Temperature effect on acetate and propionate consumption by sulfate-reducing bacteria in saline wastewater

Seawater toilet flushing, seawater intrusion in the sewerage, and discharge of sulfate-rich industrial effluents elevates sulfate content in wastewater. The application of sulfate-reducing bacteria (SRB) in wastewater treatment is very beneficial; as for example, it improves the pathogen removal and reduces the volume of waste sludge, energy requirement and costs. This paper evaluates the potential to apply biological sulfate reduction using acetate and propionate to saline sewage treatment in moderate climates. Long-term biological sulfate reduction experiments at 10 and 20 °C were conducted in a sequencing batch reactor with synthetic saline domestic wastewater. Subsequently, acetate and propionate (soluble organic carbon) conversion rate were determined in both reactors, in the presence of either or both fatty acids. Both acetate and propionate consumption rates by SRB were 1.9 times lower at 10 °C than at 20 °C. At 10 °C, propionate was incompletely oxidized to acetate. At 10 °C, complete removal of soluble organic carbon requires a significantly increased hydraulic retention time as compared to 20 °C. The results of the study showed that biological sulfate reduction can be a feasible and promising process for saline wastewater treatment in moderate climate.     

Sulfidogenic fluidized-bed treatment of metal-containing wastewater at low and high temperatures

The applicability of a fluidized-bed reactor (FBR)-based sulfate reducing bioprocess was investigated for the treatment of iron-containing (40-90 mg/L) acidic wastewater at low (8 degrees C) and high (65 degrees C) temperatures. The FBRs operated at low and high temperatures were inoculated with cultures of sulfate-reducing bacteria (SRB) originally enriched from arctic and hot mining environments, respectively. Ethanol was supplemented as carbon and electron source for SRB. At 8 degrees C, ethanol oxidation and sulfate reduction rates increased steadily and reached 320 and 265 mg/L.day, respectively, after 1 month of operation. After this point, the rates did not change significantly during 130 days of operation. Despite the complete ethanol oxidation and iron precipitation, the average sulfate reduction efficiency was 35 +/- 4% between days 30 and 130 due to the accumulation of acetate. At 65 degrees C, a rapid startup was observed as 99.9, 46, and 29% ethanol, sulfate, acetate removals, in respective order, were observed after 6 days. The feed pH was decreased gradually from its initial value of 6 to around 3.7 during 100 days of operation. The wastewater pH of 4.3-4.4 was neutralized by the alkalinity produced in acetate oxidation and the average effluent pH was 7.8 +/- 0.8. As in the low temperature FBR, acetate accumulated. Hence, the oxidation of acetate is the rate-limiting step in the sulfidogenic ethanol oxidation by thermophilic and psychrotrophic SRB. The sulfate reduction rate is three times and acetate oxidation rate is four times higher at 65 degrees C than at 8 degrees C.     

Neural network prediction of thermophilic (65 degrees C) sulfidogenic fluidized-bed reactor performance for the treatment of metal-containing wastewater

The performance of a fluidized-bed reactor (FBR) based sulfate reducing bioprocess was predicted using artificial neural network (ANN). The FBR was operated at high (65 degrees C) temperature and it was fed with iron (40-90 mg/L) and sulfate (1,000-1,500 mg/L) containing acidic (pH = 3.5-6) synthetic wastewater. Ethanol was supplemented as carbon and electron source for sulfate reducing bacteria (SRB). The wastewater pH of 4.3-4.4 was neutralized by the alkalinity produced in acetate oxidation and the average effluent pH was 7.8 +/- 0.8. The oxidation of acetate is the rate-limiting step in the sulfidogenic ethanol oxidation by thermophilic SRB, which resulted in acetate accumulation. Sulfate reduction and acetate oxidation rates showed variation depending on the operational conditions with the maximum rates of 1 g/L/d (0.2 g/g volatile solids (VS)/d) and 0.3 g/L/d (0.06 g/g VS/d), respectively. This study presents an ANN model predicting the performance of the reactor and determining the optimal architecture of this model; such as best back-propagation (BP) algorithm and neuron numbers. The Levenberg-Marquardt algorithm was selected as the best of 12 BP algorithms and optimal neuron number was determined as 20. The developed ANN model predicted acetate (R=0.91), sulfate (R=0.95), sulfide (R=0.97), and alkalinity (R=0.94) in the FBR effluent. Hence, the ANN based model can be used to predict the FBR performance, to control the operational conditions for improved process performance.     

