Metatranscriptomic Analysis Reveals the Coexpression of Hydrogen-Producing and Homoacetogenesis Genes in Dark Fermentative Reactors Operated at High Substrate Loads.

Microbial communities in dark fermentation continuous systems are affected by substrate type, concentration, and product accumulation (e.g., H2 and CO2). Metatranscriptomics and quantitative PCR (qPCR) were used to assess how high organic loading rates (OLR) from 60 to 160 g total carbohydrates (TC)/L-d modify the microbial community diversity and expression of key dark fermentative genes. Overall, the microbial communities were composed of H2-producing bacteria (Clostridium butyricum), homoacetogens (Clostridium luticellarii), and lactic acid bacteria (Enteroccocus gallinarum and Leuconostoc mesenteroides). Quantification through qPCR showed that the abundance of genes encoding the formyltetrahydrofolate synthetase (fthfs, homoacetogens) and hydrogenase (hydA, H2-producing bacteria) was strongly associated with the OLR and H2 production performance. Similarly, increasing the OLR influenced the abundance of the gene transcripts responsible for H2 production and homoacetogenesis. To evaluate the effect of decreasing the H2 partial pressure, silicone oil was added to the reactor at an OLR of 138 and 160 g TC/L-d, increasing the production of H2, the copies of genes codifying for hydA and fthfs, and the genes transcripts related to H2 production and homoacetogenesis. Moreover, the metatranscriptomic analysis also showed that lactate-type fermentation and dark fermentation simultaneously occurred without compromising the reactor performance for H2 production.

Acetotrophic sulfate-reducing consortia develop active biofilms on zeolite and glass beads in batch cultures at initial pH 3.

Sulfate-reducing microbial communities remain a suitable option for the remediation of acid mine drainage using several types of carrier materials and appropriate reactor configurations. However, acetate prevails as a product derived from the incomplete oxidation of most organic substrates by sulfate reducers, limiting the efficiency of the whole process. An established sulfate-reducing consortium, able to degrade acetate at initial acidic pH (3.0), was used to develop biofilms over granular activated carbon (GAC), glass beads, and zeolite as carrier materials. In batch assays using glycerol, biofilms successfully formed on zeolite, glass beads, and GAC with sulfide production rates of 0.32, 0.26, and 0.14 mmol H2S/L·d, respectively, but only with glass beads and zeolite, acetate was degraded completely. The planktonic and biofilm communities were determined by the 16S rRNA gene analysis to evaluate the microbial selectivity of the carrier materials. In total, 46 OTUs (family level) composed the microbial communities. Ruminococcaceae and Clostridiaceae families were present in zeolite and glass beads, whereas Peptococcaceae was mostly enriched on zeolite and Desulfovibrionaceae on glass beads. The most abundant sulfate reducer in the biofilm of zeolite was Desulfotomaculum sp., while Desulfatirhabdium sp. abounded in the planktonic community. With glass beads, Desulfovibrio sp. dominated the biofilm and the planktonic communities. Our results indicate that both materials (glass beads and zeolite) selected different key sulfate-reducing microorganisms able to oxidize glycerol completely at initial acidic pH, which is relevant for a future application of the consortium in continuous bioreactors to treat acidic streams. KEY POINTS: • Complete consumption of glycerol and acetate at acidic pH by sulfate reduction. • Glass beads and zeolite are suitable materials to form sulfate-reducing biofilms. • Acetotrophic sulfate-reducing bacteria attached to zeolite preferably.

Knowing the enemy: homoacetogens in hydrogen production reactors.

