Effect of medium composition and sludge removal on the production, composition, and architecture of thermophilic (55 degrees C) acetate-utilizing granules from an upflow anaerobic sludge blanket reactor.

A thermophilic upflow anaerobic sludge blanket (UASB) reactor degrading acetate was started by applying published methods (W. M. Wiegant and A. W. A. de Man, Biotechnol. Bioeng. 28:718-77, 1986) for production of granules dominated by Methanothrix spp. The reactor was inoculated with thermophilic digested sludge. No granules were observed during the first 7 months of start-up of the UASB reactor. However, after the concentrations of potassium, phosphate, ammonium, and magnesium in the medium were gradually increased, granules developed, indicating that there was a critical concentration of one or more of the ions required for production of granules from the starting material. After several years of stable operation, the effect of removing 60% of the granular sludge was investigated. Immunologic qualitative and quantitative studies showed that removal of the granular sludge resulted in an increase in the number of the predominant methanogens, antigenically related to Methanosarcina thermophila TM-1 and Methanosarcina mazeii S-6, and Methanobacterium thermoautotrophicum delta H and GC1. These changes were accompanied by modifications of the microanatomy of the granules, as demonstrated histochemically and immunohistochemically. The results indicated that different catabolic pathways dominated in different regions of the granules, i.e., acetate oxidation in the middle of the granules, where there is a low acetate concentration, and an aceticlastic reaction in the outer surfaces, with a high acetate concentration. The results also showed that removal of granules from a UASB reactor which has been under steady-state operation for a long period can improve the reactor's performance via formation of denser and larger granules with improved microbial activities.     

Granular sludge formation in upflow anaerobic sludge blanket (UASB) reactors

The state of the art for upflow anaerobic sludge blanket (UASB) reactors is discussed, focusing on the microbiology of immobilized anaerobic bacteria and the mechanism of granule formation. The development of granular sludge is the key factor for successful operation of the UASB reactors. Criteria for determining if granular sludge has developed in a UASB reactor is given based on the densities and diameters of the granular sludge. The shape and composition of granular sludge can vary significantly. Granules typically have a spherical form with a diameter from 0.14 to 5 mm. The inorganic mineral content varies from 10 to 90% of the dry weight of the granules, depending on the wastewater composition etc. The main components of the ash are calcium, potassium, and iron. The extracellular polymers in the granular sludge are important for the structure and maintenance of granules, while the inorganic composition seems to be of less importance. The extracellular polymer content varies between 0.6 and 20% of the volatile suspended solids and consists mainly of protein and polysaccharides. Both Methanosaeta spp. (formerly Methanothrix) and Methanosarcina spp. have been identified as important aceticlastic methanogens for the initial granulation and development of granular sludge. Immunological methods have been used to identify other methanogens in the granules. The results have showed that, besides the aceticlastic methanogens Methanosaeta spp. and Methanosarcina spp., hydrogen and formate utilizing bacteria are also present, e.g., Methanobacterium formicicum, Methanobacterium thermoautotrophicum, and Methanobrevibacter spp. Microcolonies of syntrophic bacteria are often observed in the granules, and the significant electron transfer in these microcolonies occurs through interspecies hydrogen transfer. The internal organization of the various groups of bacteria in the granules depends on the wastewater composition and the dominating metabolic pathways in the granules. Internal organization is observed in granules where such an arrangement is beneficial for an optimal degradation of the wastewater. A four-step model is given for the initial development of granular sludge. (c) 1996 John Wiley & Sons, Inc.     

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.     

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.     

Acidification of methanol-fed anaerobic granular sludge bioreactors by cobalt deprivation: Induction and microbial community dynamics

The acidification of mesophilic (30 degrees C) methanol-fed upflow anaerobic sludge bed (UASB) reactors induced by cobalt deprivation from the influent was investigated by coupling the reactor performance (pH 7.0; organic loading rate 4.5 g COD . L(-1) . d(-1)) to the microbial ecology of the bioreactor sludge. The latter was investigated by specific methanogenic activity (SMA) measurements and fluorescence in situ hybridization (FISH) to quantify the abundance of key organisms over time. This study hypothesized that under cobalt limiting conditions, the SMA on methanol of the sludge gradually decreases, which ultimately results in methanol accumulation in the reactor effluent. Once the methanol accumulation surpasses a threshold value (about 8.5 mM for the sludge investigated), reactor acidification occurs because acetogens outcompete methylothrophic methanogens at these elevated methanol concentrations. Methanogens present in granular sludge at the time of the acidification do not use methanol as the direct substrate and are unable to degrade acetate. Methylotrophic/acetoclastic methanogenic activity was found to be lost within 10 days of reactor operation, coinciding with the disappearance of the Methanosarcina population. The loss of SMA on methanol can thus be used as an accurate parameter to predict reactor acidification of methanol-fed UASB reactors operating under cobalt limiting conditions.     