固定载体卧式厌氧反应器处理糖蜜废水的快速启动

为高效处理高浓度有机废水而设计了固定载体卧式厌氧反应器R1和R2, 它是厌氧折流板反应器(ABR)的改进, 以活性炭纤维作为生物膜载体固定并充当反应器的折流板, 在实验室规模上对R1和R2处理糖蜜废水进行快速启动运行。HRT和ORL是影响R1和R2稳定高效运行及启动的2个重要工艺参数。实验证明: HRT为2 d时, 反应器运行最佳。在第30天时, R1的COD去除率达到84.88%, R2达到81.72%。随着进水ORL由1.25 kg/(m3·d)提升到10 kg/(m3·d), 沼气容积产气率由0.35 L/(L·d)逐渐增加到4.98 L/(L·d)。进水pH值为3.9?4.5之间, 整个启动运行过程中, 未调节pH值, R1和R2的出水pH值均在6.7?7.6之间, 2个反应器均有较强的抗酸能力, R1的pH波动更为平缓。在整个实验过程中, 污泥流失量小, 没有发生堵塞现象, 在处理酸性高浓度有机废水时, 2个反应器均表现出较强的抗负荷冲击能力。     

Effects of alternative carbon sources on biological transformation of nitrophenols

The removal of nitrophenols under denitrifying conditions was studied in bench-scale upflow anaerobic sludge blanket (UASB) reactors (R1, R2, R3 and R4) using three different carbon sources. Initially acetate was used as carbon source (substrate) in all the four reactors followed by glucose and methanol. Reactor R1 was kept as control and R2, R3, R4 were fed with 30 mg/l concentration of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively. Throughout the study the hydraulic retention time (HRT) and COD/NO₃ ^-–N ratio were kept as 24 h and 10, respectively. 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found as the major intermediate metabolites of 2-NP, 4-NP and 2,4-DNP degradation, respectively. Methanol was found to be a better carbon source for 4-NP and 2,4-DNP degradation as compared to acetate and glucose, while 2-NP degradation was not influenced much by the change of substrate. Nitrate nitrogen removal was always more than 99%. COD removal efficiency of the nitrophenol fed reactors varied from 85.7% to 97.7%. The oxidation-reduction potential (ORP) inside the reactors dropped, up to –300 mv, with glucose as carbon source. As the reactors were switched over to methanol, ORP increased to –190 mv. The granular sludge developed inside the reactors was light brown in colour when acetate and glucose were used as substrate, which turned dark brown to black at the end of methanol run. Biomass yield in terms of volatile suspended solids was observed as 0.15, 0.089 and 0.14 g per gram of COD removal for acetate, glucose and methanol, respectively.     

Enhancing denitrification using a carbon supplement generated from the wet oxidation of waste activated sludge

This study compared the effect of four pure carbon supplements on biological denitrification to a liquor derived as a by-product from the wet oxidation (WO) of waste activated sludge. Sequencing batch reactors were used to acclimate sludge biomass, which was used in batch assays. Acetate, WO liquor and ethanol-supplementation generated the fastest denitrification rates. Acetate and WO liquor were efficiently utilised by all acclimated biomass types, while poor rates were achieved with methanol and formate. When comparing an inoculum from an ethanol-supplemented and non-supplemented wastewater treatment plant (WWTP), the ethanol-acclimated sludge obtained superior denitrification rates when supplemented with ethanol. Similarly high nitrate removal rates were achieved with both sludge types with acetate and WO liquor supplementation, indicating that WO liquors could achieve excellent rates of nitrate removal. The performance of the WO liquor was attributed to the variety of organic carbon substrates (particularly acetic acid) present within the liquor.     