One of the bottlenecks of the hydrogen production by dark fermentation is the low yields obtained because of the homoacetogenesis persistence, a metabolic pathway where H2 and CO2 are consumed to produce acetate. The central reactions of H2 production and homoacetogenesis are catalyzed by enzyme hydrogenase and the formyltetrahydrofolate synthetase, respectively. In this work, genes encoding for the formyltetrahydrofolate synthetase (fthfs) and hydrogenase (hydA) were used to investigate the diversity of homoacetogens as well as their phylogenetic relationships through quantitative PCR (qPCR) and next-generation amplicon sequencing. A total of 70 samples from 19 different H2-producing bioreactors with different configurations and operating conditions were analyzed. Quantification through qPCR showed that the abundance of fthfs and hydA was strongly associated with the type of substrate, organic loading rate, and H2 production performance. In particular, fthfs sequencing revealed that homoacetogens diversity was low with one or two dominant homoacetogens in each sample. Clostridium carboxivorans was detected in the reactors fed with agave hydrolisates; Acetobacterium woodii dominated in systems fed with glucose; Blautia coccoides and unclassified Sporoanaerobacter species were present in reactors fed with cheese whey; finally, Eubacterium limosum and Selenomonas sp. were co-dominant in reactors fed with glycerol. Altogether, quantification and sequencing analysis revealed that the occurrence of homoacetogenesis could take place due to (1) metabolic changes of H2-producing bacteria towards homoacetogenesis or (2) the displacement of H2-producing bacteria by homoacetogens. Overall, it was demonstrated that the fthfs gene was a suitable marker to investigate homoacetogens in H2-producing reactors. KEY POINTS: • qPCR and sequencing analysis revealed two homoacetogenesis phenomena. • fthfs gene was a suitable marker to investigate homoacetogens in H2 reactors.

Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor.

The hydrogen (H2) production efficiency in dark fermentation systems is strongly dependent on the occurrence of metabolic pathways derived from the selection of microbial species that either consume molecular H2 or outcompete hydrogenogenic bacteria for the organic substrate. In this study, the effect of organic loading rate (OLR) on the H2 production performance, the metabolic pathways, and the microbial community composition in a continuous system was evaluated. Two bacterial genera, Clostridium and Streptococcus, were dominant in the microbial community depending on the OLR applied. At low OLR (14.7-44.1 gLactose/L-d), Clostridium sp. was dominant and directed the system towards the acetate-butyrate fermentation pathway, with a maximum H2 yield of 2.14 molH2/molHexose obtained at 29.4 gLactose/L-d. Under such conditions, the volumetric hydrogen production rate (VHPR) was between 3.2 and 11.6 LH2/L-d. In contrast, relatively high OLR (58.8 and 88.2 gLactose/L-d) favored the dominance of Streptococcus sp. as co-dominant microorganism leading to lactate production. Under these conditions, the formate production was also stimulated serving as a strategy to dispose the surplus of reduced molecules (e.g., NADH2+), which theoretically consumed up to 5.72 LH2/L-d. In such scenario, the VHPR was enhanced (13.7-14.5 LH2/L-d) but the H2 yield dropped to a minimum of 0.74 molH2/molHexose at OLR = 58.8 gLactose/L-d. Overall, this research brings clear evidence of the intrinsic occurrence of metabolic pathways detrimental for biohydrogen production, i.e., lactic acid fermentation and formate production, suggesting the use of low OLR as a strategy to control them.

Immobilization of biogenic Pd(0) in anaerobic granular sludge for the biotransformation of recalcitrant halogenated pollutants in UASB reactors.

The capacity of anaerobic granular sludge to reduce Pd(II), using ethanol as electron donor, in an upflow anaerobic sludge blanket (UASB) reactor was demonstrated. Results confirmed complete reduction of Pd(II) and immobilization as Pd(0) in the granular sludge. The Pd-enriched sludge was further evaluated regarding biotransformation of two recalcitrant halogenated pollutants: 3-chloro-nitrobenzene (3-CNB) and iopromide (IOP) in batch and continuous operation in UASB reactors. The superior removal capacity of the Pd-enriched biomass when compared with the control (not exposed to Pd) was demonstrated in both cases. Results revealed 80 % of IOP removal efficiency after 100 h of incubation in batch experiments performed with Pd-enriched biomass whereas only 28 % of removal efficiency was achieved in incubations with biomass lacking Pd. The UASB reactor operated with the Pd-enriched biomass achieved 81 ± 9.5 % removal efficiency of IOP and only 61 ± 8.3 % occurred in the control reactor lacking Pd. Regarding 3-CNB, it was demonstrated that biogenic Pd(0) promoted both nitro-reduction and dehalogenation resulting in the complete conversion of 3-CNB to aniline while in the control experiment only nitro-reduction was documented. The complete biotransformation pathway of both contaminants was proposed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis evidencing a higher degree of nitro-reduction and dehalogenation of both contaminants in the experiments with Pd-enriched anaerobic sludge as compared with the control. A biotechnological process is proposed to recover Pd(II) from industrial streams and to immobilize it in anaerobic granular sludge. The Pd-enriched biomass is also proposed as a biocatalyst to achieve the biotransformation of recalcitrant compounds in UASB reactors.

