Localization and quantification of extracellular polymers in methanogenic granular sludge

Summary Extracellular polymers were localized and quantitatively analysed in methanogenic granular sludge cultivated on either propionate or ethanol in laboratory upflow anaerobic sludge-blanket (UASB) reactors. Electron microscopical analysis of ultrathin sections of the two sludge types stained with ruthenium red revealed the presence of extracellular polymers with different densities and structures. For quantification, granular sludge from a large-scale UASB reactor at a liquid sugar plant was also included in this study. A three-step physical disintegration procedure was used to extract water-soluble extracellular material from the granules. After each disintegration step the extracts were analysed for polysaccharides and proteins. Cell damage and thus the contribution of intracellular proteins and polysaccharides was estimated simultaneously by the determination of free DNA and free ATP in the extracts. After two extraction steps, up to 3.5 mg polysaccharides/g organic material and 5.5 mg protein/g organic material were extracted, whereas no significant increase in DNA was detected. The role of extracellular polymers in granular stability is discussed. Offsprint requests to: A. J. B. Zehnder     

Effect of ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid (EGTA) on stability and activity of methanogenic granular sludge

Summary The effect of the calcium-specific chelant ethylene glycol-bis(\-aminoethyl ether)-N,N-tetraacetic acid (EGTA) on methanogenic granular sludge from a laboratory-scale upflow anaerobic sludge-blanket (UASB) reactor fed propionate and from a full-scale reactor treating paper-mill waste-water was studied. Upon treatment with EGTA both sludge types showed a decrease in the calcium and phosphorus content and a release of protein and polysaccharides, leading to a decrease in strength of papermill granular sludge and a disintegration of propionate-grown granules. After treatment of propionate-grown granular sludge with high EGTA concentrations, the methanogenic activity with propionate and acetate as test substrates decreased by 88 and 33%, respectively. The marked reduction in propionate oxidation activity may be caused by a disruption of the special juxtapositioning of bacteria in the granules. Offsprint requests to: A. J. B. Zehnder     

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.     

Isolation and characterization of Veillonella sp. from methanogenic granular sludge

An anaerobic, propionate-producing, mesophilic, Gram-negative, non-spore forming, non-motile, coccoid-shaped bacterium (strain S119) was isolated from methanogenic granular sludge of an upflow anaerobic sludge blanket reactor. Based on morphology and cytological and physiological properties the isolate was assigned to the genus Veillonella. Strain S119 forms spherical monospecies biofilms (granules), 1.0–3.0 mm in diameter, when grown in continuously mixed medium with sodium lactate as the sole carbon source and powdered activated carbon as biofilm support particles. The granules attained concentrations of volatile suspended solids up to 38 mg/cm³. Veillonella sp. strain S119 has a highly hydrophobic cell surface and produces extracellular slime, which contains polysaccharide fractions. Growth characteristics and adhesion properties of the isolated microorganisms suggest its participation in the formation of granular sludge. Correspondence to: W. Verstraete     

Methanogenic granular sludge formation in an upflow anaerobic sludge blanket reactir treating synthetic methanolic waste

A laboratory upflow anaerobic sludge blanket reactor, seeded with fine, suspended, bacterial floc with 1.76 g volatile suspended solids/l, was used to treat synthetic methanolic waste. After 180 days of continuous peration, granular sludge with discrete granules of 1 to 2 mm diam. was formed, with 52 g volatile suspended solids/l. Granules were brown, relatively soft and had a settling velocity of 1.61 cm/s. Extracellular polymeric matter extracted from the granular sludge had high carbohydrate content but low nucleic acid content. The ash of the granular sludge contained Na⁺, K⁺ and Mg²⁺ up to 15.0, 11.7 and 3.75 mg/g, respectively. Scanning and transmission electron microscopy revealed that the granular sludge was dominated by methanogens resembling Methanosarcina.The authors are with the Department of Environmental Engineering, Faculty of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan     

Granulation of biomass in thermophilic upflow anaerobic sludge blanket reactors treating acidified wastewaters

The development of granular sludge in thermophilic (55 degrees C) upflow anaerobic sludge blanket reactors was investigated. Acetate and a mixture of acetate and butyrate were used as substrates, serving as models for acidified waste-waters. Granular sludge with either Methanothrix or Methanosarcina as the predominant acetate utilizing methanogen was cultivated by allowing the loading rate to increase whenever the acetate concentration in the effluent dropped below 200 and 700 mg COD/L, respectively. The highest methane generation rates, up to 162 kg CH(4)-COD/m(3) day, or 2.53 mole CH(4)/L day, were achieved at hydraulic retention times down to 21 min, with granules consisting of Methanothrix. The formation of Methanothrix granules did not depend on the type of seed material, nor on the addition of inert support particles. The growth of granules proceeded rapidly with adapted seed material, even when the reactors were inoculated with low concentrations. With mesophilic seed materials growth of granules took much longer. Thermophilic Methanothrix granules strongly resemble mesophilic granules of the "filamentous" type. Some factors governing the thermophilic granulation process are discussed.     

