Biological conversion of anglesite (PbSO(4)) and lead waste from spent car batteries to galena (PbS)

Lead paste, a solid mixture containing PbSO(4), PbO(2), PbO/Pb(OH)(2) precipitate, and elemental Pb, is one of the main waste fractions from spent car batteries. Biological sulfidation represents a new process for recovery of lead from this waste. In this process the lead salts in lead paste are converted to galena (PbS) by sulfate-reducing bacteria. This paper investigates a continuous process for sulfidation of anglesite (PbSO(4)), the main constituent of lead paste, and lead paste, consisting of a laboratory-scale gas-lift bioreactor to which a slurry of anglesite or lead paste was supplied. Sulfate or elemental sulfur was added as an additional sulfur source. Hydrogen gas served as an electron donor for the biological reduction of sulfate and elemental sulfur to sulfide by sulfate- and sulfur-reducing bacteria. Anglesite was almost completely converted to galena at a loading rate of 19 kg of PbSO(4) m(-)(3) day(-)(1), producing a sludge of which the crystalline lead phases consisted of >98% PbS (galena) and 1-2% elemental Pb. With lead paste, stable sulfidation rates of up to 17 kg of lead paste m(-)(3) day(-)(1) were demonstrated, producing a sludge of which the crystalline lead phases consisted of an estimated >96% PbS, 1-2% elemental Pb, and 1-2% PbO(2).     

Anaerobic degradation of methylmercaptan and dimethyl sulfide by newly isolated thermophilic sulfate-reducing bacteria.

The complete oxidation of methylmercaptan (MSH) and dimethyl sulfide (DMS) with sulfate or nitrate as electron acceptors was observed in enrichment cultures and dilution series using thermophilic fermentor sludge as the inoculum. Three new strains of thermophilic sulfate reducers were isolated in pure culture (strains MTS5, TDS2, and SDN4). Strain MTS5 grew on MSH and strain TDS2 grew on DMS whereas strain SDN4 grew on either MSH or DMS. The cellular growth yields were 2.57 g (dry weight)/mol of MSH for strain MTS5 and 6.02 g (dry weight)/mol of DMS for strain TDS2. All strains used sulfate, sulfite, or thiosulfate as electron acceptors, but only strain SDN4 used nitrate. DMS and MSH were oxidized to CO2 and sulfide with either sulfate or nitrate as the electron acceptor. Sulfate was stoichiometrically reduced to sulfide while nitrate was reduced to ammonium. All strains were motile rods, required biotin for growth, lacked desulfoviridin, had DNA with G+C contents of 48 to 57 mol% and probably belonged to the genus Desulfotomaculum. This is the first report of the oxidation of MSH and DMS by pure cultures of sulfate-reducing bacteria.     

Geochemical, Microbiological, and Geophysical Assessments of Anaerobic Immobilization of Heavy Metals

Bioremediation methods that precipitate contaminants in situ as solid (mineral) phases can provide cost-effective options for removing dissolved metals in contaminated groundwater. The current field-scale experiments demonstrate that indigenous bacteria can be stimulated to remove metals by injection of electron-donating substrates and nutrients into a contaminated aquifer. Groundwater at the investigation site is aerobic and contains high levels of lead, cadmium, zinc, copper, and sulfuric acid (pH = 3.1) derived from a car-battery recycling plant. During the experiments, lead, cadmium, zinc, and copper were almost completely removed by precipitation of solid sulfide phases, as pH increased from 3 to ∼ 5 and Eh dropped from +400 mV to -150 mV. X-ray and transmission electron microscopy (TEM) analyses of filtered material from the treated groundwater indicated the presence of newly formed nanocrystalline metal sulfides. Genetic sequencing indicated that the principal species of sulfate-reducing bacteria involved in the bioremediation process was Desulfosporosinus orientis. Geochemical modeling shows that oxidation of added substrates and subsequent bacterial sulfate reduction produced desired geochemical conditions (i.e., decreasing Eh and increasing pH) for the precipitation and sorption of metal sulfides. Geophysical survey results suggest that bioremediation lowers electrical conductance of groundwater and possibly increases the magnetic susceptibility of porous media. This study demonstrates that integrated geochemical, geophysical, and microbiological analyses, combined with theoretical modeling, can successfully track and predict the progress of subsurface bioremediation.     

