Microbial segregation within anaerobic digester granules treating brewery wastewater

Microbial segregation was observed within granules from a laboratory scale upflow anaerobic sludge blanket (UASB) digester, using fluorescence and electron microscopy. A shift in distribution of methanogens occurred upon alteration of the feed flow rate.     

Image analysis to quantify and measure UASB digester granules

Two-dimensional image analysis was applied to counting, sizing, and density determinations of granules in full-scale and laboratory-scale upflow anaerobic sludge blanket (UASB) digesters. An advantage of this technique for monitoring laboratory-scale digester sludge is the small amount of material required for analysis. Quantification of number of granules using this method correlated well with dry weight determinations (r = 0.989). Distinguished granule size increased with time throughout the digestion process, supported by dry weight determinations which indicated an increase in biomass. The monitoring of granule density may reveal subtleties of the selection pressure placed on granules not noticed previously. (c) 1993 John Wiley & Sons, Inc.     

An integrated UASB-sludge digester system for raw domestic wastewater treatment in temperate climates

To improve the performance of an upflow anaerobic sludge blanket (UASB) reactor treating raw domestic wastewater under temperate climates conditions, the addition of a sludge digester to the process was investigated. With the decrease in temperature, the COD removal decreased from 78% at 28 °C to 42% at 10 °C for the UASB reactor operating alone at a hydraulic retention time of 6 h. The decrease was attributed to low hydrolytic activity at lower temperatures that reduced suspended matter degradation and resulted in solids accumulation in the top of the sludge blanket. Solids removed from the upper part of the UASB sludge were treated in an anaerobic digester. Based on sludge degradation kinetics at 30 °C, a digester of 0.66 l per liter of UASB reactor was design operating at a 3.20 days retention time. Methane produced by the sludge digester is sufficient to maintain the temperature at 30 °C.     

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.     

The effect of iron on anaerobic digestion

Summary The ability of methanogenic bacteria to adapt to high concentrations of iron was investigated using a 9l Upflow Anaerobic Sludge Blanket (UASB) reactor fed semi-continuously with a synthetic waste containing glucose as the organic carbon source. It was found that an iron concentration of up to 5 650 mg/l, which is well in excess of previously reported toxicity levels, had no inhibitory effects on anaerobic digestion, with the exception of a decrease in biogas production. The iron precipitated out and accumulated in the sludge bed of the digester, resulting in very low concentrations of iron in the digester effluent.     

Digestion of sand-laden manure slurry in an upflow anaerobic solids removal (UASR) digester

Studies on the performance of a laboratory scale upflow anaerobic solids removal (UASR) digester were carried out using sand-laden cow manure slurries having total solids (TS) concentration as 50 and 100 g/l. Hydraulic retention time (HRT) was maintained as 32.4 days, which resulted in the volatile solids (VS) loading rates of 1 and 1.64 g/l d. The UASR system was designed to remove sand from the manure slurry, while anaerobically digesting biodegradable solids inside a single reactor. To enhance the contact of microorganisms and substrate, the liquor from the top of the digester was recirculated through the bed of settled solids at its bottom. Volatile solids reduction through this process was observed to be 62% and 68% in the case of feed slurries having TS concentration as 50 and 100 g/l (referred in the text as 5% and 10% feed slurries), respectively. The methane production rates were observed to be 0.22 and 0.38 l/l d, while methane yield was 0.21 and 0.27 l CH₄/g VS loaded, for 5% and 10% feed slurries, respectively. This indicates that the increase in the VS loading had a positive impact on methane production rate and methane yield. It would be of interest to study the performance of a UASR digester at higher solids loadings and with longer solids retention times. Nonetheless, the presented study showed that sand-laden manure slurries can be successfully digested in a UASR digester producing methane energy equivalent to 4 kW h per m³ of digester volume per day.     

Phenylacetic acid in an anaerobic swine manure digester

Summary The presence of phenylacetic acid (PAA) in an anaerobic swine manure digester was determined by gas chromatography of the butyl ester and confirmed by mass spectroscopy. PAA concentration increased during start-up of a digester and with low carbon, high nitrogen loading. Unlike acetate, propionate and butyrate, the concentration of PAA varied little through the day in a stable digester loaded once per day. The laboratory scale digester was loaded at 4 g of swine manure solids/liter digester volume per day. The retention time and temperature were 15 days and 37°C. PAA is a microbial intermediate which is produced by one group of anaerobic bacteria and converted to methane by other members of the bacterial community in the digester. As such, it may be a useful indicator of the relative metabolic activity of the bacterial groups and thus of the overall stability of the anaerobic process.     

