Syngas as Electron Donor for Sulfate and Thiosulfate Reducing Haloalkaliphilic Microorganisms in a Gas-Lift Bioreactor.

Biodesulfurization processes remove toxic and corrosive hydrogen sulfide from gas streams (e.g., natural gas, biogas, or syngas). To improve the efficiency of these processes under haloalkaline conditions, a sulfate and thiosulfate reduction step can be included. The use of H2/CO mixtures (as in syngas) instead of pure H2 was tested to investigate the potential cost reduction of the electron donor required. Syngas is produced in the gas-reforming process and consists mainly of H2, carbon monoxide (CO), and carbon dioxide (CO2). Purification of syngas to obtain pure H2 implies higher costs because of additional post-treatment. Therefore, the use of syngas has merit in the biodesulfurization process. Initially, CO inhibited hydrogen-dependent sulfate reduction. However, after 30 days the biomass was adapted and both H2 and CO were used as electron donors. First, formate was produced, followed by sulfate and thiosulfate reduction, and later in the reactor run acetate and methane were detected. Sulfide production rates with sulfate and thiosulfate after adaptation were comparable with previously described rates with only hydrogen. The addition of CO marginally affected the microbial community in which Tindallia sp. was dominant. Over time, acetate production increased and acetogenesis became the dominant process in the bioreactor. Around 50% of H2/CO was converted to acetate. Acetate supported biomass growth and higher biomass concentrations were reached compared to bioreactors without CO feed. Finally, CO addition resulted in the formation of small, compact microbial aggregates. This suggests that CO or syngas can be used to stimulate aggregation in haloalkaline biodesulfurization systems.

Immobilization of sulfate and thiosulfate-reducing biomass on sand under haloalkaline conditions.

Biological sulfate and thiosulfate reduction under haloalkaline conditions can be applied to treat waste streams from biodesulfurization systems. However, the lack of microbial aggregation under haloalkaline conditions limits the volumetric rates of sulfate and thiosulfate reducing bioreactors. As biomass retention in haloalkaline bioreactors has not been studied before, sand was chosen as a biomass carrier material to increase cell retention and consequently raise the volumetric rates. The results showed that ~10 fold higher biomass concentrations could be achieved with sand, compared to previous studies without carrier addition. The volumetric rates of sulfate/thiosulfate reduction increased approximately 4.5 times. Biomass attachment to the sand was restricted to cavities within the sand particles. Acetate produced by acetogenic bacteria from H2 and CO2 was used as carbon source for biomass growth, while formate that was also produced from H2 and CO2 enhanced sulfate reduction. The microbial community composition was analyzed by 16S rRNA gene amplicon sequencing, and Tindallia related bacteria were probably responsible for formate formation from hydrogen. The community attached to the sand particles was similar to the suspended fraction, but the relative abundance of sequences most closely related to Desulfohalobiaceae was much higher in the attached fraction compared to the suspended fraction (30% and 13%, respectively). The results indicated that even though the biomass attachment to sand was poor, it still increased the biomass concentration and consequently the sulfate and thiosulfate reduction volumetric rates.

Thiosulfate Conversion to Sulfide by a Haloalkaliphilic Microbial Community in a Bioreactor Fed with H2 Gas.

In industrial gas biodesulfurization systems, where haloalkaline conditions prevail, a thiosulfate containing bleed stream is produced. This bleed stream can be treated in a separate bioreactor by reducing thiosulfate to sulfide and recycling it. By performing treatment and recycling of the bleed stream, its disposal decreases and less caustics are required to maintain the high pH. In this study, anaerobic microbial thiosulfate conversion to sulfide in a H2/CO2 fed bioreactor operated at haloalkaline conditions was investigated. Thiosulfate was converted by reduction to sulfide as well as disproportionation to sulfide and sulfate. Formate production from H2/CO2 was observed as an important reaction in the bioreactor. Formate, rather than H2, might have been used as the main electron donor by thiosulfate/sulfate-reducing bacteria. The microbial community was dominated by bacteria belonging to the family Clostridiaceae most closely related to Tindallia texcoconensis. Bacteria phylogenetically related to known haloalkaline sulfate and thiosulfate reducers, thiosulfate-disproportionating bacteria, and remarkably sulfur-oxidizing bacteria were also detected. On the basis of the results, two approaches to treat the biodesulfurization waste stream are proposed: (i) addition of electron donor to reduce thiosulfate to sulfide and (ii) thiosulfate disproportionation without the need for an electron donor. The concept of application of solely thiosulfate disproportionation is discussed.

