An internal sedimentation bioreactor for laboratory-scale removal of toxic metals from soil leachates using biogenic sulphide precipitation

An internal-sedimentation bioreactor was employed to provide biomass feedback and process intensification in a laboratory-scale sulphide-bioprecipitation system for toxic metals (Cd, Cr, Cu, Mn, Ni, Zn) present in acid leachates from metal-contaminated soil. Biomass feedback was improved by addition of a cationic polymer flocculant and the activity of the sulphate-reducing bacterial culture was increased by the addition of cornsteep in addition to the ethanol used as carbon/energy substrate. A mass-balance was carried out for carbon and sulphur in the system. Sulphate reduction in the reactor was able to remove acidity at moderate sulphate concentrations up to 50 mM although it was insufficient at the highest levels tested. When presented with a simulated toxic metal-containing leachate, the reactor was able to precipitate metals efficiently under all of the conditions of sulphate concentration and pH tested, producing an effluent with metal concentrations suitable for environmental discharge. Received 29 October 1996/ Accepted in revised form 31 March 1997     

Characterization of sulphate-reducing bacterial populations within marine and estuarine sediments with different rates of sulphate reduction

Viable counts of sulphate-reducing bacteria, able to use a range of different growth substrates were determined in sediments from two Sea Lochs (Etive and Eil) and an estuarine site (Tay), in Scotland. The composition of the sulphate-reducing bacterial population, in terms of substrate utilization, broadly corresponded to the in situ substrates for sulphate reduction and concentration of substrates at each site. Addition of acetate, lactate, propionate, butyrate, hydrogen and glutamate/serine (20 mM) to replicate slurries from each site resulted in stimulation of the corresponding population of sulphate-reducing bacteria and the in situ rates of sulphate reduction. The metabolism of the added substrates and changes in bacterial phospholipid fatty acids (PLFA) were quantified. With the exception of acetate and hydrogen, added substrates were incompletely oxidised, producing a mixture of further substrates, which predominantly were sequentially oxidised, and resulted in the stimulation of a mixed population of sulphate-reducing bacteria. There were significant changes in the PLFA of slurries with added substrate compared to controls. Acetate was completely removed at all sites and the small increase in even chain PLFA together with the absence of stimulation of any other biomarker, indicated that acetate was oxidised by sulphate-reducing bacteria distinctly different from those using other substrates. A biomarker for Desulfobacter, 10 Methyl 16:0, was not stimulated in any of the acetate slurries or in slurries where acetate was produced. Biomarkers for the propionate utilizing Desulfobulbus sp (17:1w6, 15:1w6) were always stimulated in propionate slurries and also in lactate slurries, where partial lactate fermentation produced propionate and acetate. In lactate and glutamate / serine slurries from the Tay estuary and lactate and hydrogen slurries from Loch Etive the biomarker for Desulfovibrio sp (i17:1w7) as well as those for Desulfobulbus were stimulated. This provides direct evidence for the significance of Desulfovibrio sp. within sediment slurries and demonstrates the competitive interaction between members of this genus and Desulfobulbus sp. for lactate, hydrogen and amino acid metabolism. At the estuarine site, sulphate reduction was limited at higher sulphate concentrations (about 3.5 mM) than the Sea Loch sites (<2 mM) and this had a significant effect on propionate and butyrate metabolism, as well as on methane production. These results demonstrate that although the sulphate-reducing bacterial population at each site could metabolise identical substrates, the types of sulphate-reducing bacteria involved and their sulphate thresholds were characteristically different.     

Lactate has the potential to promote hydrogen sulphide formation in the human colon

High concentrations of sulphide are toxic for the gut epithelium and may contribute to bowel disease. Lactate is a favoured cosubstrate for the sulphate-reducing colonic bacterium Desulfovibrio piger , as shown here by the stimulation of sulphide formation by D. piger DSM749 by lactate in the presence of sulphate. Sulphide formation by D. piger was also stimulated in cocultures with the lactate-producing bacterium Bifidobacterium adolescentis L2-32. Other lactate-utilizing bacteria such as the butyrate-producing species Eubacterium hallii and Anaerostipes caccae are, however, expected to be in competition with the sulphate-reducing bacteria (SRB) for the lactate formed in the human colon. Strains of E. hallii and A. caccae produced 65% and 96% less butyrate from lactate, respectively, in a coculture with D. piger DSM749 than in a pure culture. In triculture experiments involving B. adolescentis L2-32, up to 50% inhibition of butyrate formation by E. hallii and A. caccae was observed in the presence of D. piger DSM749. On the other hand, sulphide formation by D. piger was unaffected by E. hallii or A. caccae in these cocultures and tricultures. These experiments strongly suggest that lactate can stimulate sulphide formation by SRB present in the colon, with possible consequences for conditions such as colitis.     

