Upflow Anaerobic Sludge Blanket Granules Research Articles

Impacts of granular activated carbon addition on anaerobic granulation in blackwater treatment

Upflow anaerobic sludge blanket (UASB) reactors, with or without granular activated carbon (GAC) amendment, were applied for blackwater treatment. The impact of GAC on the formation of granules and biomethane recovery was assessed. High organic loading rates (OLRs) up to 15.7 ± 2.1 kg COD/(m3d) were achieved with both reactors. Similar chemical oxygen demand (COD) removal and methane production rate were observed with OLRs ranging from 5.1 ± 1.0 to 9.3 ± 1.5 kg COD/(m3d). Under higher OLR conditions (13.6 ± 1.1 to 15.7 ± 2.1 kg COD/(m3d)), the GAC-amended UASB achieved a higher COD reduction than the UASB without GAC addition. Interestingly, volatile suspended solids (VSS) concentrations, granule size, and extracellular polymeric substance concentrations were lower in the GAC-amended UASB reactor as compared to the UASB without GAC. The methanogenesis activity of the granules in the GAC-amended UASB reactor was significantly higher than the methanogenesis activity of the UASB granules. The microbes o_Bacteroidales and Syntrophus were predominant in both reactors. The acetoclastic methanogens dominated in the UASB reactor without GAC addition; while hydrogenotrophic methanogens dominated in the GAC-UASB reactor. A phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) indicated that syntrophic acetate oxidation improved with GAC addition. The co-occurrence network indicated that interactions between dominant bacteria and archaea were higher in the GAC-amended UASB reactor than in the UASB reactor without GAC addition. This study demonstrated the improved blackwater treatment performance as a result of granulation in UASB with the addition of GAC.

Microbial community changes in methanogenic granules during the transition from mesophilic to thermophilic conditions.

Upflow anaerobic sludge blanket (UASB) reactor is one of the most applied technologies for various high-strength wastewater treatments. The present study analysed the microbial community changes in UASB granules during the transition from mesophilic to thermophilic conditions. Dynamicity of microbial community in granules was analysed using high-throughput sequencing of 16S ribosomal RNA gene amplicons, and the results showed that the temperature strictly determines the diversity of the microbial consortium. It was demonstrated that most of the microbes which were present in the initial mesophilic community were not found in the granules after the transition to thermophilic conditions. More specifically, only members from family Anaerolinaceae managed to tolerate the temperature change and contributed in maintaining the physical integrity of granular structure. On the contrary, new hydrolytic and fermentative bacteria were quickly replacing the old members in the community. A direct result from this abrupt change in the microbial diversity was the accumulation of volatile fatty acids and the concomitant pH drop in the reactor inhibiting the overall anaerobic digestion process. Nevertheless, by maintaining deliberately the pH levels at values higher than 6.5, a methanogen belonging to Methanoculleus genus emerged in the community enhancing the methane production.

Fingerprinting and Identification of Bacteria Present in UASB Granules Used to Treat Winery, Brewery, Distillery or Peach-lye Canning Wastewater

The effective operation of the anaerobic digestion process in an upflow anaerobic sludge blanket (UASB) bioreactor is dependent on the microbial composition of the UASB granules. The granules contain a consortium of bacteria, with a specific metabolic function for each group, contributing to the overall efficiency and stability of the bioreactor. The aim of this study was to fingerprint and identify the bacteria present in four different types of South African UASB granules that are used to treat winery, brewery, distillery and peach-lye canning wastewaters. This was done by combining conventional microbiological platings with PCR-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. Each granule type showed distinct PCR-based DGGE fingerprints with unique bands, while other bands were found to be present in all the granules, regardless of the wastewater being treated. Sixty-eight different bacteria (40 pure isolates and 28 clones) were partially sequenced and identified from the winery, brewery, distillery and peach-lye canning granules. Thirty-five percent of the identified bacteria represented the unculturable bacteria and 65% represented the culturable bacteria, which included members of the following genera: Bacillus, Pseudomonas, Bacteroides, Enterococcus, Alcaligenes, Clostridium, Shewanella, Microbacterium, Leuconostoc, Sulfurospirillum, Acidaminococcus, Vibrio, Aeromonas, Nitrospira, Synergistes, Rhodococcus, Rhodocyclus and Syntrophobacter. A DGGE marker was successfully constructed, representing members of the bacterial consortium in UASB granules.

Biochemical hydrogen and methane potential of sugarcane syrup using a two-stage anaerobic fermentation process