Filamentous granular sludge bulking in a laboratory scale UASB reactor

Filamentous bulking was observed in a lab scale upflow anaerobic sludge blanket (UASB) reactor. Granules failed to settle normally and disintegrated. The characteristics of the granules in structure and microbial composition during the granulation process were investigated by means of scanning electron microscopy (SEM) and denaturing gradient gel electrophoresis (DGGE) technique. Granules with high porosity instead of compact ones were developed in the reactor and Methanosaeta concilii and Methanobacterium formicicum were identified as the predominant methanogens present in granules. The excess growth of the filamentous bacteria could be the contributing factor causing floatation and disintegration.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/ COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

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.     

Reactivation of effluent granular sludge from a high-rate Anammox reactor after storage

In this study, effluent sludge from a high-rate Anammox reactor was used to re-start new Anammox reactors for the reactivation of Anammox granular sludge. Different start-up strategies were evaluated in six upflow anaerobic sludge blanket (UASB) reactors (R₁–R₆) for their effect on nitrogen removal performance. Maximal nitrogen removal rates (NRRs) greater than 20 kg N/m³/day were obtained in reactors R₃–R₅, which were seeded with mixed Anammox sludge previously stored for approximately 6 months and 1 month. A modified Boltzmann model describing the evolution of the NRR fit the experimental data well. An amount of sludge added to the UASB reactor or decreasing the loading rate proved effective in relieving the substrate inhibition and increasing the NRR. The modified Stover–Kincannon model fit the nitrogen removal data in the Anammox reactors well, and the simulation results showed that the Anammox process has great nitrogen removal potential. The observed inhibition in the Anammox reactors may have been caused by high levels of free ammonia. The sludge used to seed the reactors did not settle well; sludge flotation was observed even after the reactors were operated for a long time at a floating upward velocity (F_s) of greater than 100 m/h. The settling sludge, however, exhibited good settling properties. Scanning electron microscopy showed that the Anammox granules consisted mainly of spherical and elliptical bacteria with abundant filaments on their surface. Hollows in the granules were also present, which may have contributed to sludge floatation.     

Stimulation of methanol degradation in UASB reactors: in situ versus pre-loading cobalt on anaerobic granular sludge

The effect of pre-loading and in situ loading of cobalt onto a cobalt-limited granular sludge on the performance of methanol fed bioreactors was investigated. One upflow anaerobic sludge bed (UASB) reactor was inoculated with cobalt pre-loaded sludge (24h; 30 degrees C; 1 mM CoCl2) and a second UASB with unloaded sludge. The UASB reactors (30 degrees C; pH 7) were operated for 77 days at 8 h hydraulic retention time and organic loading rates ranging from 5 to 20 g COD.L reactor(-1).d(-1). Cobalt pre-loading clearly stimulated the methanogenic activity of the sludge with methanol as the substrate, e.g., after 30 days of reactor operation this activity was 5.8 times higher than that of the cobalt unloaded sludge. During the experiment, part of the cobalt leached from the pre-loaded sludge, i.e., 54% of the cobalt content was lost during the 77 days of reactor operation. Sequential metal extraction showed that losses mainly occurred from the exchangeable and carbonate fraction and in the sludge remaining cobalt was mainly present in the organic/sulfide fraction of the sludge. In situ loading of cobalt in the unloaded UASB reactor on day 57 by adding 31 microM cobalt to the influent for a 24-h period (16% of the cobalt present in the loaded sludge at day 11) resulted in a 4 time increase of the methanogenic activity of the sludge with methanol as the substrate at the end of the reactor experiment, while the accumulated amount of cobalt in the sludge only amounted to 6% of the cobalt accumulated in the loaded sludge (on day 11). This study showed that both pre-loading sludge and in situ loading are adequate for achieving an increased reactor performance of methanol fed UASB reactors operating under cobalt limitation. However, the in situ dosing procedure needs substantially lower amounts of cobalt, while it also gives significantly smaller losses of cobalt with the effluent.     

Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD_soluble/ COD_total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82% and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated withMethanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together withM. concilii.     

Zinc deprivation of methanol fed anaerobic granular sludge bioreactors

The effect of omitting zinc from the influent of mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactors, and latter zinc supplementation to the influent to counteract the deprivation, was investigated by coupling the UASB reactor performance to the microbial ecology of the bioreactor sludge. Limitation of the specific methanogenic activity (SMA) on methanol due to the absence of zinc from the influent developed after 137 days of operation. At that day, the SMA in medium with a complete trace metal solution except Zn was 3.4 g CH4-COD g VSS(-1) day(-1), compared to 4.2 g CH4-COD g VSS(-1) day(-1) in a medium with a complete (including zinc) trace metal solution. The methanol removal capacity during these 137 days was 99% and no volatile fatty acids accumulated. Two UASB reactors, inoculated with the zinc-deprived sludge, were operated to study restoration of the zinc limitation by zinc supplementation to the bioreactor influent. In a first reactor, no changes to the operational conditions were made. This resulted in methanol accumulation in the reactor effluent after 12 days of operation, which subsequently induced acetogenic activity 5 days after the methanol accumulation started. Methanogenesis could not be recovered by the continuous addition of 0.5 microM ZnCl2 to the reactor for 13 days. In the second reactor, 0.5 microM ZnCl2 was added from its start-up. Although the reactor stayed 10 days longer methanogenically than the reactor operated without zinc, methanol accumulation was observed in this reactor (up to 1.1 g COD-MeOH L(-1)) as well. This study shows that zinc limitation can induce failure of methanol fed UASB reactors due to acidification, which cannot be restored by resuming the continuous supply of the deprived metal.     

Introduction of a de novo bioremediation ability, aryl reductive dechlorination, into anaerobic granular sludge by inoculation of sludge with Desulfomonile tiedjei.

Methanogenic upflow anaerobic granular-sludge blanket (UASB) reactors treat wastewaters at a high rate while simultaneously producing a useful product, methane; however, recalcitrant environmental pollutants may not be degraded. To impart 3-chlorobenzoate (3-CB)-dechlorinating ability to UASB reactors, we inoculated granular sludge in UASB reactors with either a pure culture of Desulfomonile tiedjei (a 3-CB-dechlorinating anaerobe) or a three-member consortium consisting of D. tiejei, a benzoate degrader, and an H2-utilizing methanogen. No degradation occurred in an uninoculated control reactor which was started with the same granular sludge, but inoculated reactors and granules from the inoculated UASB systems rapidly transformed 3-CB (54 mumol/day/g of granule biomass). After several months at a hydraulic retention time of 0.5 day, much shorter than the generation time of D. tiedjei, the reactors still dechlorinated 3-CB. This indicated that the bacteria were immobilized in the reactor granules, and by using an antibody probe for D. tiedjei, we demonstrated that this microorganism had colonized the sludge granules. These results represent the first addition of a pure culture or a defined microbial mixture to a viable waste treatment process to introduce a specific de novo degradative pathway into a granular-sludge consortium.     

Introduction of a de novo bioremediation ability, aryl reductive dechlorination, into anaerobic granular sludge by inoculation of sludge with Desulfomonile tiedjei.

Methanogenic upflow anaerobic granular-sludge blanket (UASB) reactors treat wastewaters at a high rate while simultaneously producing a useful product, methane; however, recalcitrant environmental pollutants may not be degraded. To impart 3-chlorobenzoate (3-CB)-dechlorinating ability to UASB reactors, we inoculated granular sludge in UASB reactors with either a pure culture of Desulfomonile tiedjei (a 3-CB-dechlorinating anaerobe) or a three-member consortium consisting of D. tiejei, a benzoate degrader, and an H2-utilizing methanogen. No degradation occurred in an uninoculated control reactor which was started with the same granular sludge, but inoculated reactors and granules from the inoculated UASB systems rapidly transformed 3-CB (54 mumol/day/g of granule biomass). After several months at a hydraulic retention time of 0.5 day, much shorter than the generation time of D. tiedjei, the reactors still dechlorinated 3-CB. This indicated that the bacteria were immobilized in the reactor granules, and by using an antibody probe for D. tiedjei, we demonstrated that this microorganism had colonized the sludge granules. These results represent the first addition of a pure culture or a defined microbial mixture to a viable waste treatment process to introduce a specific de novo degradative pathway into a granular-sludge consortium.     

PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules

Methane is produced by various methanogenic bacteria present in upflow anaerobic sludge blanket (UASB) bioreactors. Methane can be used to predict and improve UASB bioreactor efficiency. The methanogen population in the granules can be influenced by the composition of the substrate. The aim of this study was to fingerprint and identify the methanogens present in three different types of UASB granules that had been used to treat winery, brewery and peach-lye canning effluents. This was done using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. The DGGE fingerprints obtained from the methanogen reference cultures of Methanosaeta concilii, Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina mazeii and Methanobacterium formicicum were compared to the DGGE profiles of the Archaea in the different granules. The positions of the DGGE bands that did not correspond well to the bands of the known species were sequenced and compared to sequences available on GenBank using the Blastn search option. The aligned DNA sequences were used to construct a phylogenetic tree. Based on the data obtained, a DGGE marker was constructed which was used to provide a quick method to identify the Archaeal members of the microbial consortium in UASB granules.     

Acetate Degradation at 70 degrees C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70 degrees C

Anaerobic acetate degradation at 70 degrees C and at 55 degrees C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70 degrees C and less than 15 days was needed at 55 degrees C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55 degrees C up to 90% of the COD was removed. Batch assays showed that sludges from two 70 degrees C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70 degrees C. Both 70 degrees C sludges, as well as the 55 degrees C sludge, produced methane at temperatures of 37 to 73 degrees C. The 55 degrees C sludge exhibited shorter lag phases than the 70 degrees C sludges and higher specific methane production rates between 37 and 65 degrees C.     

Characteristics of methanogenic granules grown on propionate in an upflow anaerobic sludge blanket (UASB) reactor

Granulation of a propionate-degrading consortia was performed with a mesophilic propionate-acclimatized sludge in an upflow anaerobic sludge blanket (UASB) reactor. The granules formed were relatively small, ranging mainly from 0.3 to 0.6 mm in diameter, but had an excellent sedimentation velocity due to a high specific gravity of 1.355 g/cm³ (ash content, 48.2%). The ash consisted mainly of calcium (30.2%), phosphorus (19.7%), and magnesium (3.95%) forming plate crystals in the granules. The populations of three bacterial trophic groups present in the granules, propionate-degraders, hydrogenotrophic and aceticlastic methanogens were 5.6 × 10⁸, 1.6 × 10¹⁰, and 2 × 10⁹ (in most probable number/g mixed-liquor volatile suspended solids [MLVSS]), respectively, while the specific utilization rates of propionate, hydrogen, and acetate of the granules were 9.4, 850, and 20.9 (mmol/g MLVSS·d), respectively. Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. Total granular sludge concentration retained in the UASB reactor during 178 d of operation was 80.0 g mixed-liquor suspended solids (MLSS)/l-reactor, corresponding to 41.4 g MLVSS/l-reactor, which realized a high-rate methanogenic fermentation of propionate of 85 g chemical oxygen demand (COD)/l-reactor·d.     

Thermophilic anaerobic treatment of sulphur rich forest industry wastewater

Thermophilic anaerobic treatment of sulphur-rich paper mill wastewater (0.8-3.1 gCOD/l, 340–850 mgSO₄/l; COD:SO₄ 3.4-5.3) was studied in three laboratory-scale, upflow anaerobic sludge blanket (UASB) reactors and in bioassays. The reactors were inoculated with non-adapted thermophilic granular sludge. In the bioassays, no inhibition of the inoculum was detected and about 62% COD removal (sulphide stripped) was obtained. About 70 to 80% of the removed COD was methanised. In the reactors, up to 60–74% COD removal (effluent sulphide stripped) was obtained at loading rates up to 10–30 kgCOD/m³d and hydraulic retention times down to 6 to 2 hours. The effluent total sulphide was up to 150–250 mg/l. Sulphide inhibition could not be confirmed from the reactor performances. The results from bioassays suggested that both the inoculum and sludge from the UASB reactor used acetate mainly for methane production, while sulphide was produced from hydrogen or its precursors.