Anaerobic degradation of naphthalene by a pure culture of a novel type of marine sulphate-reducing bacterium

Incubation of marine sediment in anoxic, sulphate-rich medium in the presence of naphthalene resulted in the enrichment of sulphate-reducing bacteria. Pure cultures with short, oval cells (1.3 by 1.3–1.9 μm) were isolated that grew with naphthalene as the only organic carbon source and electron donor for sulphate reduction to sulphide. One strain, NaphS2, was characterized. It affiliated with completely oxidizing sulphate-reducing bacteria of the δ-subclass of the Proteobacteria, as revealed by 16S rRNA sequence analysis. 2-Naphthoate, benzoate, pyruvate and acetate were used in addition to naphthalene. Quantification of substrate consumption, sulphide formation and formed cell mass revealed that naphthalene was completely oxidized with sulphate as the electron acceptor.     

In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area

Anaerobic oxidation of methane (AOM) and sulphate reduction were examined in sediment samples from a marine gas hydrate area (Hydrate Ridge, NE Pacific). The sediment contained high numbers of microbial consortia consisting of organisms that affiliate with methanogenic archaea and with sulphate-reducing bacteria. Sediment samples incubated under strictly anoxic conditions in defined mineral medium (salinity as in seawater) produced sulphide from sulphate if methane was added as the sole organic substrate. No sulphide production occurred in control experiments without methane. Methane-dependent sulphide production was fastest between 4 degree C and 16 degree C, the average rate with 0.1 MPa (approximately 1 atm) methane being 2.5 micro mol sulphide day(-1) and (g dry mass sediment)(-1). An increase of the methane pressure to 1.1 MPa (approximately 11 atm) resulted in a four to fivefold increase of the sulphide production rate. Quantitative measurements using a special anoxic incubation device without gas phase revealed continuous consumption of dissolved methane (from initially 3.2 to 0.7 mM) with simultaneous production of sulphide at a molar ratio of nearly 1:1. To test the response of the indigenous community to possible intermediates of AOM, molecular hydrogen, formate, acetate or methanol were added in the absence of methane; however, sulphide production from sulphate with any of these compounds was much slower than with methane. In the presence of methane, such additions neither stimulated nor inhibited sulphate reduction. Hence, the experiments did not provide evidence for one of these compounds acting as a free extracellular intermediate (intercellular shuttle) during AOM by the presently investigated consortia.     

Sulphate reduction in oxic and sub-oxic North-East Atlantic sediments

Abstract Oxic and sub-oxic N.-E. Atlantic sediments were examined for sulphate-reducing activity. Oxygen and/or nitrate reduction are probably the dominant mineralisation processes in the abyssal plain sediment studied. A low rate of sulphate reduction (0.1 nmol SO²⁻₄/ml/day) was recorded in the surface 5 cm of the continental slope sediment, together with the presence of a range of sulphate-reducing bacteria (SRB). A higher activity of sulphate reduction (2.2 nmol SO²⁻₄/ml/day) occurred in the continental shelf sediment which led to a small decrease in pore water sulphate and an increase in titration alkalinity. This sediment contained approx. 10²–10³ acetate, lactate and propionate oxidising SRB/ml. No low- M _r organic acids were detected in these sediments. However, amendment with 75 μM acetate stimulated sulphate-reducing activity in the shelf sediment.     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

Reduction and precipitation of chromate by mixed culture sulphate-reducing bacterial biofilms