Effect of loading rate on the granulation process and granular activity in a bench scale UASB reactor

The effect of organic loading rate (OLR) on the granulation process was evaluated using upflow anaerobic sludge blanket (UASB) reactors running under different conditions. Results showed that increase of OLR, extracellular polymer (ECP) content and granulation were closely related to one another. ECP in the sludge accumulated over a short period under overloading conditions, which greatly enhanced the granulation process. Treatment performance could be recovered after the ECP accumulation when the overloading was suitably exerted. However, too high loading rate should be avoided because it could cause the unrecoverable decay of methanogenic activity and the serious unbalance between the feedstuff and biological requirement.     

High rate performance and characterization of granular methanogenic sludges in upflow anaerobic sludge blanket reactors fed with various defined substrates

High rate granular methanogenic fermentations were performed in one-phase upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewaters containing starch, sucrose, ethanol, and butyrate plus propionate. All granules formed showed high settling velocities which enabled high cell mass retention and accommodation of high loading rates. The maximum COD removal rates (g COD/l-reactor·d) obtained after 500-d operations were 7.6 for starch, 10.5 for sucrose, 32.1 for ethanol, and 42.6 for butyrate-propionate. Long-term growth on various defined substrates altered the population of bacterial trophic groups and overall characteristics of granules. The starch- and sucrose-grown granules were characterized by larger size and more abundant extracellular polymeric substances (EPS) than the ethanol- or fatty acids-grown granules. The fatty acids-grown granules contained a considerable amount of inorganic salts (ash content: 56 to 63%) but a small amount of EPS, and showed a denser ultrastructure than the other three types of granules. The granules grown on ethanol under slightly acidic conditions showed the lowest specific gravity and volatile suspended solids (VSS) density as well as ash content among all of the granules. As aceticlastic methanogens, Methanothrix spp. were predominant in the starch-, sucrose-, and fatty acids-grown granules, whereas comparable numbers of Methanosarcina spp. were observed only in the ethanol-grown granules. The populations of hydrogenotrophic methanogens were the largest of all bacterial trophic groups in the respective granules. The data confirm that the prevalence of Methanothrix spp. and high methanogenic activity for H₂ are general characteristics of methanogenic granucles and that EPS and inorganic deposits contribute chemically to the enhancement of structural stability and mechanical strength of granules.     

Anaerobic digestion of long-chain fatty acids in UASB and expanded granular sludge bed reactors

Solutions of sodium caprate and sodium laurate were digested in upflow anaerobic sludge bed (UASB) reactors inoculated with granular sludge and in expanded granular sludge bed (EGSB) reactors. UASB reactors are unsuitable if lipids contribute 50% or more to the COD of waste water: the gas production rate required to obtain sufficient mixing and contact cannot be achieved. At lipid loading rates exceeding 2–3 kg COD m⁻³ day⁻¹, total sludge wash-out occurred. At lower loading rates the system was unreliable, due to unpredictable sludge flotation. EGSB reactors do fulfil the requirements of mixing and contact. They accommodate space loading rates up to 30 kg COD m⁻³ day⁻¹ during digestion of caprate or laurate as sole substrate, at COD removal efficiencies of 83–91%, and can be operated at hydraulic residence times of 2 h without any problems. Augmentation of granular sludge in lab-scale EGSB reactors was demonstrated. The new granules had excellent settling properties. Floating layer formation, as well as mixing characteristics in full-scale EGSB reactors require further research.     

Ruthenium red adsorption method for measurement of extracellular polysaccharides in sludge flocs

A nondestructive method of measuring extracellular polysaccharides (ECP) in activated sludge floes using Ruthenium Red dye adsorption was developed at the Environmental Engineering Laboratory at the University of Colorado at Boulder. The effects of pH, buffer solution, dye concentration, sludge mass, temperature, and incubation time on dye adsorption was determined. Ruthenium Red dye adsorption to bacterial floes was found to fit a Brunauer-Emmett-Teller (BET) isotherm model. Of the other environmental conditions in the system, pH was found to have the strongest effect on dye adsorption to bacterial flocs. The amount of extra cellular polysaccharides (ECP) measured by Ruthenium Red adsorption was compared with extracellular polysaccharides measured by two chemical extraction methods. Of all methods considered Ruthenium Red dye adsorption measured the highest amount of extracellular polysaccharide with the lowest amount of bacterial cell disruption. Thus, Ruthenium Red dye adsorption was more effective than extraction procedures for measurement of extracellular polysaccharides in activated sludge flocs.     

