ARTICLE

Hydrogen Sulfide Production From ElementalSulfur by Desulfovibrio desulfuricans in anAnaerobic Bioreactor

Claudio Escobar, Loreto Bravo, Jose Hernandez, Leandro Herrera

Chemical Engineering and Biotechnology Department, Universidad de Chile,

Beaucheff 861, Santiago 8370456, Region Metropolitana, Chile;

telephone: 56-2-9784291; fax: 56-2-6991084; e-mail: cescobar@ing.uchile.cl

Received 26 December 2006; accepted 29 March 2007

Published online 9 April 2007 in Wiley InterScience (www.interscience.wiley.com). DO

I 10.1002/bit.21457

ABSTRACT: Feasibility of elemental sulfur reduction byDesulfovibrio desulfuricans in anaerobic conditions in astirred reactor was studied. Hydrogen was used as energysource, whereas the carbonated species were bicarbonate andyeast extract. Attention was paid to reactor engineeringaspects, biofilm formation on the sulfur surface, hydrogensulfide formation rate and kinetics limitations of the sulfurreduction. D. desulfuricans formed stable biofilms on thesulfur surface. It was found that active sulfur surface avail-ability limits the reaction rate. The reaction rate was firstorder with respect to sulfur and hydrogen velocity had noeffect in the reaction rate for the range 8.2� 10�2 to4.1� 10�1 Nm3 m�2 min�1. At a superficial gas velocity(uG)¼ 3.1� 10�2 Nm3 m�2 min�1, H2S(g) productionrate decreased due to a deficient H2S stripping. A maxi-mum H2S(g) production rate of 2.1 g H2S L�1 d�1 wasachieved during 5 days with an initial sulfur density of4.7% (w/v).

Biotechnol. Bioeng. 2007;98: 569–577.

� 2007 Wiley Periodicals, Inc.

KEYWORDS: sulfur availability; biofilm; H2S(g) productionrate; H2S stripping

Introduction

Hydrogen sulfide can be produced by the catalytic reductionof elemental sulfur with hydrogen at 5008C and highpressure. It can also be produced by reaction of sulfuric acidand iron sulfide. These methods are relatively expensive andneeds extensive safety precautions.

Hydrogen sulfide can also be produced by the biologicalsulfate reduction, using lactate, acetate or hydrogen aselectron donor (Foucher et al., 2001; Gilbert et al., 2002;Herrera et al., 1997; Jong and Parry, 2003; Luptakova andKusnierova, 2005). Elemental sulfur reduction by bacteria

Correspondence to: C. Escobar

� 2007 Wiley Periodicals, Inc.

(X) is advantageous, since the H2S production by thismethod (Eq. 1) requires only 1/4 of the amount of electrondonor in comparison with the biological reduction of sulfate(Eq. 2) (Herrera et al., 1997; Sorensen et al., 1981; vanHouten et al., 1994):

H2 þ S �!X H2S (1)

4H2 þH2SO4 �!X

H2SþH2O (2)

The bacterial reduction of elemental sulfur to H2S, was firstreported in 1937 (Starkey, 1937) and the first genericdescription of a dissimilatory sulfur-reducing bacteriumappeared in Pfennig and Biebl (1976). In 1977, Biebl andPfenning found that certain sulfate-reducing bacteria growwith sulfur as electron acceptor. In the same year, Wolfe andPfennig described that Sulfospirillum can grow by theanaerobic oxidation of hydrogen and the reduction of S0.They obtained a moderated cell yield.

Since then, elemental sulfur bioreduction has beenintensively studied. By large, most of these studies havedealt with thermophilic bacteria. Only few studies con-centrate on mesophilic conditions (Belyakova et al., 2006;Caccavo et al., 1994; Mogensen et al., 2005; Nakagawa et al.,2005; Thabet et al., 2004). Even less literature can be foundon reactors for the biological elemental sulfur reduction andlittle is known about kinetic limitations; reactor parametersor coefficients; the ability of sulfate-reducing bacteria toform biofilms on sulfur surface in turbulent three-phasesystem; and mass and energy transport phenomena.

The purpose of the present study was to investigatefeasibility of elemental sulfur reduction in stirred reactorsat mesophilic conditions using a culture of a sulfate

Biotechnology and Bioengineering, Vol. 98, No. 3, October 15, 2007 569

reducing bacteria: Desulfovibrio desulfuricans. Particularattention was paid to reactor engineering aspects, biofilmformation on the sulfur surface, hydrogen sulfide formationrate and kinetics limitations of the sulfur reduction as aprocess.