The ability of sulphate-reducing bacterial biofilms to reduce hexavalent chromium (Cr(VI)) to insoluble Cr(III), a process of environmental and biotechnological significance, was investigated. The reduction of chromate to insoluble form has been quantified and the effects of chromate on the carbon source utilization and sulphate-reducing activity of the bacterial biofilms evaluated. Using lactate as the carbon/energy source and in the presence of sulphate, reduction of 500 micromol l-1 Cr(VI) was monitored over a 48-h period where 88% of the total chromium was removed from solution. Mass balance calculations showed that ca 80% of the total chromium was precipitated out of solution with the bacterial biofilm retaining less than 10% of the chromium. Only ca 12% of the chromate added was not reduced to insoluble form. Although Cr(VI) did not have a significant effect on C source utilization, sulphate reduction was severely inhibited by 500 micromol-1 Cr(VI) and only ca 10% of the sulphate reducing activity detected in control biofilms occurred in the presence of Cr(VI). Low levels of sulphide were also produced in the presence of chromate, with control biofilms producing over 10-times more sulphide than Cr(VI)-exposed biofilms. Sulphide- or other chemically-mediated Cr(VI) reduction was not detected. The biological mechanism of Cr(VI) reduction is likely to be similar to that found in other sulphate-reducing bacteria.     

Sulphate-reducing bacteria in deep aquifer sediments of the London Basin: their role in anaerobic mineralization of organic matter

Sulphate-reducing potential was measured in sandy aquifer sediments of the London Basin. Sulphate reduction could be stimulated in the laboratory by saturating the sands with groundwater, and creating an anaerobic environment. The stimulation of vigorous sulphate reduction through the addition of an external substrate was associated with an increase in Fe_T concentration. Molybdate and selenate were added to sediment/groundwater slurries as specific inhibitors of sulphate-reducing bacteria. Under sulphate-reducing conditions acetate accumulated, but was inhibited by molybdate and selenate. ¹⁴C-acetate was used to measure the rate of acetate metabolism in the sediments.     

Isolation and characterization of sulphate-reducing bacteria <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> from Vajreshwari thermal springs in Maharashtra,India

Sulphate-reducing bacteria (SRB) in the thermal springs of Vajreshwari were investigated with combined microbiological and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 25 to 55 °C and could use pyruvate, lactate and ethanol as electron donors. Desulfoviridin was detected in all the isolates. The partial 16S rRNA and dissimilatory sulphite reductase (DSR) gene sequences of five representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris.     

Corroding iron as a hydrogen source for sulphate reduction in growing cultures of sulphate-reducing bacteria

Summary In anaerobic corrosion experiments, hydrogenase-positiveDesulfovibrio strains, grown with limiting lactate concentrations in the presence of steel wool, formed more sulphide than expected or observed with lactate alone. The additional sulphide obviously originated from sulphate reduction with cathodically formed hydrogen from the steel surface. The hydrogenasenegativeD. sapovorans did not produce additional sulphide. The observations agree with the theory of von Wolzogen Kühr and van der Vlugt (1934) that explains anaerobic corrosion as a cathodic depolarization of iron surfaces by hydrogen-consuming sulphate-reducing bacteria. The influence of the iron surface area, the salt concentration and the pH-value on the utilization of cathodically formed hydrogen was investigated. The significance of an additional organic electron donor for the corrosion of iron in aqueous environments is discussed.     

The presence and activity of Desulfotomaculum spp in sulphate-limited freshwater sediments

Abstract Addition of pyrophosphate stimulated sulphate uptake and utilization of volatile fatty acids by anaerobic freshwater sediments. Population estimates of sulphate-reducing bacteria demonstrated that a larger number were capable of utilizing acetate then lactate. Estimates increased when pyrophosphate was added to the medium and decreased by the same degree when the sediment was heated. Addition of molybdate to the sulphate-limited sediments stimulated rather than inhibited methanogenesis. The results suggest that the sediment contains a metabolically active population of acetate-utilizing members of the genus Desulfotomaculum .     