Isolation of saccharolytic dissimilatory sulfate-reducing bacteria

Abstract Four unidentified saccharolytic dissimilatory sulfate-reducing strains were isolated from an anaerobic digester. Cells were Gram-negative, motile, nonsporulating rods which differ markedly from known sulfate reducers especially with respect to carbon source utilisation and sulfur sources which can be reduced. The strains were capable of metabolising at least 26 out of 50 carbohydrates tested. Carbohydrates were, in the absence of exogenous sulfate, fermented to acetate, ethanol, lactate, carbon dioxide and hydrogen. In the presence of excess sulfate carbohydrates were fermented to acetate, ethanol, carbon dioxide, hydrogen and hydrogen sulfide, but lactate was not detected. An oxidized organic or inorganic sulfur source, including elemental sulfur, was not required as a prerequisite for growth on carbohydrates, Lactate was, in the presence of sulfate, converted to acetate, ethanol, carbon dioxide, hydrogen and hydrogen sulfide. In the absence of sulfate no lactate was utilised and no growth was observed.     

High-rate sulfate reduction at high salinity (up to 90 mS.cm(-1)) in mesophilic UASB reactors

Sulfate reduction in salt-rich wastewaters using unadapted granular sludge was investigated in 0.9 L UASB reactors (pH 7.0 +/- 0.2; hydraulic retention time from 8-14 h) fed with acetate, propionate, or ethanol at organic loading rates up to 10 gCOD x L(-1) x day(-1) and in excess sulfate (COD/SO(4) (2-) of 0.5). High-rate sulfate reduction rates (up to 3.7 gSO(4) (2-) x L(-1).day(-1)) were achieved at salinities exceeding 50 gNaCl.L(-1) and 1 gMgCl(2) x L(-1). Sulfate reduction proceeded at a salinity of up to 70 gNaCl x L(-1) and 1 gMgCl(2) x L(-1) (corresponding to a conductivity of about 85-90 mS x cm(-1)), although at lower rates compared to a conductivity of 60-70 mS x cm(-1). Ethanol as well as propionate were suitable substrates for sulfate reduction, with acetate and sulfide as the end products. The successful high-rate treatment was due to the proliferation of a halotolerant incomplete oxidizing SRB population present in the unadapted inoculum sludge. Bioaugmentation of this sludge with the acetate oxidizing halotolerant SRB Desulfobacter halotolerans was unsuccessful, as the strain washed out from the UASB reactor without colonizing the UASB granules.     

Microbial sulfate reduction at low (8 °C) temperature using waste sludge as a carbon and seed source

Biological treatment of sulfate and metal-containing wastewater (such as acid mine drainage) is a viable option due to lower cost and better sludge quality compared to conventional chemical treatment. Although several substrates can be used as carbon source, a low-cost substrate is required for large scale applications. This study was conducted to investigate the suitability of waste sludge as a carbon and seed source for sulfate reduction at 8 °C in batch bioassays. Around 7 mmol of sulfate was reduced when the waste sludge mixture (WS) (6700 mg SS l^?1) from primary and secondary settling tank was supplemented as a carbon and seed source. However, only 1.6 mmol of sulfate was reduced with anaerobic digester effluent (ADS) (5300 mg SS l^?1). The produced H₂S from 1 g VSS l^?1 WS and ADS oxidation can theoretically precipitate around 90 and 35 mg Fe²⁺, respectively. Both WS and ADS oxidized ethanol to acetate at similar rates. It appears that WS is a good candidate for carbon and start-up seed source of sulfate reduction at 8 °C, whereas sulfidogenic acetate oxidation was the limiting step. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that both sludge sources contain Desulfomicrobium apsheronum strain.     

Chemical Characterisation of Organic Electron Donors For Sulfate Reduction For Potential Use in Acid Mine Drainage Treatment