Materials and Methods

Experimental Set-Up

The experimental set-up was as shown in Figure 1. Thesystem was composed of a nonsterile bioreactor, with a gasdiffuser, mechanically stirred by magnetic bar of 6 cmat 270 rpm. The bioreactor is a cylindrical vessel of9.8� 10�2 m diameter and 1.9� 10�1 m height and thevolume of the liquid phase ranged from 1 to 1.15 L. Two gaswashing bottles (NaOH solutions, see Fig. 1) were used tocapture the hydrogen sulfide produced in the reactor. ACuSO4 trap was used as indicator of NaOH saturation. Thereactor temperature was automatically controlled at 308C,temperature that Okabe and Characklis (1992) recommendfor cultivating D. desulfuricans. Hydrogen was suppliedto the reactor, from a cylinder. The reactor was batch forthe liquid and the solid phases, but continuous for thegaseous phase (H2, H2S(g), H2O(vap)). The oxidation–reduction potential (Eh) was registered with an ORPelectrode connected to a voltmeter. The optimal pH forD. desulfuricans growth is 7.0 (Reis et al., 1992), but thereactor increases alkalinity as sulfide is produced. Therefore,pH was kept constant automatically at 7.0� 0.2. Theparticular acid source was part of this study, because the useof an inorganic acid (generally HCl) would continuously

Figure 1. Experimental reactor and equipment. CV, solenoid valve controller; CT, temp

A/D, analog to digital converter.

570 Biotechnology and Bioengineering, Vol. 98, No. 3, October 15, 2007

modify the ionic strength of the culture media(increasing Cl� ion), on account of its liquid phase batchnature. The reactor, with 1 L of medium, was inoculatedwith 100 mL of a pregrown culture, incubated at 308C.Elemental sulfur was then added to the reactor in the form ofcrystaline sulfur flowers. The reactor was sealed, and kept ata 2 psig pressure, regulating it automatically with hydrogen.After 2 days, the reactor was operated with continuoushydrogen flow, maintaining the same pressure.

Gas flow of H2 was monitored with amass flow controller.The range of H2 flow was 0.23–3.1 N L min�1 (3.1� 10�2 to4.1� 10�1 Nm3m�2 min�1) [N refers to normal conditions,i.e., 101,325 Pa and 273.15 K].

The range of sulfur densities used in this work was 4–5%(w/v).

Bacteria and Culture Conditions

A culture of D. desulfuricans in yeast extract enrichedautotrophic medium was used. Earlier studies demonstratethat in the absence of hydrogen, little or no growth occurs onyeast extract. In strictly autotrophic medium without yeastextract and in the presence of hydrogen, growth also ismarginal (Mechalas and Rittenberg, 1960). Therefore yeastextract acts as a growth factor, incorporating amino acids tothe cells, that they cannot synthesize. Hydrogen andelemental sulfur were used as energy source and electronacceptor, respectively.

The medium had the following composition (per litreof distilled water): 0.2 g KH2PO4; 0.3 g NH4Cl;3.0 g MgCl2�6H2O; 0.5 g KCl; 0.15 g CaCl2�2H2O; 1�10�3 g Resazurin (Redox indicator); 1.0 g yeast extract; 2.5 g

erature controller; TT, temperature transmitter; TpH, pH transmitter; TEh, Eh transmitter;

DOI 10.1002/bit

NaHCO3 (carbon source); 1 mL of trace elementssolution (Widdel et al., 1983); 10 mL of Wolfe’sVitamin solution (Atlas, 1997); 1 mL of a solutioncomprising 3 mg Na2SeO3�5H2O in a litre of NaOH 0.01 M; and solid sulfur according to experimentaldesign.

Analytical Methods

Hydrogen sulfide production was determined by themethylene blue method (Clesceri et al., 1998) from thegas washing bottles (NaOH 0.1 N). The methylene bluemethod was also used to measure total sulfide concentrationin the reactor.

Suspended cells were counted in an aliquot from thereactor, using a counting chamber and a conventionaloptical microscope.

The biofilm formation in the sulfur surface, was examinedby scanning electron microscope (SEM) in a LEO Electronmicroscope, Leo 1420 VP model. For energy dispersive X-ray spectroscopy (EDS), a detector EDS OXFORDINSTRUMENTS, EDS 7424 model was used.