Anaerobic treatment of fish meal process waste-water in a UASB reactor at high pH

Summary The influence of high pH on anaerobic degradation of fish process waste-water with a high total ammonia concentration was investigated in an upflow anaerobic sludge blanket (UASB) reactor. More than 99% of volatile fatty acids and trimethylamine in the process waste-water were degraded up to pH 7.9. Above pH 7.9 only the conversion of acetate was slightly decreased. At pH 8.3 serious disintegration of the granules occurred leading to process failure. Increasing the pH changed the physical characteristics of the granules leading to decreased density, size, and volatile solids content. After 4 months of acclimatization to high pH in the reactor, the specific methanogenic activity of the granular biomass was the same from pH 7.1 to 8.5. At pH 8.3 and 8.5, acclimatization had improved the specific activity by 25 and 50%, respectively. However, the acclimatized biomass generally showed a decreased activity (60%) at all pH values tested below the acclimatization pH.Offprint requests to: B. K. Ahring     

Extraction of extracellular polymer from anaerobic sludges

Summary Two types of anaerobic sludge were analyzed for ECP (extracellular polymers) content under five extraction conditions. Results showed that EDTA was more effective than formaldehyde as an extractant. Increase of temperature and addition caustic also enhanced the extraction. The ratio between carbohydrate and protein fractions of ECP for both acetate- and benzoate-degrading sludge was 0.16–0.18. The former sludge had only 40–45% of ECP as in the latter sludge.     

保藏温度对厌氧氨氧化颗粒污泥特性的影响

为考察保藏温度对厌氧氨氧化污泥颗粒特性的影响,同时优化保藏厌氧氨氧化颗粒污泥温度参数,本试验首先通过HRT调控进水基质负荷培养厌氧氨氧化颗粒污泥,并采用KHCO3和Na HCO3交替提供无机碳源。然后分别在–40℃、4℃、(27±4)℃室温和35℃条件下避光保藏。结果表明,Na HCO3可代替KHCO3作为厌氧氨氧化菌生长的无机碳源。相比于其他保藏温度,4℃保藏能够较好地维持生物量和生物活性,同时能较好地维持颗粒污泥的沉降性能、颗粒污泥和细胞结构完整性。在保藏过程中,一阶衰减指数模型可拟合厌氧氨氧化颗粒污泥生物量及活性的衰减过程,衰减指数与胞溶程度正相关,而且生物量的衰减比活性的衰减更快。同时,颗粒污泥胞外聚合物中蛋白质与多糖的比值(PN/PS)和血红素不能有效指示保藏过程中颗粒污泥沉降性能和活性的变化,而生物活性与胞溶程度呈负相关。     

Characteristics of granular methanogenic sludge grown on glucose in a UASB reactor

The formation of granules grown on glucose in an upflow anaerobic sludge blanket (UASB) reactor was investigated. Total granular sludge concentration retained in the UASB reactor was 34.5 g MLSS/l (30.0 g MLVSS/l) during 240 d operation on glucose minimum medium with the supplementation of 1.07 g NaHCO₃ per 1 g glucose. This realized a high-rate methanogenic fermentation of glucose of 17.6 g COD/l-reactor-d at 3.4 d⁻¹ of space velocity. The granules formed were relatively small, ranging mainly from 0.4 to 0.5 mm, had a relatively low cell density of 0.0542–0.0560 g MLVSS/ml, and had low specific gravity (0.97–1.19) due to very low ash content (11–13%). Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by glucose, acetate, and propionate.     

Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

Changes in the composition of extracellular polymeric substances in activated sludge during anaerobic storage

Changes in the chemical composition of organic compounds in total activated sludge, activated sludge extracellular polymeric substances (EPS), and sludge bulk water during anaerobic storage (12 days) were studied. The background for the study was that anaerobic storage of activated sludge, which often takes place at wastewater treatment plants before dewatering, causes a deterioration of the dewaterability. The reasons are not known at present, but may be related to changes in exopolymer composition of the flocs. The results showed that a fast decrease in total sludge protein and carbohydrate took place within 3 days of anaerobic storage as a result of degradation processes, which accounted for approximately 20% of the organic fraction. The amount of uronic acids and humic compounds remained almost constant in the sludge. The EPS were extracted from the floc matrix using a cationexchange resin. In the EPS matrix a similar initial (2–3 days) degradation of proteins and carbohydrate took place, whereas the content of DNA and uronic acids showed minor changes. The extractability of humic compounds increased during the first 3 days of storage. No changes in extractability of the carbohydrate were observed. A fraction of the EPS protein was found to be difficult to extract but was observed to be degraded during the anaerobic storage. The EPS composition was further characterized by high-performance size-exclusion chromatography analysis obtained by on-line UV detection and post-column detection of proteins, carbohydrates, humic compounds and DNA. Four fractions of polysaccharides were found, of which only one was responsible for the decrease in the carbohydrate content observed with storage time. The fraction was presumably of low molecular mass. Humic compounds and volatile fatty acids (acetate and propionate) were released to the bulk water from the flocs during the storage. A possible mechanism to explain the reduced dewaterability developed during anaerobic storage, partly because of the observed changes in EPS, is discussed.     

Properties of anaerobic granular sludge as affected by yeast extract,cobalt and iron supplements

The effect of yeast extract (YE), iron (Fe) and cobalt (Co) on anaerobic bacterial granules grown in eight laboratory-scale upflow anaerobic sludge blanket reactors was investigated using a factorial design. The experiment was performed in three periods in which different chemical oxygen demand (COD) loading rates were applied to the reactors. The COD digestion rate and the specific activity of the bacteria were positively affected by supplementation of Fe and Co in the feed under a high COD loading rate. YE had a strong positive effect on the bacterial growth rate, but no significant effect on the specific activity of the bacteria. With Fe supplementation, an excellent COD digestion rate was maintained in the reactors, and addition of YE in the feed was not necessary. Granules with better settling properties were developed under a relatively low bacterial growth rate. With the increase in COD loading rate, the percentage of calcium increased rapidly in the granules from the reactors without YE supplementation. The bacteria grown under high COD loading rate and without an Fe supplement could be Fe-deficient. Methanothrix-like rod-shaped bacteria were dominant in the granules from all reactors. Correspondence to: N. Kosaric     

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.     

Anammox反应器启动过程中颗粒污泥性状变化特性

以厌氧颗粒污泥作为接种物,通过185 d的运行,成功启动了上流式厌氧氨氧化污泥床(Upflow anaerobic sludge blanket,UASB)反应器。反应器的进水氨氮与亚硝氮浓度分别提升至224 mg/L和255 mg/L,容积氮去除速率提升至3.76 kg/(m3·d)。采用红外光谱、扫描电镜和透射电镜等对厌氧氨氧化颗粒污泥的性状进行观察,发现颗粒污泥在启动过程中经历了污泥颗粒裂解到污泥颗粒重组的过程,且厌氧氨氧化颗粒污泥表面含有丰富的官能团,说明厌氧氨氧化颗粒污泥可能具有良好的吸附性能。采用宏基因组测序的方法对启动前后颗粒污泥的生态结构进行分析,发现原接种污泥优势菌群(变形菌门、厚壁菌门、拟杆菌门)丰度大幅减少,厌氧氨氧化菌所属的浮霉状菌门丰度则由1.59%提升到23.24%。     

Bonding Form Analysis of Metals and Sulfur Fractionation in Methanol‐Grown Anaerobic Granular Sludge

This study investigates the metal and sulfur bonding form distribution in mesophilic (30 °C, pH 7) methanol‐grown anaerobic granular sludge from upflow anaerobic sludge bed reactors operating at an organic loading rate of 3.8 g CH₃OH‐COD/L d. This was achieved by applying a modified Tessier sequential extraction scheme to investigate the metal bonding forms and a sequential extraction scheme for sulfur and simultaneously extracted metals to granular sludge samples of the reactors after 0, 22, 35 and 43 days of operation. Metals were also determined in the sulfur extracts. Co and Ni predominated in their oxidizable bonding forms, which increased together with the pseudo‐total content during reactor operation. An omission of Co and Ni from the influent led to only a minor decline of the pseudo‐total content in the sludge, mainly from the acid‐soluble fraction. The ratio of simultaneously extracted metals (Co, Fe, Mn, Ni) to acid‐volatile sulfides was lower than 1, indicating that the sludge contained sufficient sulfide to bind the metals as metal monosulfides. The bioavailability of metals in the methanol‐grown anaerobic granular sludge investigated is therefore mainly controlled by sulfide formation/dissolution.