Selection and Application of Sulfide Oxidizing Microorganisms Able to Withstand Thiols in Gas Biodesulfurization Systems.

After the first commercial applications of a new biological process for the removal of hydrogen sulfide (H2S) from low pressure biogas, the need arose to broaden the operating window to also enable the removal of organosulfur compounds from high pressure sour gases. In this study we have selected microorganisms from a full-scale biodesulfurization system that are capable of withstanding the presence of thiols. This full-scale unit has been in stable operation for more than 10 years. We investigated the microbial community by using high-throughput sequencing of 16S rRNA gene amplicons which showed that methanethiol gave a competitive advantage to bacteria belonging to the genera Thioalkalibacter (Halothiobacillaceae family) and Alkalilimnicola (Ectothiorhosdospiraceae family). The sulfide-oxidizing potential of the acclimatized population was investigated under elevated thiol loading rates (4.5-9.1 mM d-1), consisting of a mix of methanethiol, ethanethiol, and propanethiol. With this biomass, it was possible to achieve a stable bioreactor operation at which 80% of the supplied H2S (61 mM d-1) was biologically oxidized to elemental sulfur. The remainder was chemically produced thiosulfate. Moreover, we found that a conventionally applied method for controlling the oxygen supply to the bioreactor, that is, by maintaining a redox potential set-point value, appeared to be ineffective in the presence of thiols.

Inhibition of a biological sulfide oxidation under haloalkaline conditions by thiols and diorgano polysulfanes.

A novel approach has been developed for the simultaneous description of reaction kinetics to describe the formation of polysulfide and sulfate anions from the biological oxidation of hydrogen sulfide (H2S) using a quick, sulfide-dependent respiration test. Next to H2S, thiols are commonly present in sour gas streams. We investigated the inhibition mode and the corresponding inhibition constants of six thiols and the corresponding diorgano polysulfanes on the biological oxidation of H2S. A linear relationship was found between the calculated IC50 values and the lipophilicity of the inhibitors. Moreover, a mathematical model was proposed to estimate the biomass activity in the absence and presence of sulfurous inhibitors. The biomass used in the respiration tests originated from a full-scale biodesulfurization reactor. A microbial community analysis of this biomass revealed that two groups of microorganism are abundant, viz. Ectothiorhodospiraceae and Piscirickettsiaceae.

Valorization of lubricant-based wastewater for bacterial neutral lipids production: Growth-linked biosynthesis.

Lipids produced by microorganisms are currently of great interest as raw material for either biofuels or oleochemicals production. Significant biosynthesis of neutral lipids, such as triacylglycerol (TAG) and wax esters (WE) are thought to be limited to a few strains. Hydrocarbonoclastic bacteria (HCB), key players in bioremediation of hydrocarbon contaminated ecosystems, are among this group of strains. Hydrocarbon rich wastewaters have been overlooked concerning their potential as raw material for microbial lipids production. In this study, lubricant-based wastewater was fed, as sole carbon source, to two HCB representative wild strains: Alcanivorax borkumensis SK2, and Rhodococcus opacus PD630. Neutral lipid production was observed with both strains cultivated under uncontrolled conditions of pH and dissolved oxygen. A. borkumensis SK2 was further investigated in a pH- and OD-controlled fermenter. Different phases were assessed separately in terms of lipids production and alkanes removal. The maximum TAG production rate occurred during stationary phase (4 mg-TAG/L h). The maximum production rate of WE-like compounds was 15 mg/L h, and was observed during exponential growth phase. Hydrocarbons removal was 97% of the gas chromatography (GC) resolved straight-chain alkanes. The maximum removal rate was observed during exponential growth phase (6 mg-alkanes/L h). This investigation proposes a novel approach for the management of lubricant waste oil, aiming at its conversion into valuable lipids. The feasibility of the concept is demonstrated under low salt (0.3%) and saline (3.3%) conditions, and presents clues for its technological development, since growth associated oil production opens the possibility for establishing continuous fermentation processes.

