Anaerobic sludge granulation.

This paper reviews different theories on anaerobic sludge granulation in UASB-reactors that have been proposed during the past two decades. The initial stages of the formation of anaerobic granules follow the same principles as biofilm formation of bacteria on solid surfaces. There exist strong evidence that inert carriers play an important positive role in granulation. Most researchers conclude that Methanosaeta concilii is a key organism in granulation. Only the Cape Town Hypothesis presumes that an autotrophic hydrogenotrophic organism, i.e., Methanobacterium strain AZ, growing under conditions of high H(2)-pressures, is the key organism in granulation. Many authors focus on the initial stage of granulation, and only a few contributions discuss the latter stages in granulation: granule maturation and multiplication. Granule enhancing factors in the latter stages predominantly rely on manipulation of the selection pressure, through which selectively heavier sludge particles are retained in the UASB reactor.

Effect of NaCl on thermophilic (55 degrees C) methanol degradation in sulfate reducing granular sludge reactors.

The effect of NaCl on thermophilic (55 degrees C) methanol conversion in the presence of excess of sulfate (COD/SO(4)(2-)=0.5) was investigated in two 6.5L lab-scale upflow anaerobic sludge bed reactors inoculated with granular sludge previously not adapted to NaCl. Methanol was almost completely used for sulfate reduction in the absence of NaCl when operating at an organic loading rate of 5 g CODL(-1)day(-1) and a hydraulic retention time of 10h. The almost fully sulfidogenic sludge consisted of both granules and flocs developed after approximately 100 days in both reactors. Sulfate reducing bacteria (SRB) outcompeted methane producing archaea (MPA) for methanol, but acetate represented a side-product, accounting for maximal 25% of the total COD converted. Either MPA or SRB did not use acetate as substrate in activity tests. High NaCl concentrations (25 gL(-1)) completely inhibited methanol degradation, whereas low salt concentrations (2.5 g NaClL(-1)) provoked considerable changes in the metabolic fate of methanol. The MPA were most sensitive towards the NaCl shock (25 gL(-1)). In contrast, the addition of 2.5 gL(-1) of NaCl stimulated MPA and homoacetogenic bacteria.

Effect of high salinity on the fate of methanol during the start-up of thermophilic (55 degrees C) sulfate reducing reactors.

Two 6.5 L lab-scale upflow anaerobic sludge bed (UASB) reactors were operated at 55 degrees C fed with methanol as the sole electron and carbon source and in excess of sulfate (COD/SO4(2-) of 0.5) in order to investigate the effect of high wastewater salinity on the start-up period. The first reactor (UASB I) was operated without NaCl addition, while the second reactor (UASB II) was fed with 25 g x L(-1) of NaCl in the first 13 days of operation. Successful start-up of UASB I was achieved, with full methanol conversion (100% elimination) to methane gas (methane production rate up to 3.66 gCOD.L(-1).day(-1)). Despite the detection of sulfide from day 15 onwards in UASB I, methane was the main mineralization product when operating at an organic loading rate (OLR) of 5 gCOD.L(-1).day(-1) and a hydraulic retention time (HRT) of 10 hours. Sulfide and acetate started to be produced after salt omission from the influent in UASB II at day 13, with no detection of methane. Acetate was the main product when operating at an OLR of 10 gCOD.L(-1).day(-1) and HRT of 6.5 hours in both reactors. Apparently, the methane producing bacteria (MPB) are the trophic group most sensible to the NaCl shock.

Competition for H2 between sulfate reducers, methanogens and homoacetogens in a gas-lift reactor.

Reported values for growth kinetic parameters show an order in competitivity of heterotrophic sulfate reducing bacteria>methanogens>homoacetogens for the substrate hydrogen. This order suggests that methanogens can succesfully compete with consortia of heterotrophic SRB and homoacetogens when H2/CO2 is present as sole substrate. However, we found in experiments using gas-lift reactors inoculated with anaerobic sludge and fed with H2/CO2 and sulfate, that heterotrophic sulfate reduction rapidly and completely outcompeted methanogenesis, whereas a low amount of acetate was formed. Thus, in disagreement with the above competitivity order, hydrogen is more readily consumed by homoacetogenesis than by methanogenesis, indicating that the competition is not kinetically determined. The superior settling velocity of sulfidogenic-acetogenic sludge compared to that of methanogenic sludge suggests that the former sludge is better retained, which can explain the predominance of sulfate reduction/homoacetogenesis over methanogenesis.