The production of acid mine drainage (AMD) containing high amounts of sulfate, heavy metals and low pH is of increasing concern. AMD is highly corrosive and results in economic and environmental problems. Organic electron donors for sulfate reduction were chemically characterised for potential use in AMD treatment. This was done in a process to develop a correlation between chemical composition and the capacity to drive sulfate reduction. Potential organic electron donors for sulfate reduction were chemically characterised in terms of dry matter content, ash content, total Kjeldahl nitrogen, lignin content, cellulose content, crude fat, crude fibre, in vitro digestibility, water-soluble carbohydrates, total non-structural carbohydrates and starch content. The chemical composition of the organic electron donors was then compared to results obtained from pilot plant studies where the organic electron donors for sulfate reduction were evaluated in terms of sulfate reduction. The chemical composition of the carbon source severely impacted its capacity to drive sulfate reduction and may be used to assist in predicting the sulfate reduction capacity of a carbon source. Organic electron donors for sulfate reduction high in protein content and low in lignin content or high in carbohydrate and crude fat content increased the capacity of a carbon source to drive sulfate reduction. The higher the fibre content of a carbon source, the lower the capacity to drive sulfate reduction. No correlation could be drawn between % dry matter, % ash content and sulfate reduction for the organic electron donors tested. Chemical characterisation can be used to assist in predicting sulfate reduction capacity of organic electron donors.     

Effect of organic carbon shock loading on endogenous denitrification in sequential batch reactors

This work was focused on the performance evaluation of sequential batch reactors (SBR) treating sewage, through a process of endogenous biological denitrification. Different operational conditions were carried out, and the behaviour under the effects of organic shock loading was examined. Three laboratory scale reactors were operated simultaneously and fed with similar wastewater. The substratum was molasses and nitrate, as carbon and nitrogen sources, respectively. The three reactors were operated during different aeration periods (0, 15 and 30 min). Sudden changes (shock loading) in organic matter concentration were performed during the experiment. Thus, influent load was quickly increased threefold in relation to the original concentration. Results indicated that SBR reactors withstand adequately moderate shock loading. With regard to substratum degradation, nitrate elimination achieved was approximately 80%, while denitrification rate was approximately 0.87 mgg(-1)h(-1).     

Influence of pH shocks on trace metal dynamics and performance of methanol fed granular sludge bioreactors

The influence of pH shocks on the trace metal dynamics and performance of methanol fed upflow anaerobic granular sludge bed (UASB) reactors was investigated. For this purpose, two UASB reactors were operated with metal pre-loaded granular sludge (1mM Co, Ni and Fe; 30°C; 96h) at an organic loading rate (OLR) of 5gCOD l reactor^–1d^–1. One UASB reactor (R1) was inoculated with sludge that originated from a full scale reactor treating alcohol distillery wastewater, while the other reactor (R2) was inoculated with sludge from a full scale reactor treating paper mill wastewater. A 30h pH shock (pH 5) strongly affected the metal retention dynamics within the granular sludge bed in both reactors. Iron losses in soluble form with the effluent were considerable: 2.3 and 2.9% for R1 and R2, respectively, based on initial iron content in the reactors, while losses of cobalt and nickel in soluble form were limited. Sequential extraction of the metals from the sludge showed that cobalt, nickel, iron and sulfur were translocated from the residual to the organic/sulfide fraction during the pH shock in R2, increasing 34, 47, 109 and 41% in the organic/sulfide fraction, respectively. This is likely due to the modification of the iron sulfide precipitate stability, which influences the extractability of iron and trace metals. Such a translocation was not observed for the R1 sludge during the first 30h pH shock, but a second 4day pH shock induced significant losses of cobalt (18%), iron (29%) and sulfur (29%) from the organic/sulfide fraction, likely due to iron sulfide dissolution and concomitant release of cobalt. After the 30h pH shock, VFA accumulated in the R2 effluent, whereas both VFA and methanol accumulated in R1 after the 4day pH shock. The formed VFA, mainly acetate, were not converted to methane due to the loss of methanogenic activity of the sludge on acetate. The VFA accumulation gradually disappeared, which is likely to be related to out-competition of acetogens by methanogens. Zinc, copper and manganese supply did not have a clear effect on the acetate removal and methanol conversion, but zinc may have induced the onset of methanol degradation after day 152 in R1.     