Layered structure of UASB granules gives microbial populations resistance to toxic chemicals

To investigate the effect of granular structure on resistance to toxic chemicals in UASB (Upflow Anaerobic Sludge Blanket) reactors, normal and broken granules were examined for their ability to degrade acetate with and without the addition of toluene or trichloroethylene as a toxic chemical. Without a toxic chemical, both normal and broken granules degraded the acetate at the same volumetric degradation rate (3.21 mM h–1). However, when 500 l l–1 of toluene or trichloroethylene was added, the acetate-degradation rate of the broken granules was about a third of the rate with normal granules. Therefore, the layered structure of the UASB granules seems to give microbial populations the ability to resist toxic chemicals.     

Granulation in thermophilic upflow anaerobic sludge blanket (UASB) reactors

The state of the art for thermophilic UASB reactors is discussed focusing on the start-up of UASB reactors, the influence of the waste water composition and temperature on the development and maintenance of thermophilic granules, and the microbial composition and structure of thermophilic granules.     

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.     

PCR-based denaturing gradient gel electrophoretic evaluation of changes in the non-methanogenic population of stressed upflow anaerobic sludge blanket granules

Summary The performance of upflow anaerobic sludge blanket (UASB) bioreactors is influenced by the composition of the substrate and the microbial species present in the granules. The aim of this study was to determine if a change in the structure of the non-methanogenic microbial community takes place when UASB brewery granules are subjected to the sudden addition of different carbon sources at different concentrations. A shift in the microbial community did occur when the granules were subjected to lactate medium. The granules that were stressed with glucose medium did not show changes in the microbial consortium regardless of the increase in the glucose concentrations. The polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) method was successfully applied to show changes in the structure of the microbes present in UASB granules that were cultivated under different environmental conditions.     

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.     

The effect of aluminium on Anaerobic digestion

Summary The ability of methanogenic bacteria to adapt to high concentrations of aluminium was investigated using a 9 Upflow Anaerobic Sludge Blanket (UASB) reactor fed semi-continuously with a synthetic waste containing glucose as the organic carbon source. It was found that an influent concentration of 2500 mg/ Al³⁺ could be tolerated by acclimatized methanogenic bacteria. However, if the concentration in the synthetic waste was further increased, inhibition of the methanogens resulted, with a corresponding decrease in digester performance. The aluminium precipitated as Al(OH)₃ and accumulated in the sludge bed of the digester.     

Preparation techniques for the electron microscopy of granular sludge from an anaerobic digester

Several fixation and dehydration techniques for scanning and transmission electron microscopy of glycocalyx and microbial populations within granules from an upflow anaerobic sludge blanket digester purifying a brewery wastewater were compared. Sputter-cryo and freeze-drying techniques prior to scanning electron microscopy (SEM) allowed viewing of the glycocalyx. In contrast standard fixation and dehydration techniques were suitable for examination of underlying microbial populations by both SEM and transmission electron microscopy.     

Feasibility studies on the treatment of dairy wastewaters with upflow anaerobic sludge blanket reactors

The feasibility of using upflow anaerobic sludge blanket (UASB) reactors for the treatment of dairy wastewaters was explored. Two types of UASBs were used--one operating on anaerobic sludge granules developed by us from digested cowdung slurry (DCDS) and the other on the granules obtained from the reactors of M/s EID Parry treating sugar industry wastewaters. The reactors were operated at HRT of 3 and 12 h and on COD loading rates ranging from 2.4 kg per m3 of digester volume, per day to 13.5 kg m(-3) d(-1). At the 3 h HRT, the maximum COD reduction in the DCDS-seeded and the industrial sludge-seeded reactors was 95.6% and 96.3%, respectively, better than at 12 h HRT (90% and 92%, respectively). In both the reactors, the maximum, the second best, and the third best COD reduction occurred at the loading rates of 10.8, 8.6 and 7.2 kg m3 d(-1), respectively. At loading rates higher than 10.8 kg, the reactor performance dropped precipitously. Whereas in the first few months the reactors operating on sludge from EID Parry achieved better biodegradation of the waste, compared to the reactors operated on DCDS, the performance of the latter gradually improved and matched with the performance of the former.     