Microbial communities from 20 different hydrogen-producing reactors studied by 454 pyrosequencing.

To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.

Direct and quinone-mediated palladium reduction by Geobacter sulfurreducens: mechanisms and modeling.

Palladium(II) reduction to Pd(0) nanoparticles by Geobacter sulfurreducens was explored under conditions of neutral pH, 30 °C and concentrations of 25, 50, and 100 mg of Pd(II)/L aiming to investigate the effect of solid species of palladium on their microbial reduction. The influence of anthraquinone-2,6-disulfonate was reported to enhance the palladium reaction rate in an average of 1.7-fold and its addition is determining to achieve the reduction of solid species of palladium. Based on the obtained results two mechanisms are proposed: (1) direct, which is fully described considering interactions of amide, sulfur, and phosphoryl groups associated to proteins from bacteria on palladium reduction reaction, and (2) quinone-mediated, which implies multiheme c-type cytochromes participation. Speciation analysis and kinetic results were considered and integrated into a model to fit the experimental data that explain both mechanisms. This work provides elements for a better understanding of direct and quinone-mediated palladium reduction by G. sulfurreducens, which could facilitate metal recovery with concomitant formation of valuable palladium nanoparticles in industrial processes.

Reduction of palladium and production of nano-catalyst by Geobacter sulfurreducens.

The present study is the first report on the ability of Geobacter sulfurreducens PCA to reduce Pd(II) and produce Pd(0) nano-catalyst, using acetate as electron donor at neutral pH (7.0 ± 0.1) and 30 °C. The microbial production of Pd(0) nanoparticles (NPs) was greatly enhanced by the presence of the redox mediator, anthraquinone-2,6-disulfonate (AQDS) when compared with controls lacking AQDS and cell-free controls. A cell dry weight (CDW) concentration of 800 mg/L provided a larger surface area for Pd(0) NPs deposition than a CDW concentration of 400 mg/L. Sample analysis by transmission electron microscopy revealed the formation of extracellular Pd(0) NPs ranging from 5 to 15 nm and X-ray diffraction confirmed the Pd(0) nature of the nano-catalyst produced. The present findings open the possibility for a new alternative to synthesize Pd(0) nano-catalyst and the potential application for microbial metal recovery from metal-containing waste streams.

Distribution of CO(2) fixation and acetate mineralization pathways in microorganisms from extremophilic anaerobic biotopes.

Extremophilic anaerobes are widespread in saline, acid, alkaline, and high or low temperature environments. Carbon is essential to living organisms and its fixation, degradation, or mineralization is driven by, up to now, six metabolic pathways. Organisms using these metabolisms are known as autotrophs, acetotrophs or carbon mineralizers, respectively. In anoxic and extreme environments, besides the well-studied Calvin-Benson-Bassham cycle, there are other five carbon fixation pathways responsible of autotrophy. Moreover, regarding carbon mineralization, two pathways perform this key process for carbon cycling. We might imagine that all the pathways can be found evenly distributed in microbial biotopes; however, in extreme environments, this does not occur. This manuscript reviews the most commonly reported anaerobic organisms that fix carbon and mineralize acetate in extreme anoxic habitats. Additionally, an inventory of autotrophic extremophiles by biotope is presented.

Continuous thermophilic hydrogen production from an enzymatic hydrolysate of agave bagasse: Inoculum origin, homoacetogenesis and microbial community analysis.

In this research, the performance of two thermophilic inocula of different origin on continuous hydrogen production from an enzymatic hydrolysate of agave bagasse were compared; one of them was obtained from a thermophilic reactor and the second one was taken from a mesophilic reactor and acclimated to thermophilic conditions. The acclimation process in one-step quickly established a high-performance hydrogen producing community, obtaining a volumetric hydrogen production rate of 3811 ± 19 mL H2/L-d with an hydrogen yield of 121 L H2/kg bagasse compared to 1473 ± 6 mL H2/L-d and 26.6 L H2/kg obtained with the thermophilic-origin inoculum. The differences in the performance of both inocula were closely linked to the profile of volatile fatty acids produced, the homoacetogenic pathway and the microbial community, the latter being the determining factor. The use of mesophilic-origin inoculum acclimated to thermophilic conditions can significantly improve the hydrogen production from lignocellulosic bagasse.