Studies of Clostridium cellulolyticum ATCC 35319 under dialysis and co-culture conditions

A. Gehin, C. Cailliez, E. Petitdemange And L. Benoit. 1996. The degradation of cellulose by Clostridium celulolyticum has been studied in several ways; (1) in batch fermentation in 50-ml sealed-cap flasks, referred to as the control; (2) in batch fermentation with pH at 7.2; (3) fermentation in dialysis which permits elimination of all the products of metabolism; (4) fermentation in dialysis with a constant bubbling of nitrogen; (5) in co-culture with Clostridium A22 in batch with and without pH regulation and with dialysis. H₂, CO₂, acetate, ethanol and lactate were the major end-products of cellobiose and cellulose fermentation. Compared to batch culture, growth of CI. cellulolyticum on cellobiose increased by a factor of 10 in dialysed culture. The end products from the dialysed culture were detected in a small range compared to the concentration for the batch culture. Related to the biomass, CMCase activities were of the same level, showing a direct relation between the biomass formation and the cellulase production. The percentage of cellulose degradation (50%) by CI. cellulolyticum was greater when dialysis of end products with a constant bubbling of nitrogen took place during the course of fermentation (6 d) in comparison with cultures in 50-ml sealedcap flasks (23%), in a fermentor (36%) or using dialysis without N₂ bubbling (40%). The presence of two micro-organisms produced no further enzyme activities and hence the percentage of cellulose degradation was quite similar in mono- and co-culture. No synergistic action was found between two cellulolytic strains.     

Effect of carbon source and competition for electrons on nitrous oxide reduction in a mixed denitrifying microbial community

The competition for electrons has been recently demonstrated to affect the reduction rates of the nitrogen oxides in a methanol enriched denitrifying community. The aim of this study was to test if electron competition also occurred when other substrates were used for denitrification and if that could have an effect on the potential nitrous oxide (N₂O) production and subsequent consumption. A denitrifying culture was developed in a sequencing batch reactor using nitrate as electron acceptor and a combination of acetate, ethanol and methanol as carbon sources. Four sets of batch tests were conducted using acetate, ethanol, methanol and a combination of the three carbon sources respectively. For each set the effect of nitrate, nitrite and nitrous oxide on each other reduction rates when present individually or in combination was assessed. Results show that reduction rates are affected by the type of substrate added, probably due to different microbial populations specialized with consuming a particular substrate. Also, N₂O reduction rate is the most reduced under the different electron competition scenarios tested, which results in N₂O accumulation in some cases. The effect of substrate limitation on N₂O reduction was also assessed.     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulfate and tetrachloroethene

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis -dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.     

The same species of sulphate-reducing Desulfomicrobium occur in different oil field environments in the north sea

Several metabolic types of sulphate-reducing bacteria, including mesophiles and thermophiles, were successfully obtained from four samples from two different North Sea oil fields. The Gram-negative, rod-shaped, sulphate-reducing strains MM6, EF2, FM2, and GF2 were isolated from drain water, and from drilling muds E, F, and G, respectively. All four isolates grew on lactate, pyruvate, glycerol, and ethanol, with optimal growth temperatures between 25 degrees C and 35 degrees C and at salinities between 0 and 5% NaCl. They were capable of using sulphate, thiosulphate or sulphite, but not nitrate, as electron acceptors. These isolates were tentatively identified to be the same species of Desulfomicrobium based on physiological and biochemical characterization, and 16S rRNA gene analysis. Therefore, the same Desulfomicrobium species was present in different samples from distant oil fields. This result suggests that these microorganisms are likely to be widespread throughout oil field systems, and possibly play an important role in the generation of sulphide.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

Biological sulphate reduction using gas-lift reactors fed with hydrogen and carbon dioxide as energy and carbon source

Feasibility and engineering aspects of biological sulphate reduction in gas-lift reactors were studied. Hydrogen and carbon dioxide were used as energy and carbon source. Attention was paid to biofilm formation, sulphide toxicity, sulphate conversion rate optimization, and gasliquid mass transfer limitations. Sulphate-reducing bacteria formed stable biofilms on pumice particles. Biofilm formation was not observed when basalt particles were used. However, use of basalt particles led to the formation of granules of sulphate-reducing biomass. The sulphate-reducing bacteria, grown on pumice, easily adapted to free H(2)S concentrations up to 450 mg/L. Biofilm growth rate then equilibrated biomass loss rate. These high free H(2)S concentrations caused reversible inhibition rather than acute toxicity. When free H(2)S concentrations were kept below 450 mg/L, a maximum sulphate conversion rate of 30 g SO(4) (2-)/L . d could be achieved after only 10 days of operation. Gas-to-liquid hydrogen mass transfer capacity of the reactor determined the maximum sulphate conversion rate. (c) 1994 John Wiley & Sons, Inc.     

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.