Sulfur X-ray diffraction patterns were measured using CuKa radiation (1.5406 A) on a Siemens D5000 difractometerwith Bragg–Brentano geometry and graphite monochro-mator.

The measurements of zeta potential were made in a ZetaMeter, model 3.0.

Determination of Kinetics Parameters

Kinetics:The kinetics of sulfur reduction are based on the following

assumptions:

� G

rowth of biomass proceeds according toMonod kineticswith two substrate simultaneous limitation:

m ¼ mmax

S0

KS1 þ S0H2

KS2 þH2ð3Þ

However, in batch culture, S0�KS1 and H2�KS2, thenm isconstant and equal to mmax.

� P

roduct formation is directly coupled to biomass prod-uction.

� S

ubstrate consumption for maintenance is incorporatedin the overall biomass yield.

� T

he term S0 refers to the active sulfur with a particle meansize distribution of 20 mm (initial mean sizecalculated by SEM).

Mass balances:For the reactor the following assumptions are made:

� G

Esc

as and liquid phase of the reactor are ideally mixed.

� G as-side mass transfer resistance and external mass

transfer limitations (around biofilms) are negligible.

The mass balances over the liquid phase of the reactor,are:

d½X�dt

¼ Xðm� TXÞ (4)

d½S0�dt

¼ �mX

YX=S0(5)

d½H2�acdt

¼ ðkLaÞH2

½H2�gHH2

� ½H2�ac� �

� mX

YX=H2

(6)

d½HS��dt

¼ k11½H2S� � k12½HS��½Hþ� þ mX

YX=S0(7)

d½H2S�acdt

¼ k12½HS��½Hþ� � k11½H2S� � ðkLaÞH2S

� ½H2S�ac �½H2S�gHH2S

� �(8)

The mass balance over the gas phase is:

d½H2S�gdt

¼ ðkLaÞH2S½H2S�ac �

½H2S�gHH2S

� �VL

VG

� �

�QG½H2S�g

VG(9)

where VG is the gas volume (0.355 L); k11 is the kineticconstant of the direct reaction of the H2S dissociation(29.9� 106 h�1); k22 is the kinetic constant of the reversereaction of the H2S dissociation (2.7� 1014 M h�1); HH2S isthe Henry’s law constant of H2S at 308C (0.4403 mol/mol);HH2 is the Henry’s law constant of H2 at 308C (52.63 mol/mol).

Growth yield coefficient (YX/S) determination was basedon the total increase in cell biomass per unit of total amountof sulfur (molar) consumed. According to the stoichiometryof the reaction (Eq. 1), the YX=H2

is equal to YX/S. Maximumspecific growth rate (mmax) and cellular decay rate (TX) wereobtained by solving the mass balance equations describedabove, and diminishing the difference between these resultsand the experimental data of cell density and sulfurconcentration.

Determination of Volumetric Mass Transfer Coefficient

The volumetric mass transfer coefficient (kL a) wasdetermined for oxygen in two systems: two phase system(liquid medium and air) and three phase system (liquid

obar et al.: Hydrogen Sulfide Production From Elemental Sulfur 571

Biotechnology and Bioengineering. DOI 10.1002/bit

medium, solid sulfur and air). The dynamic method (Dunnand Einsele, 1975) was used for different superficial gasvelocities. No biomass was present in the reactor. Thetransfer properties for hydrogen and hydrogen sulfide werecalculated using the diffusion coefficients.

Figure 2. H2S(g) production rate for uG¼ 1.3� 10�1 Nm3 m�2 min�1. Arrow 1

indicates that 18 g of sulfur were added to the reactor.

Results

The pH was successful controlled automatically with CO2

injection. The use of this gas in liquid phase batch is betterthan the use of an inorganic acid, because this one wouldcontinuously increase the ionic strength of the culturemedia.

The results of the ðkLaÞH2measurements are shown in

Table I. No significant differences between both systemswere observed. In both systems, kL a increases with thesuperficial gas velocity.