Effect of the liquid upflow velocity on thermophilic sulphate reduction in acidifying granular sludge reactors.

The effect of the superficial liquid upflow velocity on the acidifying and sulfate reducing capacity of thermophilic (55 degrees C; pH 6.0) granular sludge bed reactors treating partly acidified wastewater was investigated. A comparison was made between a UASB and an EGSB reactor, operated at an upflow velocity of 1 m.h-1 and 6.8 m.h-1, respectively. Both reactors were inoculated with a mixture of mesophilic sulphidogenic, thermophilic sulphidogenic and thermophilic methanogenic sludge (ratio 2:1:1). They were fed a synthetic wastewater containing starch, sucrose, lactate, propionate and acetate and a low sulphate concentration (COD/SO4(2-) ratio of 10). At the end of the experiment, the sulphate level of the influent was slightly increased to a COD/SO4(2-) ratio of 8. The reactors were operated at a hydraulic retention time of about 5 h and the imposed volumetric organic loading rates (OLR) ranged from 4.9 to 40.0 g COD l-1d-1. When imposing an OLR of 40.0 g COD l-1d-1, the acidification efficiency dropped to 80% and the sulphate reduction efficiency decreased to 50% in the UASB reactor. In the EGSB reactor, the sulphate reduction efficiency dropped to 30% directly following the OLR increase to 40 g COD l-1d-1, but recovered rapidly to 100% (at an OLR of 35 g COD l-1d-1) until the end of the experiment. In the UASB reactor, there was a net acetate and propionate production. At the higher organic loading rates, propionate was converted to n-butyrate and n-valerate. These back reactions did not occur in the EGSB reactor, in which an active methanogenic population developed, leading to a net acetate removal (up to 50%) and a high gas loading rate (up to 8.5 l l-1d-1). In both reactors, the effluent sulphide concentration was always below 200 mg l-1, of which about 90% was present as undissociated H2S (under the given conditions--pH 5.8-6.1 and 55 degrees C). The biogas (including CH4 and CO2) production rates in the UASB were very low, i.e. < 31 biogas l-1 reactor d-1, resulting in negligible amounts (< 20%) of H2S stripped from the reactor liquid. In the EGSB reactor, the biogas production rates reached up to 8.5 l l-1d-1, resulting in H2S stripping efficiencies up to 75%.

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor.

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.

Performance of a thermophilic sulfate and sulfite reducing high rate anaerobic reactor fed with methanol.

Thermophilic sulfate and sulfite reduction was studied in lab-scale Expanded Granular Sludge Bed (EGSB) reactors operated at 65 degrees C and pH 7.5 with methanol as the sole carbon and energy source for the sulfate- and sulfite-reducing bacteria. At a hydraulic retention time (HRT) of 10 h, maximum sulfite and sulfate elimination rates of 5.5 g SO3(2-) L(-1) day(-1) (100% elimination) and 5.7 g SO4(2-) L(-1) day(-1) (55% elimination) were achieved, resulting in an effluent sulfide concentration of approximately 1800 mg S L(-1). Sulfate elimination was limited by the sulfide concentration, as stripping of H2S from the reactor with nitrogen gas was found to increase the sulfate elimination rate to 9.9 g SO4(2-) L(-1) day(-1) (100% elimination). At a HRT of 3 h, maximum achievable sulfite and sulfate elimination rates were even 18 g SO3(2-) L(-1) day(-1) (100% elimination) and 11 g SO4(2-) L(-1) day(-1) (50% elimination). At a HRT of 3 h, the elimination rate was limited by the biomass retention of the system. 5.5 +/- 1.8% of the consumed methanol was converted to acetate, which was not further degraded by sulfate reducing bacteria present in the sludge. The acetotrophic activity of the sludge could not be stimulated by cultivating the sludge for 30 days under methanol-limiting conditions. Omitting cobalt as trace element from the influent resulted in a lower acetate production rate, but it also led to a lower sulfate reduction rate. Sulfate degradation in the reactor could be described by zeroth order kinetics down to a threshold concentration of 0.05 g L(-1), while methanol degradation followed Michaelis-Menten kinetics with a Km of 0.037 g COD L(-1).