Standardized protocol for determination of biohydrogen potential.

Biohydrogen production potential (BHP) depends on several factors like inoculum source, substrate, pH, among many others. Batch assays are the most common strategy to evaluate such parameters, where the comparison is a challenging task due to the different procedures used. The present method introduces the first internationally validated protocol, evaluated by 8 independent laboratories from 5 different countries, to assess the biohydrogen potential. As quality criteria, a coefficient of variation of the cumulative hydrogen production (H max) was defined to be <15 %. Two options to run BHP batch tests were proposed; a manual protocol with periodic measurements of biogas production, needing conventional laboratory materials and analytical equipment for biogas characterization; and an automatic protocol, which is run in a device developed for online measurements of low biogas production. The detailed procedures for both protocol options are presented, as well as data validating them. The validation showed acceptable repeatability and reproducibility, measured as intra- and inter-laboratory coefficient of variation, which can be reduced up to 9 %.

Precipitation and recovery of metal sulfides from metal containing acidic wastewater in a sulfidogenic down-flow fluidized bed reactor.

This study reports the feasibility of recovering metal precipitates from a synthetic acidic wastewater containing ethanol, Fe, Zn, and Cd at an organic loading rate of 2.5 g COD/L-day and a COD to sulfate ratio of 0.8 in a sulfate reducing down-flow fluidized bed reactor. The metals were added at increasing loading rates: Fe from 104 to 320 mg/L-day, Zn from 20 to 220 mg/L-day, and Cd from 5 to 20 mg/L-day. The maximum COD and sulfate removals attained were 54% and 41%, respectively. The biofilm reactor was operated at pH as low as 5.0 with stable performance, and no adverse effect over COD consumption or sulfide production was observed. The metals precipitation efficiencies obtained for Fe, Zn, and Cd exceeded 99.7%, 99.3%, and 99.4%, respectively. The total recovered precipitate was estimated to be 90% of the theoretical mass expected as metal sulfides. The precipitate was mainly recovered from the bottom of the reactor and the equalizer. The analysis of the precipitates showed the presence of pyrite (FeS2), sphalerite (ZnS) and greenockite (CdS); no metal hydroxides or carbonates in crystalline phases were identified. This study is the first in reporting the feasibility to recover metal sulfides separated from the biomass in a sulfate reducing process in one stage.

Characterization of sulfate-reducing bacteria dominated surface communities during start-up of a down-flow fluidized bed reactor.

An anaerobic down-flow fluidized bed reactor was inoculated with granular sludge and started-up with sulfate containing synthetic wastewater to promote the formation of a biofilm enriched in sulfate-reducing bacteria (SRB), to produce biogenic sulfide. The start-up was done in two stages operating the reactor in batch for 45 days followed by 85 days of continuous operation. Low-density polyethylene was used as support. The biofilm formation was followed up by biochemical and electron microscopy analyses and the composition of the community was examined by 16S rDNA sequence analysis. Maximum immobilized volatile solids (1.2 g IVS/L(support)) were obtained after 14 days in batch regime. During the 85 days of continuous operation, the reactor removed up to 80% of chemical oxygen demand (COD), up to 28% of the supplied sulfate and acetate was present in the effluent. Sulfate-reducing activity determined in the biofilm with ethanol or lactate as substrate was 11.7 and 15.3 g COD/g IVS per day, respectively. These results suggested the immobilization of sulfate reducers that incompletely oxidize the substrate to acetate; the phylogenetic analysis of the cloned 16S rDNA gene sequences showed high identity to the genus Desulfovibrio that oxidizes the substrates incompletely. In contrast, in the granular sludge used as inoculum a considerable number of clones showed homology to Methanobacterium and just few clones were close to SRB. The starting-up approach allowed the enrichment of SRB within the diverse community developed over the polyethylene support.

Mineralization of methyl tert-butyl ether and other gasoline oxygenates by Pseudomonads using short n-alkanes as growth source.