Involvement of Sulfur- and Iron-Transforming Bacteria in Heaving of House Foundations

About 1,000 houses built on excavated nonweathered mudstone sediments, originally deposited in the Neogene, have been damaged by microbially induced heaving of foundations. The maximal height of the heaving was 48 cm. The presence of sulfate-reducing, sulfur-oxidizing, and acidophilic iron-oxidizing bacteria in the mudstone indicated that the joint activity of these three types of bacteria could account for the heaving. A hypothesis is presented in which, first, the temperature of the newly exposed mudstone sediments increased above 25 °C, which stimulated the sulfate-reducing bacteria in the mudstone to actively reduce sulfate to hydrogen sulfide. The mudstone sediments under the houses gradually dried, and became permeable to air. Consequently, sulfur-oxidizing bacteria oxidized the hydrogen sulfide to sulfuric acid and the environmental pH decreased to approximately 3. Next, the acidophilic iron-oxidizing bacteria actively oxidized the sulfur in pyrite to produce much more acid. The resulting sulfuric acid reacted with calcium carbonate and with ferric and potassium ions to produce gypsum and jarosite, respectively. A combination of the increased volume of gypsum and jarosite crystals and the production of CO 2 as a by-product of their formation made the mudstone sediments bulky. The end result was widespread heaving.     

Elemental sulfur as an intermediate of sulfide oxidation with oxygen byDesulfobulbus propionicus

The sulfate-reducing bacteriumDesulfobulbus propionicus oxidized sulfide, elemental sulfur, and sulfite to sulfate with oxygen as electron acceptor. Thiosulfate was reduced and disproportionated exclusively under anoxic conditions. When small pulses of oxygen were added to washed cells in sulfide-containing assays, up to 3 sulfide molecules per O₂ disappeared transiently. After complete oxygen consumption, part of the sulfide reappeared. The intermediate formed was identified as elemental sulfur by chemical analysis and turbidity measurements. When excess sulfide was present, sulfur dissolved as polysulfide. This process was faster in the presence of cells than in their absence. The formation of sulfide after complete oxygen consumption was due to a disproportionation of elemental sulfur (or polysulfide) to sulfide and sulfate. The uncoupler tetrachlorosalicylanilide (TCS) and the electron transport inhibitor myxothiazol inhibited sulfide oxidation to sulfate and caused accumulation of sulfur. In the presence of the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), sulfite and thiosulfate were formed. During sulfur oxidation at low oxygen concentrations, intermediary formation of sulfide was observed, indicating disproportionation of sulfur also under these conditions. It is concluded that sulfide oxidation inD. propionicus proceeds via oxidation to elemental sulfur, followed by sulfur disproportionation to sulfide and sulfate. Dedicated to Prof. Dr. Dr. h.c. Norbert Pfennig on the occasion of his 70th birthday     

Methanogenic potential of tailings samples from oil sands extraction plants

Approximately 20% of Canada's oil supply now comes from the extraction of bitumen from the oil sands deposits in northeastern Alberta. The oil sands are strip-mined, and the bitumen is typically separated from sand and clays by an alkaline hot water extraction process. The rapidly expanding oil sands industry has millions of cubic metres of tailings for disposal and large areas of land to reclaim. There are estimates that the consolidation of the mature fine tails (MFT) in the settling ponds will take about 150 years. Some of the settling ponds are now evolving microbially produced methane, a greenhouse gas. To hasten consolidation, gypsum (CaSO4 x 2H2O) is added to MFT, yielding materials called consolidated or composite tailings (CT). Sulfate from the gypsum has the potential to stimulate sulfate-reducing bacteria (SRB) to out-compete methanogens, thereby stopping methanogenesis. This investigation examined three MFT and four CT samples from three oil sands extractions companies. Each was found to contain methanogens and SRB. Serum bottle microcosm studies showed sulfate in the CT samples stopped methane production. However, if the microcosms were amended with readily utilizable electron donors, the sulfate was consumed, and when it reached approximately 20 mg/L, methane production began. Some unamended microcosms were incubated for 372 days, with no methane production detected. This work showed that each MFT and CT sample has the potential to become methanogenic, but in the absence of exogenous electron donors, the added sulfate can inhibit methanogenesis for a long time.     