Bioreduction kinetics was studied at a 5% (w/v) sulfurdensity, for three superficial gas velocities (1.3� 10�1,2.3� 10�1 and 4.1� 10�1 Nm3 m�2 min�1) in threedifferent experiments. A hydrogen sulfide production rateof 2.3 g H2S L

�1 d�1 was reached after 2 h for 2.3� 10�1 and4.1� 10�1 Nm3 m�2 min�1, and after 14 h for 1.3� 10�1

Nm3m�2 min�1. Therefore, a superficial gas velocity (uG) of1.3� 10�1 Nm3 m�2 min�1 was selected for a moreextensive experiment at 4% (w/v) sulfur. In the threeexperiments, Eh remained constant, at around �300 mV.

The results of the first 10 days of the experiment at 4%(w/v) sulfur are shown in Figure 2. A gradual increase ofthe H2S(g) production rate was observed with time, untilreaching a maximum value of 1.8 g H2S L

�1 d�1 at day 2.5.Hydrogen sulfide production rate remained constant untilday 4.5. After 5.4 days, it decreased to 1.5 g H2S L�1 d�1.After 7.5 days, the H2S(g) production rate decreasedprogressively until day 9.13. At that time, 18 g of sulfurwere added to the reactor (see Arrow 1 in Fig. 2), so asulfur density of 4.7% (w/v) was reached in the reactor. After3 h, a H2S(g) production rate of 2.1 g H2S L�1 d�1 wasreached. Therefore, sulfur availability was probably limitingkinetics. Between the 2.5 and 4.5 days, a total sulfideconcentration of 4.73� 0.12 mg L�1 was reached in thereactor. At the days 3.0, 5.3 and 7.0, 100 mL of mediumwithout yeast extract were added to the reactor. No changein the H2S(g) production rate and in the biomass

Table I. Mass transfer coefficients for hydrogen as a function of

superficial gas velocity (uG).

uG (Nm3 m�2 min�1)

kL a (two phase

system) (s�1)

kL a (three phase

system) (s�1)

3.1� 10�2 5.6� 10�3 N.D.

5.1� 10�2 6.7� 10�3 7.0� 10�3

1.3� 10�1 9.3� 10�3 8.2� 10�3

2.3� 10�1 1.6� 10�2 1.7� 10�2

4.1� 10�1 2.9� 10�2 N.D.

N.D., not determined.

572 Biotechnology and Bioengineering, Vol. 98, No. 3, October 15, 2007

concentration occurred, for the days 3.0 and 5.3. Thebiomass concentration increased from 7.7� 106 to 1.1� 107

cel mL�1, 10 h after the last addition of medium, as can beseen in Figure 5. During these 10 days, the oxidation–reduction potential decreased from �350 to �500 mV andthe biomass reached a maximum concentration of 1.2�107 cel mL�1.

The results of the following days are shown in Figure 3.After 12.2 days of operation the superficial gas velocity wasdecreased to 8.2� 10�2 Nm3 m�2 min�1. However, nochange in H2S(g) production rate occurred and the totalsulfide concentration remained at 7.64� 0.42 mg L�1. Atday 14.0 the superficial gas velocity was decreased to3.1� 10�2 Nm3 m�2 min�1. Between that time and day15.3, the H2S(g) production rate decreased from 2.04 to1.42 g H2S L�1 d�1 and the total sulfide concentrationincreased from 9.93 to 20.85 mg L�1. From day 15.3, boththe total sulfide concentration and the H2S(g) productionrate decreased progressively. At day 17.2 the superficialgas velocity was increased to 8.2� 10�2 Nm3 m�2 min�1.However, both the total sulfide concentration andthe H2S(g) production rate continued decreasing. At day 18,400 mg of yeast extract were added to the reactor (see Arrow1 in Fig. 3), and an increase in the H2S(g) production rateoccurred. No change in the biomass concentration occurred.Afterwards, the H2S(g) production rate decreased until theday 19.3, when 16 g of sulfur were added to the reactor (seeArrow 2 in Fig. 3), so a sulfur density of 4.7% (w/v) wasreached again in the reactor. After 7.5 h, the H2S(g)production rate increased to 2.1 g H2S L�1 d�1. Again, thesulfur availability limits the reaction. At the days 13.5 and16.4, 100 mL of medium without yeast extract were added tothe reactor. No change in the H2S(g) production rateoccurred. The biomass concentration increased from2.0� 106 to 4.0� 106 cel mL�1, after 10 h of the firstaddition of medium and increased from 4.7� 106 to

DOI 10.1002/bit

Figure 3. H2S(g) production rate for 3.1� 10�2, 8.2� 10�2, and 1.3� 10�1 Nm3 m�2 min�1. Arrow 1 indicates that 400 mg of yeast extract were added to the reactor. Arrow

2 indicates that 16 g of sulfur were added to the reactor.