Electricity generation from an inorganic sulfur compound containing mining wastewater by acidophilic microorganisms.

Sulfide mineral processing often produces large quantities of wastewaters containing acid-generating inorganic sulfur compounds. If released untreated, these wastewaters can cause catastrophic environmental damage. In this study, microbial fuel cells were inoculated with acidophilic microorganisms to investigate whether inorganic sulfur compound oxidation can generate an electrical current. Cyclic voltammetry suggested that acidophilic microorganisms mediated electron transfer to the anode, and that electricity generation was catalyzed by microorganisms. A cation exchange membrane microbial fuel cell, fed with artificial wastewater containing tetrathionate as electron donor, reached a maximum whole cell voltage of 72 ± 9 mV. Stepwise replacement of the artificial anolyte with real mining process wastewater had no adverse effect on bioelectrochemical performance and generated a maximum voltage of 105 ± 42 mV. 16S rRNA gene sequencing of the microbial consortia resulted in sequences that aligned within the genera Thermoplasma, Ferroplasma, Leptospirillum, Sulfobacillus and Acidithiobacillus. This study opens up possibilities to bioremediate mining wastewater using microbial fuel cell technology.

Influence of methanethiol on biological sulphide oxidation in gas treatment system.

Inorganic and organic sulphur compounds such as hydrogen sulphide (H2S) and thiols (RSH) are unwanted components in sour gas streams (e.g. biogas and refinery gases) because of their toxicity, corrosivity and bad smell. Biological treatment processes are often used to remove H2S at small and medium scales (<50 tons per day of H2S). Preliminarily research by our group focused on achieving maximum sulphur production from biological H2S oxidation in the presence of methanethiol. In this paper the underlying principles have been further studied by assessing the effect of methanethiol on the biological conversion of H2S under a wide range of redox conditions covering not only sulphur but also sulphate-producing conditions. Furthermore, our experiments were performed in an integrated system consisting of a gas absorber and a bioreactor in order to assess the effect of methanethiol on the overall gas treatment efficiency. This study shows that methanethiol inhibits the biological oxidation of H2S to sulphate by way of direct suppression of the cytochrome c oxidase activity in biomass, whereas the oxidation of H2S to sulphur was hardly affected. We estimated the kinetic parameters of biological H2S oxidation that can be used to develop a mathematical model to quantitatively describe the biodesulphurization process. Finally, it was found that methanethiol acts as a competitive inhibitor; therefore, its negative effect can be minimized by increasing the enzyme (biomass) concentration and the substrate (sulphide) concentration, which in practice means operating the biodesulphurization systems under low redox conditions.

Ecology and application of haloalkaliphilic anaerobic microbial communities.

Haloalkaliphilic microorganisms that grow optimally at high-pH and high-salinity conditions can be found in natural environments such as soda lakes. These globally spread lakes harbour interesting anaerobic microorganisms that have the potential of being applied in existing technologies or create new opportunities. In this review, we discuss the potential application of haloalkaliphilic anaerobic microbial communities in the fermentation of lignocellulosic feedstocks material subjected to an alkaline pre-treatment, methane production and sulfur removal technology. Also, the general advantages of operation at haloalkaline conditions, such as low volatile fatty acid and sulfide toxicity, are addressed. Finally, an outlook into the main challenges like ammonia toxicity and lack of aggregation is provided.

Effect of Methanethiol Concentration on Sulfur Production in Biological Desulfurization Systems under Haloalkaline Conditions.