Sulfate reduction and anaerobic glycerol degradation by a mixed microbial culture

Anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water, was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2. In the absence of sulfate, glycerol utilization was found to be characterized by the transient formation of 1,3-propanediol prior to propionate and acetate accumulation. In the presence of sulfate, 1,3-propanediol production was minor, and the carbon balance reflected a considerable accumulation of intermediate(s). A study of the role of sulfate reduction and methanogenesis on anaerobic 1,3-propanediol degradation showed that consumption of this substrate by the mixed microbial culture required a terminal electron acceptor. The number of fermentative and sulfate-reducing bacteria with glycerol or 1,3-propanediol as carbon and energy source revealed that sulfate-reducing bacteria outcompete fermentative bacteria for these substrates. The possible ecological role of sulfate-reducing bacteria in the metabolism of these reduced substrates is discussed.     

Characterization of 16S rRNA genes from oil field microbial communities indicates the presence of a variety of sulfate-reducing, fermentative, and sulfide-oxidizing bacteria.

Oil field bacteria were characterized by cloning and sequencing of PCR-amplified 16S rRNA genes. A variety of gram-negative, sulfate-reducing bacteria was detected (16 members of the family Desulfovibrionaceae and 8 members of the family Desulfobacteriaceae). In contrast, a much more limited number of anaerobic, fermentative, or acetogenic bacteria was found (one Clostridium sp., one Eubacterium sp., and one Synergistes sp.). Potential sulfide oxidizers and/or microaerophiles (Thiomicrospira, Arcobacter, Campylobacter, and Oceanospirillum spp.) were also detected. The first two were prominently amplified from uncultured production water DNA and represented 28 and 47% of all clones, respectively. Growth on media containing sulfide as the electron donor and nitrate as the electron acceptor and designed for the isolation of Thiomicrospira spp. gave only significant enrichment of the Campylobacter sp., which was shown to be present in different western Canadian oil fields. This newly discovered sulfide oxidizer may provide a vital link in the oil field sulfur cycle by reoxidizing sulfide formed by microbial sulfate or sulfur reduction.     

Sulfate reduction from phosphogypsum using a mixed culture of sulfate-reducing bacteria

Phosphogypsum (CaSO₄), a primary by-product of phosphoric acid production, is accumulated in large stockpiles and occupies vast areas of land. It poses a severe threat to the quality of water and land in countries producing phosphoric acid. In this study, the potential of sulfate-reducing bacteria for biodegradation of this sulfur-rich industrial solid waste was assessed. The effect of phosphogypsum concentration, carbon and nitrogen sources, temperature, pH and stirring on the growth of sulfate-reducing bacteria was investigated. Growth of sulfate-reducing bacteria was monitored by measuring sulfide production. Phosphogypsum was shown to be a good source of sulfate, albeit that the addition of organic carbon was necessary for bacterial growth. Biogenic sulfide production occurred with phosphogypsum up to a concentration of 40 g L⁻¹, above which no growth of sulfate-reducing bacteria was observed. Optimal growth was obtained at 10 g L⁻¹ phosphogypsum. Both the gas mixture H₂/CO₂ and lactate supported high amounts of H₂S formation (19 and 11 mM, respectively). The best source of nitrogen for sulfate-reducing bacteria was yeast extract, followed by ammonium chloride. The presence of nitrate had an inhibitory effect on the process of sulfate reduction. Stirring the culture at 150 rpm slightly stimulated H₂S formation, probably by improving sulfate solubility.     