6.5� 106 cel mL�1, 5 h after the last addition (see Fig. 4).During the last 10 days, the biomass reached a maximumconcentration of 7.4� 106 cel mL�1.

At day 8.5 of the experiment biofilm formation wasexamined by SEM (Fig. 5). Microphotographs indicated that

Figure 4. Suspended cells density in the reactor. Arrows 1, 3 and 4 indicate that 100

400 mg of yeast extract were added to the reactor. Arrows 2 and 6 indicate that 18 and

bacteria adhere favourably to sulfur. However, it was notpossible to determine when biofilm begins to form orwhether the bacteria were active in the surface.Figure 5b clearly shows a film adhering to the sulfur, andbacteria adhering to this film. An EDS analysis was

mL of medium without yeast extract were added to the reactor. Arrow 5 indicates that

16 g of sulfur were added to the reactor, respectively.

Escobar et al.: Hydrogen Sulfide Production From Elemental Sulfur 573

Biotechnology and Bioengineering. DOI 10.1002/bit

Figure 5. Biofilm formation on elemental sulfur. Pictures taken at day 8.5. a and b: Represent two different views.

Figure 6. Zeta potential of sulfur particles for different pH values. Each value is

the mean value of three determinations. The isoelectric points in both samples are

indicated.

performed with the image shown in Figure 5b. Carbon,oxygen and sulfur were the main elements found on thesurface, with atomic percentages of 58.3%, 28.5% and3.11%, respectively. The surface occupied by the bacteria issmall compared with the analysed total surface (Fig. 5b).Therefore, the result of the EDS indicates that the film is richin carbon and oxygen.

For the determination of kinetics parameters, only resultsof the five first days of experiment were considered (beforethe sulfur limitation appeared). A maximum specificgrowth rate of 4.3� 10�2 h�1 and a cellular decay rate of8.31� 10�5 h�1 were obtained. A growth yield coefficient of1.58� 1010 cel (mol S0)�1 was reached.

A proportional increase in the H2S(g) production rate wasobtained when the initial sulfur density was increased.Hydrogen velocity has no effect in the H2S(g) productionrate for uG greater than 3.1� 10�2 Nm3 m�2 min�1. For asulfur density range of 4–5% (w/v), and for uG range of8.2� 10�2 to 4.1� 10�1 Nm3 m�2 min�1, the followingexpression was found:

rH2S ¼ 4:07� 10�1½S0�1:07 (10)

The reaction rate was first order with respect to sulfur,with a correlation coefficient (r) of 0.995. The term ofhydrogen concentration appear implicit in the kineticconstant.

These experiments show that sulfur availability dimin-ished along time, but do not explain its nature ormechanism. In order to explain this phenomenon, an X-ray analysis was made to a blank sulfur particles before thereaction and also to a sample of reacted sulfur particles. Thesample of reacted sulfur was taken after 25 days of the lastaddition of sulfur to the reactor (Arrow 2 in Fig. 3). Nostructural differences were observed. In both cases the X-raydiffraction patterns correspond to an orthorhombic a-S8with density equal to 2.070 g mL�1 and with cell: a¼ 10.45,b¼ 12.84, c¼ 24.46.

Since no change was observed with the X-rays analysis, itwas necessary to analyse the sulfur at superficial level.

574 Biotechnology and Bioengineering, Vol. 98, No. 3, October 15, 2007

Therefore, the isoelectric point of sulfur blank particles wascompared with reacted sulfur particles (25 days of the lastaddition of sulfur to the reactor). This was made bymeans ofzeta potential determination for different pH values. Theresults are shown in Figure 6. The figure clearly shows asignificative difference between both samples. The isoelectricpoint of sulfur blank particles was 3.92, whereas theisoelectric point of reacted sulfur particles was 4.70.

Discussion

The determination of kL a was done without the presence ofbiomass, although Galaction et al. (2004) found thatbiomass concentration increases the media viscosity. Thisinduces two direct major effects on mass transfer coefficient:

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the reduce of turbulence and the perturbation ofbubbles dispersion-coalescence equilibria. These effectswere observed with biomass concentration between 30.5and 120.5 g L�1 dry weight. In this study a maximumbiomass concentration of 1.2� 107 cel mL�1 was reached.This value corresponds to 2.4� 10�3 g L�1 dry weight,assuming that 1 g of biomass contains 1012 cells and that acell has a 80% humidity (Bailey and Ollis, 1986). Thisconcentration is considerably lower than those employed byGalaction et al. therefore in this work it was assumed that kLa is not affected by the biomass. Moreover, for bacteriaconcentration lower than 1� 109 cel mL�1, the effect onmedia viscosity is negligible (Bailey and Ollis, 1986).