High-rate treatment of terephthalate in anaerobic hybrid reactors.

The anaerobic degradation of terephthalate as sole substrate was studied in three anaerobic upflow reactors. Initially, the reactors were operated as upflow anaerobic sludge bed (UASB) reactors and seeded with suspended methanogenic biomass obtained from a full-scale down-flow fixed film reactor, treating wastewater generated during production of purified terephthalic acid. The reactors were operated at 30, 37, and 55 degrees C. The terephthalate removal capacities remained low in all three reactors (<4 mmolxL-1xday-1, or 1 g of chemical oxygen demand (COD)xL-1xday-1) due to limitations in biomass retention. Batch experiments with biomass from the UASB reactors revealed that, within the mesophilic temperature range, optimal terephthalate degradation is obtained at 37 degrees C. No thermophilic terephthalate-degrading culture could be obtained in either continuous or batch cultures. To enhance biomass retention, the reactors were modified to anaerobic hybrid reactors by introduction of two types of reticulated polyurethane (PUR) foam particles. The hybrid reactors were operated at 37 degrees C and seeded with a mixture of biomass from the UASB reactors operated at 30 and 37 degrees C. After a lag period of approximately 80 days, the terephthalate conversion capacity of the hybrid reactors increased exponentially at a specific rate of approximately 0.06 day-1, and high removal rates were obtained (40-70 mmolxL-1xday-1, or 10-17 g of CODxL-1xday-1) at hydraulic retention times between 5 and 8 h. These high removal capacities could be attributed to enhanced biomass retention by the development of biofilms on the PUR carrier material as well as the formation of granular biomass. Biomass balances over the hybrid reactors suggested that either bacterial decay or selective wash-out of the terephthalate fermenting biomass played an important role in the capacity limitations of the systems. The presented results suggest that terephthalate can be degraded at high volumetric rates if sufficiently long sludge ages can be maintained, and the reactor pH and temperature are close to their optima.

Anaerobic degradation of phthalate isomers by methanogenic consortia.

Three methanogenic enrichment cultures, grown on ortho-phthalate, iso-phthalate, or terephthalate were obtained from digested sewage sludge or methanogenic granular sludge. Cultures grown on one of the phthalate isomers were not capable of degrading the other phthalate isomers. All three cultures had the ability to degrade benzoate. Maximum specific growth rates (microseconds max) and biomass yields (YXtotS) of the mixed cultures were determined by using both the phthalate isomers and benzoate as substrates. Comparable values for these parameters were found for all three cultures. Values for microseconds max and YXtotS were higher for growth on benzoate compared to the phthalate isomers. Based on measured and estimated values for the microbial yield of the methanogens in the mixed culture, specific yields for the phthalate and benzoate fermenting organisms were calculated. A kinetic model, involving three microbial species, was developed to predict intermediate acetate and hydrogen accumulation and the final production of methane. Values for the ratio of the concentrations of methanogenic organisms, versus the phthalate isomer and benzoate fermenting organisms, and apparent half-saturation constants (KS) for the methanogens were calculated. By using this combination of measured and estimated parameter values, a reasonable description of intermediate accumulation and methane formation was obtained, with the initial concentration of phthalate fermenting organisms being the only variable. The energetic efficiency for growth of the fermenting organisms on the phthalate isomers was calculated to be significantly smaller than for growth on benzoate.

The role of benzoate in anaerobic degradation of terephthalate.

The effects of acetate, benzoate, and periods without substrate on the anaerobic degradation of terephthalate (1, 4-benzene-dicarboxylate) by a syntrophic methanogenic culture were studied. The culture had been enriched on terephthalate and was capable of benzoate degradation without a lag phase. When incubated with a mixture of benzoate and terephthalate, subsequent degradation with preference for benzoate was observed. Both benzoate and acetate inhibited the anaerobic degradation of terephthalate. The observed inhibition is partially irreversible, resulting in a decrease (or even a complete loss) of the terephthalate-degrading activity after complete degradation of benzoate or acetate. Irreversible inhibition was characteristic for terephthalate degradation only because the inhibition of benzoate degradation by acetate could well be described by reversible noncompetitive product inhibition. Terephthalate degradation was furthermore irreversibly inhibited by periods without substrate of only a few hours. The inhibition of terephthalate degradation due to periods without substrate could be overcome through incubation of the culture with a mixture of benzoate and terephthalate. In this case no influence of a period without substrate was observed. Based on these observations it is postulated that decarboxylation of terephthalate, resulting in the formation of benzoate, is strictly dependent on the concomitant fermentation of benzoate. In the presence of higher concentrations of benzoate, however, benzoate is the favored substrate over terephthalate, and the culture loses its ability to degrade terephthalate. In order to overcome the inhibition of terephthalate degradation by benzoate and acetate, a two-stage reactor system is suggested for the treatment of wastewater generated during terephthalic acid production.

