Hydrogen Sulfide Oxidation by a Microbial Consortium in a Recirculation Reactor System: Sulfur Formation under Oxygen Limitation and Removal of Phenols SERGIO ALCA Ä NTARA, † ANTONIO VELASCO, ‡ ANA MUN ˜ OZ, † JUAN CID, † SERGIO REVAH, ‡ AND ELI Ä AS RAZO-FLORES* ,† Program a de Biotecnologı ´a, Instituto Mexicano del Petro ´leo, Eje Central La ´zaro Ca ´rdenas 152, C.P. 07730, Me ´xico D.F., and Departamento de Ingenierı ´a de Procesos, Universidad Auto ´noma Metropolitana-Iztapalapa, Apdo. Postal 55-534, C.P. 09340, Me ´xico D.F. Wastewater from petroleum refining may contain a number of undesirable contaminants including sulfides, phenolic compounds, and ammonia. The concentrations of these compounds must be reduced to acceptable levels before discharge. Sulfur formation and the effect of selected phenolic compounds on the sulfide oxidation were studied in autotrophic aerobic cultures. A recirculation reactor system was implemented to improve the elemental sulfur recovery. The relation between oxygen and sulfide was determined calculating the O 2 /S 2- loading rates (Q O2 / Q S 2- ) R mt ), which adequately defined the operation conditions to control the sulfide oxidation. Sulfur-producing steady states were achieved at R mt ranging from 0.5 to 1.5. The maximum sulfur formation occurred at R mt of 0.5 where 85% of the total sulfur added to the reactor as sulfide was transformed to elemental sulfur and 90% of it was recovered from the bottom of the reactor. Sulfide was completely oxidized to sulfate (R mt of 2) in a stirred tank reactor, even when a mixture of phenolic compounds was present in the medium. Microcosm experiments showed that carbon dioxide production increased in the presence of the phenols, suggesting that these compounds were oxidized and that they may have been used as carbon and energy source by heterotrophic microorganisms present in the consortium. Introduction A petroleum refinery is a complex combination of inter- dependent industrial processes that generate effluents containingboth organicand inorganiccompounds.The term “sour”was originated to describe a waste contaminated with sulfide (1).Sour water streams in the refineries are generated from sour steam condensates from distillation, thermal or hydrogen cracking operations, and product heating process (2).Dependingon wherethesourcondensatesareproduced, the sulfide, phenols, and ammonia concentrations (mg L -1 ) present in these streams can range from 10 to 5000, from 5 to 300, and from 10 to 3000, respectively. Because of its high sulfide,phenols,and ammoniacontent,thesourwaterstream must be treated before its release into the environment. Sour waste streams,includingsour water,sour gases,and refineryspent-sulfidiccaustics,have been successfullytreated using Thiobacillus denitrificans, while organic compounds such as benzene, toluene, and phenol are biodegraded by heterotrophic bacteria grown in co-culture with T. denitri- ficans (1, 3). T.denitrificans strain F,isolated bySublette and Woolsey (4), was used to treat sour effluents because of its higher sulfide tolerance in comparation with other facultative strains. Strain F tolerates sulfide concentrations up to 56 mg L -1 .RecentlyMcComaset al.(5)characterized a novelsystem for sulfide oxidation usingan enrichment culture dominated by Thiom icrospira sp. The consortium showed similar oxidation activities as compared to T. denitrificans under anaerobic conditions; however, it was more tolerant to extreme culture conditions such as pH (5.6-10.4), temper- ature (up to 46 °C), and salt concentration (10% of NaCl). The authorsconcluded that the processdescribed wasshown to be a more robust biocatalyst system for sulfide oxidation than the systems using T. denitrificans. Other species ofthiobacillihave been studied to promote the sulfur production from partially reduced sulfur com- pounds oxidation (6-8). Sulfur production (eq 1) from the partial oxidation of sulfide instead of a complete oxidation to sulfate (eq 2) presents environmental implications as elemental sulfur can be removed by sedimentation. Ad- ditionally, lower energy consumption is required because the oxidation to sulfur requires 4-fold less oxygen: According to the stoichiometry of the aerobic biological sulfide oxidation, oxygen is the key parameter that controls the level of oxidation (7). Buisman et al. (9) reported that, at sulfide concentrations below 20 mg L -1 , the oxygen con- centration should be kept low (below of 1 mg L -1 ) to limit the sulfur oxidation to sulfate. Thereby at sulfide concentra- tions higher than 20 mg L -1 , the sulfur formation is independent ofthe oxygen concentration.Accordingto these data, Janssen et al. (10) found that the optimal oxygen to sulfide molar ratio to improve the sulfur production was about 0.7. The same authors described the performance of a sulfide-oxidizing expanded-bed reactor that was designed forelementalsulfurformation (7).In thisreactor,the aeration ofthe liquid phase and the oxidation ofsulfide were spatially separated.Sulfur sludge with good settlingproperties,which consisted mainlyofelementalsulfur(92%)and biomass(2%), was obtained. Stefess et al. (11) reported that Thiobacillus o and Thiobacillus neapolitanus produced elemental sulfur from partialoxidation ofhydrogen sulfide orthiosulfate.They also found that elemental sulfur was formed under oxygen limitation (0.1%saturation)or at high substrate loadingrates (Q ) 18 mmol L -1 h -1 ). Sulfide and thiosulfate thus proved to be interchangeable substrates for chemolithotrophic bacteria producingsulfur and sulfate.In the same way,Visser et al. (8) reported that sulfur formation in a Thiobacillus sp. *Corresponding author telephone: +(52) 55 91 75 69 13; fax: +(52) 55 91 75 77 05; e-mail: erazo@imp.mx. † Instituto Mexicano del Petro ´leo. ‡ Universidad Auto ´noma Metropolitana-Iztapalapa. 2HS - + O 2 f 2S 0 + 2OH - ∆G°′ )-129.50 kJ m ol -1 (1) 2HS - + 4O 2 f 2SO 4 2- + 2H + ∆G°′ )-732.58 kJ m ol -1 (2) Environ. Sci. Technol. 2004, 38, 918-923 918 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 3, 2004 10.1021/es034527y CCC: $27.50  2004 American Chemical Society Published on Web 12/23/2003