Low pH (6, 5 and 4) sulfate reduction during the acidification of sucrose under thermophilic (55 °C) conditions

The effect of a low pH (6, 5 and 4) and different COD/SO₄²⁻ ratios (9 and 3.5) on thermophilic (55 °C) sulfate reduction and acidification of sucrose was investigated using three upflow anaerobic sludge bed reactors fed with sucrose at an organic loading rate of 3.5 gCOD (l_reactor d)⁻¹. The three reactors showed nearly 100% acidification of sucrose for all pH values and COD/SO₄²⁻ ratios investigated. Sulfate reduction was complete at pH 6 and a COD/SO₄²⁻ ratio of 9. At pH 5, sulfate reduction efficiencies were 80–95% for both COD/SO₄²⁻ ratios (9 and 3.5). At pH 4, sulfate reduction efficiencies further dropped to 55–65% at a COD/SO₄²⁻ ratio of 9 and 30–40% at a COD/SO₄²⁻ ratio of 3.5. The pH decrease from 6 to 5 or 4 caused a shift in the acidification products from mainly acetate to butyrate, as well as a higher production of ethanol, especially at pH 4. At pH 4, propionate and methane were not formed and hydrogen concentrations in the biogas reached 50%, equivalent to a hydrogen yield of 1.3 mol H₂ (mol glucose)⁻¹. This study shows that sulfate reduction is possible in the acidification phase of anaerobic wastewater treatment at pH values as low as 6 till 4 and that the pH strongly affects both the acidification pathways and the sulfate reduction efficiencies.     

Thermophilic (55 degrees C) conversion of methanol in methanogenic-UASB reactors: influence of sulphate on methanol degradation and competition

Two upflow sludge bed reactors (UASB) were operated for 80 days at 55 degrees C with methanol as the substrate with an organic loading rate (OLR) of about 20 g CODl(-1) per day and a hydraulic retention time (HRT) of 10 h. One UASB was operated without sulphate addition (control reactor-R1) whereas the second was fed with sulphate at a COD:SO4(2-) ratio of 10 (sulphate-fed reactor-R2), providing an influent sulphate concentration of 0.6 g l(-1). For both reactors, methanogenesis was the dominant process with no considerable accumulation of acetate. The methanol removal averaged 93% and 83% for R1 and R2, respectively, and total sulphate removal was achieved in the latter. The pathway of methanol conversion for both sludges was investigated by measuring the fate of carbon in the presence and absence of bicarbonate or specific inhibitors for a sludge sample collected at day 72. In both sludges, about 70% of the methanol was syntrophically converted to methane and/or sulphide, via the intermediate H2/CO2. A strong competition between methanogens and sulphidogens took place in the R2 sludge with half of the methanol-COD being used by methane-producing bacteria and the other half by sulphate-reducing bacteria. Acetate was not an important intermediate for both sludges, and played a slightly more important role for the sulphate-adapted sludge (R2), sustained by the higher amount of bicarbonate produced during sulphate-reduction. The pathway study indicates that, although acetate does not represent an important intermediate, the system is susceptible to its accumulation.     

Bioremediation of synthetic high–chemical oxygen demand wastewater using microalgal species Chlorella pyrenoidosa

The microalgal species Chlorella pyrenoidosa was cultivated in synthetic wastewater of initial chemical oxygen demand (COD), nitrate, and phosphate concentrations of 5000, 100, and 40 mg/L, respectively. The aim of the study was to find out the tolerance of microalgae to different COD concentrations and the extent of COD degradation at those concentrations. Three dilutions of wastewater (initial COD concentrations 5000, 3000, and 1000 mg/L) and three inoculum sizes (0.1, 0.2, and 0.3 g/L) were considered for the study. The experimental parameters such as total organic carbon, total inorganic carbon, COD, optical density, total solids, nitrate, and phosphate were measured on a daily basis. Biodegradation kinetics was determined for all cases using first-order reaction and Monod degradation equations. Optimal results showed that up to 90% reduction in TOC was obtained for 1000 COD wastewater while only 38% reduction in total organic carbon (TOC) was achieved for 5000 COD wastewater. Over 95% reduction in nitrate and nearly 90% removal of phosphate were obtained with the lowest microalgal inoculum concentration (i.e., 0.1 g/L) for all COD dilutions. This study showed that microalgal species C. pyrenoidosa can successfully degrade the organic carbon source (i.e., acetate) with significant removal efficiencies for nitrate and phosphate.     

Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysis

AIMS: The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. METHODS AND RESULTS: Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37 degrees C) treatment of high-strength [10 g chemical oxygen demand (COD) l(-1)] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum- and Methanococcus-like organisms and uncultured Crenarchaeota in R1. CONCLUSIONS: The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities.     