Isolation of highly performant sulfate reducers from sulfate-rich environments

Eleven pure strains of sulfate-reducing bacteria have been isolated from lab-scale bioreactors or gypsum disposal sites, all featuring relatively high concentrations of sulfate, and from natural environments in order to produce sulfide from gypsum using hydrogen as energy source. The properties of the eleven strains have been investigated and compared to these of three collection strains i.e. Desulfovibrio desulfuricans and Dv. vulgaris and Desulfotomaculum orientis. Particular attention was paid to the volumetric and specific sulfide production rate and to the hydrogen sulfide inhibition level. By comparison to the three collection strains, a 75% higher production rate and a hydrogen sulfide inhibition level about twice as high i.e. 25.1 mM have been achieved with strains isolated from sulfate-rich environments. The strain selection, particularly from sulfate-rich environments, should be considered as an optimization factor for the sulfate reduction processes.     

Metabolic Engineering of an Aerobic Sulfate Reduction Pathway and Its Application to Precipitation of Cadmium on the Cell Surface

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.     

Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.     

Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Sulfidogenesis from 2-aminoethanesulfonate (taurine) fermentation by a morphologically unusual sulfate-reducing bacterium, Desulforhopalus singaporensis sp. nov.

A pure culture of an obligately anaerobic marine bacterium was obtained from an anaerobic enrichment culture in which taurine (2-aminoethanesulfonate) was the sole source of carbon, energy, and nitrogen. Taurine fermentation resulted in acetate, ammonia, and sulfide as end products. Other sulfonates, including 2-hydroxyethanesulfonate (isethionate) and cysteate (alanine-3-sulfonate), were not fermented. When malate was the sole source of carbon and energy, the bacterium reduced sulfate, sulfite, thiosulfate, or nitrate (reduced to ammonia) but did not use fumarate or dimethyl sulfoxide as a terminal electron acceptor for growth. Taurine-grown cells had significantly lower adenylylphosphosulfate reductase activities than sulfate-grown cells had, which was consistent with the notion that sulfate was not released as a result of oxidative C-S bond cleavage and then assimilated. The name Desulforhopalus singaporensis is proposed for this sulfate-reducing bacterium, which is morphologically unusual compared to the previously described sulfate-reducing bacteria by virtue of the spinae present on the rod-shaped, gram-negative, nonmotile cells; endospore formation was not discerned, nor was desulfoviridin detected. Granules of poly-beta-hydroxybutyrate were abundant in taurine-grown cells. This organism shares with the other member of the genus Desulforhopalus which has been described a unique 13-base deletion in the 16S ribosomal DNA. It differs in several ways from a recently described endospore-forming anaerobe (K. Denger, H. Laue, and A. M. Cook, Arch. Microbiol. 168:297-301, 1997) that reportedly produces thiosulfate but not sulfide from taurine fermentation. D. singaporensis thus appears to be the first example of an organism which exhibits sulfidogenesis during taurine fermentation. Implications for sulfonate sulfur in the sulfur cycle are discussed.     

Ferrihydrite-Dependent Growth of Sulfurospirillum deleyianum through Electron Transfer via Sulfur Cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Microbial sulfate-reducing activities in anoxic sediment from Marine Lake Grevelingen: screening of electron donors and acceptors

Sulfate-reducing bacteria in marine sediments mainly utilize sulfate as a terminal electron acceptor with different organic compounds as electron donors. This study investigated microbial sulfate-reducing activity of coastal sediment from Marine Lake Grevelingen (MLG), the Netherlands using different electron donors and electron acceptors. All four electron donors (ethanol, lactate, acetate and methane) showed sulfate-reducing activity with sulfate as electron acceptor, suggesting the presence of an active sulfate-reducing bacterial population in the sediment, even at dissolved sulfide concentrations exceeding 12 mM. Ethanol showed the highest sulfate reduction rate of 55 µmol g _VSS ^?1 day^?1 compared to lactate (32 µmol g _VSS ^?1 day^?1), acetate (26 µmol g _VSS ^?1 day^?1) and methane (4.7 µmol g _VSS ^?1 day^?1). Sulfide production using thiosulfate and elemental sulfur as electron acceptors and methane as the electron donor was observed, however, mainly by disproportionation rather than by anaerobic oxidation of methane coupled to sulfate reduction. This study showed that the MLG sediment is capable of performing sulfate reduction by using diverse electron donors, including the gaseous and cheap electron donor methane.     