Biodegradation of methyl tert-butyl ether (MTBE) by cometabolism has shown to produce recalcitrant metabolic intermediates that often accumulate. In this work, a consortium containing Pseudomonads was studied for its ability to fully degrade oxygenates by cometabolism. This consortium mineralized MTBE and TBA with C3-C7 n-alkanes. The highest degradation rates for MTBE (75 +/- 5 mg g(protein) (-1) h(-1)) and TBA (86.9 +/- 7.3 mg g(protein) (-1) h(-1)) were obtained with n-pentane and n-propane, respectively. When incubated with radiolabeled MTBE and n-pentane, it converted more than 96% of the added MTBE to (14)C-CO(2). Furthermore, the consortium degraded tert-amyl methyl ether, tert-butyl alcohol (TBA), tert-amyl alcohol, ethyl tert-butyl ether (ETBE) when n-pentane was used as growth source. Three Pseudomonads were isolated but only two showed independent MTBE degradation activity. The maximum degradation rates were 101 and 182 mg g(protein) (-1) h(-1) for Pseudomonas aeruginosa and Pseudomonas citronellolis, respectively. The highest specific affinity (a degrees (MTBE)) value of 4.39 l g(protein) (-1) h(-1) was obtained for Pseudomonas aeruginosa and complete mineralization was attained with a MTBE: n-pentane ratio (w/w) of 0.7. This is the first time that Pseudomonads have been reported to fully mineralize MTBE by cometabolic degradation.

Improvement of continuous hydrogen production using individual and binary enzymatic hydrolysates of agave bagasse in suspended-culture and biofilm reactors.

Continuous hydrogen (H2) production from individual (Stonezyme, IH) and binary (Celluclast-Viscozyme, BH) enzymatic hydrolysates of agave bagasse was evaluated in continuous stirred-tank reactors (CSTR) and trickling bed reactors (TBR). The volumetric H2 production rates (VHPR) in CSTR were 13 and 2.25 L H2/L-d with BH and IH, respectively. Meanwhile, VHPR of 5.76 and 2.0 L H2/L-d were obtained in the TBR configuration using BH and IH, respectively. Differences on VHPR between reactors could be explained by substrate availability, which is intrinsic to the growth mode of each reactor configuration; while differences of VHPR between hydrolysates were possibly related to the composition of enzymatic hydrolysates. Furthermore, homoacetogenesis was strongly influenced by H2 and substrate transfer conditions. Considering VHPR, H2 yields, and costs of hydrolysis, hydrogen production from binary hydrolysates of agave bagasse was identified as the most promising alternative evaluated with scale-up potential for the production of energy biofuels.

Continuous hydrogen and methane production from Agave tequilana bagasse hydrolysate by sequential process to maximize energy recovery efficiency.

Continuous H2 and CH4 production in a two-stage process to increase energy recovery from agave bagasse enzymatic-hydrolysate was studied. In the first stage, the effect of organic loading rate (OLR) and stirring speed on volumetric hydrogen production rate (VHPR) was evaluated in a continuous stirred tank reactor (CSTR); by controlling the homoacetogenesis with the agitation speed and maintaining an OLR of 44 g COD/L-d, it was possible to reach a VHPR of 6 L H2/L-d, equivalent to 1.34 kJ/g bagasse. In the second stage, the effluent from CSTR was used as substrate to feed a UASB reactor for CH4 production. Volumetric methane production rate (VMPR) of 6.4 L CH4/L-d was achieved with a high OLR (20 g COD/L-d) and short hydraulic retention time (HRT, 14 h), producing 225 mL CH4/g-bagasse equivalent to 7.88 kJ/g bagasse. The two-stage continuous process significantly increased energy conversion efficiency (56%) compared to one-stage hydrogen production (8.2%).

Chemolithotrophic denitrification with elemental sulfur for groundwater treatment.

Denitrification for the treatment of nitrates in wastewater typically relies on organic electron donating substrates. However, for groundwater treatment, inorganic compounds such as elemental sulfur (S0) are being considered as alternative electron donors in order to overcome concerns that residual organics can cause biofouling. In this study, a packed-bed bioreactor supplied with S0:limestone granules (1:1, v/v) was started up utilizing a chemolithotrophic denitrifying enrichment culture in the form of biofilm granules that was pre-cultivated on thiosulfate. The granular enrichment culture enabled a rapid start-up of the bioreactor. A nearly complete removal of nitrate (7.3 mM) was NO3- attained by the bioreactor at nitrate loading rates of up to 21.6 mmol/(L(reactor)d). With lower influent concentrations (1.3 mM nitrate) comparable to those found in contaminated groundwater, high nitrate loads of 18.1 mmol/(L(reactor)d) were achieved with an average nitrate removal efficiency of 95.9%. The recovery of nitrogen as benign N2 gas was nearly stoichiometric. The concentration of undesirable products from S0-based denitrification such as nitrite and sulfide were low. Comparison of bioreactor results with batch kinetic studies revealed that denitrification rates were dependent on the surface area of the added S0. The surface area normalized denitrification rate was determined to be 26.4 mmol /(m2 S0 d).