Desulforhabdus amnigenus gen. nov. sp. nov., a sulfate reducer isolated from anaerobic granular sludge

From granular sludge of an upflow anaerobic sludge bed (UASB) reactor treating paper-mill wastewater, a sulfate-reducing bacterium (strain ASRB1) was isolated with acetate as sole carbon and energy source. The bacterium was rod-shaped, (1.4–1.9×2.5–3.4 μm), nonmotile, and gram-negative. Optimum growth with acetate occurred around 37°C in freshwater medium (doubling time: 3.5–5.0 days). The bacterium grew on a range of organic acids, such as acetate, propionate, and butyrate, and on alcohols, and grew autotrophically with H₂, CO₂ and sulfate. Fastest growth occurred with formate, propionate, and ethanol (doubling time: approx. 1.5 days). Strain ASRB1 clusters with the delta subdivision of Proteobacteria and is closely related toSyntrophobacter wolinii a syntrophic propionate oxidizer. Strain ASRB1 was characterized as a new genus and species:Desulforhabdus amnigenus.     

Consortium diversity of a sulfate‐reducing biofilm developed at acidic pH influent conditions in a down‐flow fluidized bed reactor

Sulfate reduction is an appropriate approach for the treatment of effluents with sulfate and dissolved metals. In sulfate‐reducing reactors, acetate may largely contribute to the residual organic matter, because not all sulfate reducers are able to couple the oxidation of acetate to the reduction of sulfate, limiting the treatment efficiency. In this study, we investigated the diversity of a bacterial community in the biofilm of a laboratory scale down‐flow fluidized bed reactor, which was developed under sulfidogenic conditions at an influent pH between 4 and 6. The sequence analysis of the microbial community showed that the 16S rRNA gene sequence of almost 50% of the clones had a high similarity with Anaerolineaceae. At second place, 33% of the 16S rRNA phylotypes were affiliated with the sulfate‐reducing bacteria Desulfobacca acetoxidans and Desulfatirhabdium butyrativorans, suggesting that acetotrophic sulfate reduction was occurring in the system. The remaining bacterial phylotypes were related to fermenting bacteria found at the advanced stage of reactor operation. The results indicate that the acetotrophic sulfate‐reducing bacteria were able to remain within the biofilm, which is a significant result because few natural consortia harbor complete oxidizing sulfate‐reducers, improving the acetate removal via sulfate reduction in the reactor.     

A comparative study of methanol as a supplementary carbon source for enhancing denitrification in primary and secondary anoxic zones

A comparative study on the use of methanol as a supplementary carbon source to enhance denitrification in primary and secondary anoxic zones is reported. Three lab-scale sequencing batch reactors (SBR) were operated to achieve nitrogen and carbon removal from domestic wastewater. Methanol was added to the primary anoxic period of the first SBR, and to the secondary anoxic period of the second SBR. No methanol was added to the third SBR, which served as a control. The extent of improvement on the denitrification performance was found to be dependent on the reactor configuration. Addition to the secondary anoxic period is more effective when very low effluent nitrate levels are to be achieved and hence requires a relatively large amount of methanol. Adding a small amount of methanol to the secondary anoxic period may cause nitrite accumulation, which does not improve overall nitrogen removal. In the latter case, methanol should be added to the primary anoxic period. The addition of methanol can also improve biological phosphorus removal by creating anaerobic conditions and increasing the availability of organic carbon in wastewater for polyphosphate accumulating organisms. This potentially provides a cost-effective approach to phosphorus removal from wastewater with a low carbon content. New fluorescence in situ hybridisation (FISH) probes targeting methanol-utilising denitrifiers were designed using stable isotope probing. Microbial structure analysis of the sludges using the new and existing FISH probes clearly showed that the addition of methanol stimulated the growth of specific methanol-utilizing denitrifiers, which improved the capability of sludge to use methanol and ethanol for denitrification, but reduced its capability to use wastewater COD for denitrification. Unlike acetate, long-term application of methanol has no negative impact on the settling properties of the sludge.     

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria.

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.