. 8, p. 2521-2530.

HUNTER, C.A. and CRECELIUS, H.G. Hydrogen sulfide studies: detection of hydrogen sulfide in cultures. Journal of Bacteriology, 1938, vol. 35, p. 185.

JIHO, M.; CHANG, W. L.; SEUNG-HYEUN, M.; LAROSSA, R. A. and MAN BOCK, G. Detection of radiation effects using recombinant bioluminescent Escherichia coli strains.Radiation and Environmental Biophysics, 2000, vol. 39, no. 1, p. 41-45.

KIM, S. and OLSON, N. Production of hydrogen sulfide in milkfat-coated microcapsules containing Brevibacterium linens and cysteine. Journal of Microencapsulation, October-December1989, vol. 6, no. 4, p. 501-513.

LEVINE, J.; ELLIS, C.; FURNE, J.; SPRINGFIELD, J. and LEVITT, M. Fecal hydrogen sulfide production in ulcerative colitis. American Journal of Gastroenterology, January 1998, vol. 93, no. 1, p. 83-87.

MAN BOCK, G.; BYOUNG, C. K.; JAEWEON, C. and HANSEN, P. D. The Continuous Monitoring of Field Water Samples With a novel multi-channel two-stage mini-bioreactor system.Environmental Monitoring and Assessment, 2001, vol. 70, no. 1-2, p. 71-81.

MAN BOCK, G.; GEUN CHEOL, G. and JOONG HYUN, K.. A two-stage minibioreactor system for continuous toxicity monitoring.Biosensors and Bioelectronics, 1999, vol. 14, no. 4, p. 355-361.

PEYTON, B.M.; TRUEX, M.J.; TOTH, J.J. Biogenic sulfide precipitation of heavy metals to recycle electroplating and electronics manufacturing process streams. Richland, WA, Batelle, Pacific Northwest Division, 1995, PNWD-2315.

SMITH, A. Harnessing nature to clean up Doñana-scientists recommend a biological solution. European Science Foundation, Strasbourg, France, 2000.

WANG, C. L.; MARATUKULAM, PRIYA D.; LUM, A. M.; CLARK, D. S. and KEASLING, J.D. Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface.Applied and Environmental Microbiology, 2000, vol. 66, no. 10, p. 4497-4502.

WHITE, C. and GADD, G.M. Accumulation and effects of cadmium on sulfate-reducing bacterial biofilms. Microbiology, 1998, vol. 144, no. 5, p. 1407-1415.

WHITE, C. and GADD, G.M. Mixed sulphate-reducing bacterial cultures for bioprecipitation of toxic metals: factorial and response-surface analysis of the effects of dilution rate, sulphate and substrate concentration. Microbiology, 1996, vol. 142, no. 8, p. 2197-2205.