Layered structure of granules in upflow anaerobic sludge blanket reactor gives microbial populations resistance to metal ions

Metal ions (Cd²⁺, Cu²⁺, Ni²⁺, Zn²⁺ and Cr³⁺) did not affect glucose degradation or the production of methane during anaerobic digestion with intact and disintegrated granules from a UASB (Upflow Anaerobic Sludge Blanket) reactor. However, when Cu²⁺ was at 500 mg g^–1 VSS (volatile suspended solids) in the media, the glucose degradation rates and methane production rates decreased by 14% and 32% in disintegrated granules, respectively, whereas, in intact granules, decreases were 3% and 14%, respectively. When various electroplating metal ions were tested, 50% inhibition of acetate degradation and methane production were produced by 210–770 mg g^–1 VSS and 120–630 mg g^–1 VSS, respectively. The relative toxicity of the electroplating metals on methane production was in the order of Zn²⁺ (most toxic) > Ni²⁺ > Cu²⁺ > Cr³⁺ > Cd²⁺ (least toxic).     

A further study of the anaerobic biotreatment of malt whisky distillery pot ale using an UASB system

Pot ale from a pilot-scale malt whisky distillery was treated using a mesophilic upflow anaerobic sludge blanket (UASB) digester. Stable operation was observed at organic loading rates (OLRs) of 5.46 kg COD/m3 day or less when the pot ale was diluted with tap water. Digester failure occurred when undiluted pot ale was used, even though OLR was less than 5 kg COD/m3 day. Overall performance was worse than that observed previously when UASB digesters were used to treat pot ale from a different source supplemented with trace elements. A substantial proportion of effluent chemical oxygen demand (COD) was present as volatile fatty acids (VFA), particularly during periods of reactor stress, indicating that overall performance was limited by the rate of VFA conversion. Wastewater alkalinity rose during digestion. The sludge which developed in the reactor was flocculent but did not form compact granules.     

An original procedure for physical simulation of upflow anaerobic sludge blanket reactors

A procedure for sludge blanket reactors physical simulation is presented. To simulate the liquid phase, a solution of 1.0 g/l of polyvinyl alcohol provided the desired rheological behavior. Biological granules presented in UASB reactors were simulated using granules of a water absorbent acrylic polymer capable to expand up to one hundred times in volume when moistened. The biochemical reactions that produces methane and carbon dioxide in anaerobic reactors were simulated reacting nitric acid and sodium bicarbonate. This procedure differs from gas injection since the gas is produced at the reactor core as it happens in the actual situation, thus the simulation is more realistic. Flow visualization and residence time distribution measurements were used to verify if the proposed procedure acceptably simulates UASB reactors treating wastewater. All tests were performed in a 10.5 l bench scale reactor and the results allow to recommend this method whenever flow visualization and reactor physical simulation are useful for reactor design and development.     

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.     

Biosorption and biodegradation of pentachlorophenol (PCP) in an upflow anaerobic sludge blanket (UASB) reactor

In order to understand the fate of PCP in upflow anaerobic sludge blanket reactor (UASB) more completely, the sorption and biodegradation of pentachlorophenol (PCP) by anaerobic sludge granules were investigated. The anaerobic granular sludge degrading PCP was formed in UASB reactor, which was seeded with anaerobic sludge acclimated by chlorophenols. At the hydraulic retention time (HRT) of 20–22 h, and PCP loading rate of 200–220 mg l⁻¹ d⁻¹, UASB reactor exhibited good performance in treating wastewater which containing 170–180 mg l⁻¹ PCP and the PCP removal rate of 99.5% was achieved. Sequential appearance of tetra-, tri-, di-, and mono-chlorophenol was observed in the reactor effluent after 20 mg l⁻¹ PCP introduction. Sorption and desorption of PCP on the anaerobic sludge granules were all fitted to the Freundlich isotherm equation. Sorption of PCP was partly irreversible. The Freundlich equation could describe the behavior of PCP amount sorbed by granular sludge in anaerobic reactor reasonably well. The results demonstrated that the main mechanism leading to removal of PCP on anaerobic granular sludge was biodegradation, not sorption or volatization.