Experimental results reveal that D. desulfuricans was ableto form a stable biofilm. Figure 5 clearly shows such biofilmformation on elemental sulfur under the experimentalflow conditions. Pure and mixed cultures of sulfate-reducing bacteria cultivated in the laboratory oftenaggregate or stick to surfaces (Nielsen, 1987). Also theycan form biofilms on inert carrier material under turbulentflow conditions (Hulshoff et al., 2004; van Houten el al.,1994; Visser et al., 1993). However, sulfate reducers abilityto form biofilms on elemental sulfur in turbulentanaerobic reactors has not been described before. Thisability is very useful for designing and operation ofcontinuous reactors, since it allows a greater number ofbacteria by reactor volume.

Figure 5b shows bacteria adhering to a film, and this filmadhering to the sulfur. The elemental composition of thisfilm was determined by EDS. Results indicate that film has agreat abundance of carbon and oxygen. Probably this film isorganic, since carbon–oxygen ratio is 2 and for inorganiccompounds a ratio lesser than 1 is expected. Typically, abiofilm is composed of the microbial cells, extracellularpolymers substances (EPS) secreted by the cells, metabolicproducts, plus a variety of colloidal and dissolvedsubstances. The film reported in the present study couldbe EPS. However, further studies are necessary to determinethe composition and nature of this film.

Experiments results show that the main rate limitingreagent of the sulfur bioreduction is the sulfuravailability itself. This was evident at days 9.13 and 19.3,when sulfur was added to the reactor and immediatelythe H2S(g) production rate increased.

Table II. Comparison of the H2S(g) production rate per gram of initial sulfu

Author HRG (mg H2S g S�1 d�1)

Stetter and Gaag (1983) 32.5

Belkin et al. (1985) 23.8

Takai et al. (2005) 23.2

Nakagawa et al. (2005) 2.12

This study 44.7

Caccavo et al. (1994) 29.1

Takai et al. (2003) 1.08

Azabou et al. (2007) 42.5

N.I., not informed.

Hydrogen velocity has no effect in the H2S(g) productionrate for uG greater than 3.1� 10�2 Nm3 m�2 min�1. Anaccumulation of total sulfide in the reactor took place foruG¼ 3.1� 10�2 Nm3 m�2 min�1 and a possible productinhibition by H2S(aq) could occur. However, the reactortotal sulfide concentration at these conditions wasbelow the concentrations reported as inhibitory (Okabeet al., 1992, 1995; Reis et al., 1992). Therefore, the decreasein the H2S(g) production rate was caused by adeficient H2S stripping. Because the volumetric masstransfer coefficient (kL a) was measured, the maximummass transfer rate of hydrogen was calculated assuming thatthe liquid bulk concentration of hydrogen was zero. ForuG¼ 1.3� 10�1 Nm3 m�2 min�1, the maximum masstransfer rate of hydrogen was 0.567 mol H2 L

�1 d�1, then amaximum H2S(g) production rate of 19.3 g H2S L�1 d�1

was expected and only a H2S(g) production rate of2.1 g H2S L�1 d�1 was obtained. This indicates that therewas not mass transfer limitation of hydrogen for uG greaterthan 3.1� 10�2 Nm3 m�2 min�1.

When medium without yeast extract was added to thereactor, no effect was observed in the sulfur reduction rate,but cell density increased. Therefore, these bacteria needed amedium renovation frequency within 2.3 days (mean valueof differences between successive additions of medium). Theaddition of yeast extract at day 18 enhanced the H2S(g)production rate, but had no effect in cell density. A commonamino acid present in yeast extract is cystein. Blumentalset al. (1990) found that this amino acid is a nucleophile thatopen the sulfur ring, enhancing the solubility of sulfur.Belkin et al. (1985) also found that presence of yeast extractenhances sulfide production rate, with an optimal con-centration of yeast extract of 1 g L�1. The reactor will need apermanent supply of yeast extract that ensures the bestdispersion of elemental sulfur in liquid media. Furtherstudies are necessary to optimise the concentration of yeastextract that maximize the H2S(g) production rate under theexperimental conditions of this study.