Bioremoval of H2S from gas streams became popular in recent years because of high process efficiency and low operational costs. To expand the scope of these processes to gas streams containing volatile organic sulfur compounds, like thiols, it is necessary to provide new insights into their impact on overall biodesulfurization process. Published data on the effect of thiols on biodesulfurization processes are scarce. In this study, we investigated the effect of methanethiol on the selectivity for sulfur production in a bioreactor integrated with a gas absorber. This is the first time that the inhibition of biological sulfur formation by methanethiol is investigated. In our reactor system, inhibition of sulfur production started to occur at a methanethiol loading rate of 0.3 mmol L(-1) d(-1). The experimental results were also described by a mathematical model that includes recent findings on the mode of biomass inhibition by methanethiol. We also found that the negative effect of methanethiol can be mitigated by lowering the salinity of the bioreactor medium. Furthermore, we developed a novel approach to measure the biological activity by sulfide measurements using UV-spectrophotometry. On the basis of this measurement method, it is possible to accurately estimate the unknown kinetic parameters in the mathematical model.

Sulfate reduction in a hydrogen fed bioreactor operated at haloalkaline conditions.

Biological sulfate reduction is used as a biotechnological process to treat sulfate rich streams. However, application of biological sulfate reduction at high pH and high salinity using H2 was not thoroughly investigated before. In this work the sulfate reduction activity, biomass growth, microbial community and biomass aggregation were investigated in a H2-fed gas lift bioreactor at haloalkaline conditions. The process was characterized by low sulfate reduction volumetric rates due to slow growth and lack of biomass aggregation. Apparently, the extreme conditions and absence of organic compounds prevented the formation of stable aggregates. The microbial community analysis revealed a low abundance of known haloalkaliphilic sulfate reducers and presence of a Tindallia sp. The identified archaea were related to Methanobacterium alcaliphilum and Methanocalculus sp. The biomass did not attach to metal sulfides, calcite and magnesite crystals. However, biofilm formation on the glass bioreactor walls showed that attachment to glass occurs.

Sulfate reduction at low pH to remediate acid mine drainage.

Industrial activities and the natural oxidation of metallic sulfide-ores produce sulfate-rich waters with low pH and high heavy metals content, generally termed acid mine drainage (AMD). This is of great environmental concern as some heavy metals are highly toxic. Within a number of possibilities, biological treatment applying sulfate-reducing bacteria (SRB) is an attractive option to treat AMD and to recover metals. The process produces alkalinity, neutralizing the AMD simultaneously. The sulfide that is produced reacts with the metal in solution and precipitates them as metal sulfides. Here, important factors for biotechnological application of SRB such as the inocula, the pH of the process, the substrates and the reactor design are discussed. Microbial communities of sulfidogenic reactors treating AMD which comprise fermentative-, acetogenic- and SRB as well as methanogenic archaea are reviewed.

Application of a 2-step process for the biological treatment of sulfidic spent caustics.

This research demonstrates the feasibility and advantages of a 2-step process for the biological treatment of sulfidic spent caustics under halo-alkaline conditions (i.e. pH 9.5; Na(+) = 0.8 M). Experiments with synthetically prepared solutions were performed in a continuously fed system consisting of two gas-lift reactors in series operated at aerobic conditions at 35 °C. The detoxification of sulfide to thiosulfate in the first step allowed the successful biological treatment of total-S loading rates up to 33 mmol L(-1) day(-1). In the second, biological step, the remaining sulfide and thiosulfate was completely converted to sulfate by haloalkaliphilic sulfide oxidizing bacteria. Mathematical modeling of the 2-step process shows that under the prevailing conditions an optimal reactor configuration consists of 40% 'abiotic' and 60% 'biological' volume, whilst the total reactor volume is 22% smaller than for the 1-step process.

Biological treatment of refinery spent caustics under halo-alkaline conditions.

The present research demonstrates the biological treatment of refinery sulfidic spent caustics in a continuously fed system under halo-alkaline conditions (i.e. pH 9.5; Na(+)= 0.8M). Experiments were performed in identical gas-lift bioreactors operated under aerobic conditions (80-90% saturation) at 35°C. Sulfide loading rates up to 27 mmol L(-1)day(-1) were successfully applied at a HRT of 3.5 days. Sulfide was completely converted into sulfate by the haloalkaliphilic sulfide-oxidizing bacteria belonging to the genus Thioalkalivibrio. Influent benzene concentrations ranged from 100 to 600 µM. At steady state, benzene was removed by 93% due to high stripping efficiencies and biodegradation. Microbial community analysis revealed the presence of haloalkaliphilic heterotrophic bacteria belonging to the genera Marinobacter, Halomonas and Idiomarina which might have been involved in the observed benzene removal. The work shows the potential of halo-alkaliphilic bacteria in mitigating environmental problems caused by alkaline waste.