Long-term competition between sulfate reducing and methanogenic bacteria in UASB reactors treating volatile fatty acids.

The competition between acetate utilizing methane-producing bacteria (MB) and sulfate-reducing bacteria (SRB) was studied in mesophilic (30 degrees C) upflow anaerobic sludge bed (UASB) reactors (upward velocity 1 m h-1; pH 8) treating volatile fatty acids and sulfate. The UASB reactors treated a VFA mixture (with an acetate:propionate:butyrate ratio of 5:3:2 on COD basis) or acetate as the sole substrate at different COD:sulfate ratios. The outcome of the competition was evaluated in terms of conversion rates and specific methanogenic and sulfidogenic activities. The COD:sulfate ratio was a key factor in the partitioning of acetate utilization between MB and SRB. In excess of sulfate (COD:sulfate ratio lower than 0.67), SRB became predominant over MB after prolonged reactor operation: 250 and 400 days were required to increase the amount of acetate used by SRB from 50 to 90% in the reactor treating, respectively, the VFA mixture or acetate as the sole substrate. The competition for acetate was further studied by dynamic simulations using a mathematical model based on the Monod kinetic parameters of acetate utilizing SRB and MB. The simulations confirmed the long term nature of the competition between these acetotrophs. A high reactor pH (+/-8), a short solid retention time (<150 days), and the presence of a substantial SRB population in the inoculum may considerably reduce the time required for acetate-utilising SRB to outcompete MB.

Anaerobic treatment of sulphate-rich wastewaters.

Until recently, biological treatment of sulphate-rich wastewater was rather unpopular because of the production of H2S under anaerobic conditions. Gaseous and dissolved sulphides cause physical-chemical (corrosion, odour, increased effluent chemical oxygen demand) or biological (toxicity) constraints, which may lead to process failure. Anaerobic treatment of sulphate-rich wastewater can nevertheless be applied successfully provided a proper treatment strategy is selected. The strategies currently available are discussed in relation to the aim of the treatment: i) removal of organic matter, ii) removal of sulphate or iii) removal of both. Also a whole spectrum of new biotechnological applications (removal of organic chemical oxygen demand, sulphur, nitrogen and heavy metals), recently developed based on a better insight in sulphur transformations, are discussed.

Biological sulfate reduction using synthesis gas as energy and carbon source.

Biological sulfate reduction was studied in laboratory-scale gas-lift reactors. Synthesis gas (gas mixtures of H(2)/CO/CO(2)) was used as energy and carbon source. The required biomass retention was obtained by aggregation and immobilization on pumice particles. Special attention was paid to the effect of CO addition on the sulfate conversion rate, aggregation, and aggregate composition.Addition of 5% CO negatively affected the overall sulfate conversion rate; i.e., it dropped from 12-14 to 6-8 g SO(2-) (4)/L day. However, a further increase of CO to 10 and 20% did not further deteriorate the process. With external biomass recycling the sulfate conversion rate could be improved to 10 g SO(2-) (4)/L day. Therefore biomass retention clearly could be regarded as the rate-limiting step. Furthermore, CO affected the aggregate shape and diameter. Scanning electron microscopy (SEM) photographs showed that rough aggregates pregrown on H(2)/CO(2) changed into smooth aggregates upon addition of CO. Addition of CO also changed the aggregate Sauter mean diameter (d(32)) from 1.7 mm at 5% CO to 2.1 mm at 20% CO. After addition of CO, a layered biomass structure developed. Acetobacterium sp. were mainly located at the outside of the aggregates, whereas Desulfovibrio sp. were located inside the aggregates.