The maximum H2S(g) production rate obtained during asignificant period of time was 2.1 g H2S L�1 d�1, for uG of1.3� 10�1 Nm3 m�2 min�1 and initial sulfur density of4.7% (w/v). A comparison of the H2S(g) production rate pergram of initial sulfur obtained by different authors isshown in Table II. Our rate is within the range obtained in

r (HRG) obtained in this work with those obtained by different authors.

Temperature (8C) pH Electron donor

65 6.5 H2

77 7.5 Acetate

50 5.2 H2

32 6.0 H2

30 7.0 H2

35 N.I. Acetate

70 5.4 H2

37 7.0 Lactate

Escobar et al.: Hydrogen Sulfide Production From Elemental Sulfur 575

Biotechnology and Bioengineering. DOI 10.1002/bit

experiments reported elsewhere. A more rigorous com-parison is based on H2S(g) production rate per superficialarea, but it was impossible to carry out, because all of therevised studies omitted the sulfur superficial area. Themmax obtained in the present work was 4.3� 10�2 h�1 and isin agreement with mmax obtained by Zavarzina et al. (2000),although these authors worked with thermophilic hetero-trophic bacteria. The results obtained in the present studydemonstrate the feasibility of elemental sulfur reduction instirred reactor at mesophilic conditions, since a good H2S(g)production rate was obtained.

The adhered biomass to sulfur may have createddiffusional limitations both to the access of the sulfur tobacteria in the upper layers, and to the access of hydrogen tothe bacteria in the lower layers, if a multilayer biofilm wasformed. However, a monolayer biofilm rather than amultilayer biofilm was formed, and not all the sulfursurface was occupied by the bacteria (Fig. 5). Moreover,when new sulfur particles was added, a larger surface areawas made available for adhesion and the suspended cellswould decrease, but this not occurred (Fig. 4). The adhesionis not necessarily related to the sulfur bioreduction, sincean increase of the H2S(g) production rate was immediatelyproduced after new sulfur surface was added to thereactor. Therefore, the diffusional limitations not seem tobe the phenomena that explain the detected sulfur limitation.

The isoelectric point of sulfur particles without reactionwas 3.92, whereas the isoelectric point of reacted sulfurparticles was 4.70. This means that for a given pH the surfacecharge of sulfur particles diminished after 25 days ofreaction. This evidence could explain the sulfur availabilitylimitation detected in the present work, since a smallersurface charge implies a smaller reactivity. The deposit of aless reactive layer, like the film (possibly EPS) described inthe present work, could be the reason of the decrease of thesulfur surface charge. Future studies are necessary to clarifythe mechanism involved in the sulfur limitation.

Conclusions

The results obtained in the present work demonstrate thefeasibility of elemental sulfur bioreduction in a stirredreactor under mesophilic conditions.

A maximum H2S(g) production rate of 2.1 g H2S L�1 d�1

was achieved during 5 days, with an initial sulfur density of4.7% (w/v).

The bacteria formed a stable biofilm on the sulfur surfaceunder turbulent flow conditions.

Availability of active sulfur surface limited the reactionrate.

The reaction was first order with respect to sulfur andhydrogen velocity had no effect in the reaction rate forthe range 8.2� 10�2 to 4.1� 10�1 Nm3 m�2 min�1. AtuG¼ 3.1� 10�2 Nm3 m�2 min�1, the H2S(g) productionrate decreased due to a deficient H2S stripping.

576 Biotechnology and Bioengineering, Vol. 98, No. 3, October 15, 2007

The kinetic parameters that described the sulfurbioreduction in this system are: a maximum specificgrowth rate of 4.3� 10�2 h�1, a cellular decay rate of8.31� 10�5 h�1 and a growth yield coefficient of 1.58� 1010

cel (mol S0)�1.After 25 days of reaction, the isoelectric point of the sulfur

particles increased from 3.92 to 4.70. This means that for agiven pH the surface charge of sulfur particles diminishedafter that period of time.

The authors wish to acknowledge CONICYT and Universidad de

Chile. The authors also wish to acknowledge the help of Mr. Patricio

Baeza with the zeta potential experiments and the help of Mr. Andres

Ibanez with the X-ray diffraction experiments.

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