Hydrogenotrophic sulfate reduction in a gas-lift bioreactor operated at 9 degrees C.

The viability of low temperature sulfate reduction with hydrogen as electron donor was studied with a bench-scale gas-lift bioreactor (GLB) operated at 9 degrees C. Prior to the GLB experiment, the temperature range of sulfate reduction of the inoculum was assayed. The results of the temperature gradient assay indicated that the inoculum was a psychrotolerant mesophilic enrichment culture that had an optimal temperature for sulfate reduction of 31 degrees C, and minimum and maximum temperatures of 7 degrees C and 41 degrees C, respectively. In the GLB experiment at 9 degrees C, a sulfate reduction rate of 500-600 mg L(-1) d(-1), corresponding to a specific activity of 173 mg SO(4)(2-) g VSS(-1) d(-1), was obtained. The electron flow from the consumed H(2)-gas to sulfate reduction varied between 27% and 52%, while the electron flow to acetate production decreased steadily from 15% to 5%. No methane was produced. Acetate was produced from CO(2) and H(2) by homoacetogenic bacteria. Acetate supported the growth of some heterotrophic sulfate-reducing bacteria. The sulfate reduction rate in the GLB was limited by the slow biomass growth rate at 9 degrees C and low biomass retention in the reactor. Nevertheless, this study demonstrated the potential sulfate reduction rate of psychrotolerant sulfate-reducing mesophiles at sub-optimal temperature.

Sulfate reduction at pH 4.0 for treatment of process and wastewaters.

Acidic industrial process and wastewaters often contain high sulfate and metal concentrations and their direct biological treatment is thus far not possible as biological processes at pH < 5 have been neglected. Sulfate-reducing bacteria convert sulfate to sulfide that can subsequently be used to recover metals as metal-sulfides precipitate. This study reports on high-rate sulfate reduction with a mixed microbial community at pH 4.0 and 4.5 with hydrogen and/or formate as electron donors. The maximum sulfate reducing activity at pH 4.0 was sustained for over 40 days with a specific activity 500-fold greater than previously reported values: 151 mmol sulfate reduced/L reactor liquid per day with a maximum specific activity of 84 mmol sulfate per gram of volatile suspended solids per day. The biomass yield gradually decreased from 38 to 0.4 g volatile suspended solids per kilogram of sulfate when decreasing the reactor pH from pH 6 to 4. The microorganisms had a high maintenance requirement probably due maintaining pH homeostasis and the toxicity of sulfide at low pH. The microbial community diversity in the pH 4.0 membrane bioreactor decreased over time, while the diversity of the sulfate reducing community increased. Thus, a specialized microbial community containing a lower proportion of microorganisms capable of activity at pH 4 developed in the reactor compared with those present at the start of the experiment. The 16S rRNA genes identified from the pH 4.0 grown mixed culture were most similar to those of Desulfovibrio species and Desulfosporosinus sp. M1.

Sulfate reduction at pH 5 in a high-rate membrane bioreactor: reactor performance and microbial community analyses.

High rate sulfate reduction under acidic conditions opens possibilities for new process flow sheets that allow the selective recovery of metals from mining and metallurgical waste and process water. However, knowledge about high-rate sulfate reduction under acidic conditions is limited. This paper investigates sulfate reduction in a membrane bioreactor at a controlled pH of 5. Sulfate and formate were dosed using a pH-auxostat system while formate was converted into hydrogen, which was used for sulfate reduction. Sulfide was removed from the gas phase to prevent sulfide inhibition. This study shows a high-rate sulfate-reducing bioreactor system for the first time at pH 5, with a volumetric activity of 188mmol SO(4)(2-)/I/d and a specific activity of 81mmol SO(4)(2-) volatile suspended. The microbial community at the end of the reactor run consisted of a diverse mixed population including sulfate-reducing bacteria.