Biogenic sulphide plays a major role on the riboflavin-mediated decolourisation of azo dyes under sulphate-reducing conditions.

The effect of high concentrations of sulphate on the reductive decolourisation of different azo dyes by anaerobic sludge was studied in batch cultures. Sludge cultures were pre-incubated under sulphate-reducing conditions prior addition of dyes. Little or no effects of sulphate (5-10 g sulphate l(-1)) on the rate of decolourisation of Reactive Orange 14 (RO14), Direct Blue 53 (DB53) and Direct Blue 71 (DB71) were observed when no external redox mediator was provided. However, an increase in sulphate concentration, in the presence of riboflavin (20 microM), enhanced the decolourisation of all dyes. The first-rate constant of decolourisation (k) was increased up to 2-, 3.6- and 2-fold for RO14, DB53 and DB71, respectively, by supplying high sulphate concentrations, compared to the controls lacking sulphate, in the presence of the redox mediator. Sulphate reduction did not take place during the course of azo reductions, but was only evident before dye addition and after complete decolourisation, suggesting azo dyes reduction out-competed sulphate reduction for the available reducing equivalents. The experimental data suggest that reduction of azo dyes by riboflavin, which had been reduced by biogenic sulphide, was the major mechanism implicated during decolourisations, which was corroborated by abiotic incubations. Riboflavin greatly accelerated the abiotic reduction of RO14, so that the k value was increased up to 44-fold compared to the control lacking riboflavin.

Recovery of palladium(II) by methanogenic granular sludge.

This is the first report that demonstrates the ability of anaerobic methanogenic granular sludge to reduce Pd(II) to Pd(0). Different electron donors were evaluated for their effectiveness in promoting Pd reduction. Formate and H2 fostered both chemically and biologically mediated Pd reduction. Ethanol only promoted the reduction of Pd(II) under biotic conditions and the reduction was likely mediated by H2 released from ethanol fermentation. No reduction was observed in biotic or abiotic assays with all other substrates tested (acetate, lactate and pyruvate) although a large fraction of the total Pd was removed from the liquid medium likely due to biosorption. Pd(II) displayed severe inhibition towards acetoclastic and hydrogenotrophic methanogens, as indicated by 50% inhibiting concentrations as low as 0.96 and 2.7 mg/L, respectively. The results obtained indicate the potential of utilizing anaerobic granular sludge bioreactor technology as a practical and promising option for Pd(II) reduction and recovery offering advantages over pure cultures.

Simultaneous biological removal of nitrogen, carbon and sulfur by denitrification.

Refinery wastewaters may contain aromatic compounds and high concentrations of sulfide and ammonium which must be removed before discharging into water bodies. In this work, biological denitrification was used to eliminate carbon, nitrogen and sulfur in an anaerobic continuous stirred tank reactor of 1.3 L and a hydraulic retention time of 2 d. Acetate and nitrate at a C/N ratio of 1.45 were fed at loading rates of 0.29 kg C/m3 d and 0.2 kg N/m3 d, respectively. Under steady-state denitrifying conditions, the carbon and nitrogen removal efficiencies were higher than 90%. Also, under these conditions, sulfide (S(2-)) was fed to the reactor at several sulfide loading rates (0.042-0.294 kg S(2-)/m3 d). The high nitrate removal efficiency of the denitrification process was maintained along the whole process, whereas the carbon removal was 65% even at sulfide loading rates of 0.294 kg S(2-)/m3 d. The sulfide removal increased up to approximately 99% via partial oxidation to insoluble elemental sulfur (S0) that accumulated inside the reactor. These results indicated that denitrification is a feasible process for the simultaneous removal of nitrogen, carbon and sulfur from effluents of the petroleum industry.