Sulfide-controlled continuous culture of sulfate-reducing bacteria

In continuous culture set-up for sulfate-reducing bacteria a sulfide electrode (made from silver wire) is used to control the electron donor supply and the medium pump. The sulfied concentration of the medium is kept at a low level by continuosly flushing out H₂S and replacing it with CO₂. The pH is controlled automatically by regulating the CO₂ content of the gas mixture flushed through the medium. With the sulfide-controlled set-up sulfate-reducing bacteria can be grown in chemostat culture under electron donor as well as electron acceptor limitation. Furthermore, by continuously washing out the culture to a preselected residual sulfide concentration, cells can be grown in sulfidostat culture under non-limiting conditions at maximal growth rate. Growth yields of Desulfotmaculum orientis, when growth in this system with hydrogen as electron donor, were considerably higher than previously reported.     

A sulfate-reducing bacterium from the oxic layer of a microbial mat from Solar Lake (Sinai), Desulfovibrio oxyclinae sp. nov.

In an investigation on the oxygen tolerance of sulfate-reducing bacteria, a strain was isolated from a 10⁷-fold dilution of the upper 3-mm layer of a hypersaline cyanobacterial mat (transferred from Solar Lake, Sinai). The isolate, designated P1B, appeared to be well-adapted to the varying concentrations of oxygen and sulfide that occur in this environment. In the presence of oxygen strain P1B respired aerobically with the highest rates [260 nmol O₂ min^–1 (mg protein)^–1] found so far among marine sulfate-reducing bacteria. Besides H₂ and lactate, even sulfide or sulfite could be oxidized with oxygen. The sulfur compounds were completely oxidized to sulfate. Under anoxic conditions, it grew with sulfate, sulfite, or thiosulfate as the electron acceptor using H₂, lactate, pyruvate, ethanol, propanol, or butanol as the electron donor. Furthermore, in the absence of electron donors the isolate grew by disproportionation of sulfite or thiosulfate to sulfate and sulfide. The highest respiration rates with oxygen were obtained with H₂ at low oxygen concentrations. Aerobic growth of homogeneous suspensions was not obtained. Additions of 1% oxygen to the gas phase of a continuous culture resulted in the formation of cell clumps wherein the cells remained viable for at least 200 h. It is concluded that strain P1B is oxygen-tolerant but does not carry out sulfate reduction in the presence of oxygen under the conditions tested. Analysis of the 16S rDNA sequence indicated that strain P1B belongs to the genus Desulfovibrio, with Desulfovibrio halophilus as its closest relative. Based on physiological properties strain P1B could not be assigned to this species. Therefore, a new species, Desulfovibrio oxyclinae, is proposed. Received: 7 August 1996 / Accepted: 29 January 1997     

Interrelations between populations of methanogenic archaea and sulfate-reducing bacteria in the human colon.

In humans, CH4 is produced in the colon by methanogenic archaea and is detected in breath samples from approximately 50% of healthy adults, identified as CH4-excretors. Methanogenesis and sulfate reduction have been described as two mutually exclusive processes, potentially regulated by sulfate availability. To determine whether microbial population balances reflected these apparently co-regulated activities, we compared sulfate-reducing bacteria, methanogenic archaea, sulfate and sulfide concentrations in faeces of 10 CH4-excretors (CH4+) and 9 non-CH4-excretors (CH4-). The mean +/- SE of the logarithm of methanogenic archaea per gram wet weight were 9.0 +/- 0.2 and 4.0 +/- 0.7 for CH4+ and CH4-, respectively (P < 0.001). Sulfate-reducing bacterial counts were 6.5 +/- 0.1 and 7.3 +/- 0.2, respectively (P < 0.001). Fecal sulfate and sulfide concentrations did not differ between groups. These results suggest that a competitive interrelation between methanogenic archaea and sulfate-reducing bacteria occurs in the human colon. However, it does not lead to a complete exclusion of the two populations.