Population dynamics of a single-stage sulfidogenic bioreactor treating synthetic zinc-containing waste streams.

Waste streams from industrial processes such as metal smelting or mining contain high concentrations of sulfate and metals with low pH. Dissimilatory sulfate reduction carried out by sulfate-reducing bacteria (SRB) at low pH can combine sulfate reduction with metal-sulfide precipitation and thus open possibilities for selective metal recovery. This study investigates the microbial diversity and population changes of a single-stage sulfidogenic gas-lift bioreactor treating synthetic zinc-rich waste water at pH 5.5 by denaturing gradient gel electrophoresis of 16S rRNA gene fragments and quantitative polymerase chain reaction. The results indicate the presence of a diverse range of phylogenetic groups with the predominant microbial populations belonging to the Desulfovibrionaceae from delta-Proteobacteria. Desulfovibrio desulfuricans-like populations were the most abundant among the SRB during the three stable phases of varying sulfide and zinc concentrations and increased from 13% to 54% of the total bacterial populations over time. The second largest group was Desulfovibrio marrakechensis-like SRB that increased from 1% to about 10% with decreasing sulfide concentrations. Desulfovibrio aminophilus-like populations were the only SRB to decrease in numbers with decreasing sulfide concentrations. However, their population was <1% of the total bacterial population in the reactor at all analyzed time points. The number of dissimilatory sulfate reductase (DsrA) gene copies per number of SRB cells decreased from 3.5 to 2 DsrA copies when the sulfide concentration was reduced, suggesting that the cells' sulfate-reducing capacity was also lowered. This study has identified the species present in a single-stage sulfidogenic bioreactor treating zinc-rich wastewater at low pH and provides insights into the microbial ecology of this biotechnological process.

Selective recovery of nickel over iron from a nickel-iron solution using microbial sulfate reduction in a gas-lift bioreactor.

Process streams with high concentrations of metals and sulfate are characteristic for the mining and metallurgical industries. This study aims to selectively recover nickel from a nickel-iron-containing solution at pH 5.0 using a single stage bioreactor that simultaneously combines low pH sulfate reduction and metal-sulfide formation. The results show that nickel was selectively precipitated in the bioreactor at pH 5.0 and the precipitates consisted of >or=83% of the nickel content. The nickel-iron precipitates were partly crystalline and had a metal/sulfur ratio of 1, suggesting these precipitates were NiS and FeS. Experiments focusing on nickel recovery at pH 5.0 and 5.5 reached a recovery of >99.9%, resulting in a nickel effluent concentration<0.05 microM. The mixed microbial population included known sulfate reducers and acetogens. This study shows that selective metal precipitation in a single stage sulfate reducing bioreactor operated at low pH has the potential to produce metal-sulfides that can be used by the metallurgical industry as a resource for metal production.

Effect of sulfide removal on sulfate reduction at pH 5 in a hydrogen fed gas-lift bioreactor.

Biotechnological treatment of sulfate- and metal-ionscontaining acidic wastewaters from mining and metallurgical activities utilizes sulfate-reducing bacteria to produce sulfide that can subsequently precipitate metal ions. Reducing sulfate at a low pH has several advantages above neutrophilic sulfate reduction. This study describes the effect of sulfide removal on the reactor performance and microbial community in a high-rate sulfidogenic gas-lift bioreactor fed with hydrogen at a controlled internal pH of 5. Under sulfide removal conditions, 99% of the sulfate was converted at a hydraulic retention time of 24 h, reaching a volumetric activity as high as 51 mmol sulfate/l/d. Under nonsulfide removal conditions, <25% of the sulfate was converted at a hydraulic retention time of 24 h reaching volumetric activities of <13mmol sulfate/l/d. The absence of sulfide removal at a hydraulic retention time of 24 h resulted in an average H2S concentration of 18.2 mM (584 mg S/l). The incomplete sulfate removal was probably due to sulfide inhibition. Molecular phylogenetic analysis identified 11 separate 16S rRNA bands under sulfide stripping conditions, whereas under nonsulfide removal conditions only 4 separate 16S rRNA bands were found. This shows that a less diverse population was found in the presence of a high sulfide concentration.