The potential of using a two-stage hydrogen and methane fermentation of sugarcane juice was studied. The effects of pure and mixed culture as inocula on hydrogen production were compared. Additionally, the effects of pure culture inocula form, i.e., free cells or immobilized cells of Clostridium butyricum TISTR1032 was compared to different forms of mixed cultures, i.e., granules or suspended cells of heat-treated upflow anaerobic sludge blanket (UASB) granules. The hydrogenogenic effluents from all treatments were used as substrates to evaluate the potential of methane production by non-pretreated UASB granules. Results showed that a pure culture gave a higher hydrogen production potential and a shorter lag time in comparison to mixed cultures. Immobilized cells of C. butyricum TISTR1032 gave a hydrogen production potential that was 1.2 times higher than that of free cells. However, there was no significant difference in hydrogen production potential of granules and suspended cells. Moreover, hydrogenogenic effluent from the first stage fermentation showed a high efficiency in methane production by non-pretreated UASB granules. Although fermentation of mixed cultures resulted in lower hydrogen yield, its hydrogenogenic effluent yielded a higher methane production than that of pure culture. Therefore, overall energy yield when using mixed cultures in hydrogen production stage was higher. The two-stage hydrogen and methane production process removed 94–95% of chemical oxygen demand (COD) resulting in energy recovery in the range of 12–13.4kJ/gCODadded. Results indicated that the COD removal efficiency and the energy recovery from two-stage hydrogen and methane production was improved 6–7 fold when compared to hydrogen production alone.

Transcriptomic and physiological insights into the robustness of long filamentous cells of Methanosaeta harundinacea, prevalent in upflow anaerobic sludge blanket granules.

Methanosaeta spp. are widely distributed in natural environments, and their filamentous cells contribute significantly to sludge granulation and the good performance of anaerobic reactors. A previous study indicated that Methanosaeta harundinacea 6Ac displays a quorum sensing-regulated morphological transition from short to long filaments, and more acetate is channeled into methane production in long filaments, whereas more is channeled into biomass synthesis in short filaments. Here, we performed transcriptomic and physiological analysis to gain insights into active methanogenesis in long filaments of M. harundinacea 6Ac. Both RNA sequencing (RNA-seq) and quantitative reverse transcription-PCR indicated that transcription of the genes involved in aceticlastic methanogenesis and energy metabolism was upregulated 1.2- to 10.3-fold in long filaments, while transcription of the genes for the methyl oxidative shunt was upregulated in short filaments. [2-(13)C]acetate trace experiments demonstrated that a relatively higher portion of the acetate methyl group was oxidized to CO2 in short filaments than in long filaments. The long filaments exhibited higher catalase activity and oxygen tolerance than the short ones, which is consistent with increased transcription of the oxidant-scavenging genes. Moreover, transcription of genes for cell surface structures was upregulated in the long filaments, and transmission electron microscopy revealed a thicker cell envelope in the filaments. RNA-seq determined a >2-fold upregulation of a variety of antistress genes in short filaments, like those encoding chaperones and DNA repair systems, which implies that the short filaments can be stressed. This study reveals the genetic basis for the prevalence of the long filamentous morphology of M. harundinacea cells in upflow anaerobic sludge blanket granules.

Correlation between microbial community and granule conductivity in anaerobic bioreactors for brewery wastewater treatment

Prior investigation of an upflow anaerobic sludge blanket (UASB) reactor treating brewery wastes suggested that direct interspecies electron transfer (DIET) significantly contributed to interspecies electron transfer to methanogens. To investigate DIET in granules further, the electrical conductivity and bacterial community composition of granules in fourteen samples from four different UASB reactors treating brewery wastes were investigated. All of the UASB granules were electrically conductive whereas control granules from ANAMMOX (ANaerobic AMMonium OXidation) reactors and microbial granules from an aerobic bioreactor designed for phosphate removal were not. There was a moderate correlation (r=0.67) between the abundance of Geobacter species in the UASB granules and granule conductivity, suggesting that Geobacter contributed to granule conductivity. These results, coupled with previous studies, which have demonstrated that Geobacter species can donate electrons to methanogens that are typically predominant in anaerobic digesters, suggest that DIET may be a widespread phenomenon in UASB reactors treating brewery wastes.

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated UASB granules as affected by organic loading rate

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated upflow anaerobic sludge blanket (UASB) granules was examined in a UASB reactor. The organic loading rate (OLR) was optimized in order to maximize the hydrogen production rate (HPR). The maximum hydrogen content (37.1% and 24.2%) and HPR (9 and 6.2 mmol H2/L h) were achieved at the optimum OLR of 50 g/L d using pure and waste glycerol as the substrate, respectively. The major soluble metabolite products (SMPs) were ethanol, 1,3-propanediol (1,3-PD), formic acid, and acetic acid. The microbial community and microbial structure, analyzed by fluorescent in situ hybridization (FISH) and scanning electron microscopy (SEM), revealed that the predominant hydrogen producers were E. aerogenes ATCC 13048 and firmicutes bacteria including Clostridium, Bacillus, and Dialister sp.

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

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.

Overview of Anaerobic Treatment: Thermophilic and Propionate Implications ‐ Keynote Address—Association of Environmental Engineering and Science Professors—78th Annual Water Environment Federation Technical Exposition and Conference, Washington, D.C., Oct. 29–Nov. 2, 2005

Difficulties in achieving low propionate concentrations in anaerobically treated effluents are frequently reported in the literature (Ahring, 1994; Kugelman and Guida, 1989; Rimkus et al., 1982), especially at thermophilic temperatures, with concentrations as high as 1000 to 9600 mg/L sometimes produced. This paper will detail the effect of several variables on the performance of both mesophilic and thermophilic regimes. Studies concerning the effect of the following four important factors on performance are included: reactor configuration, inorganic nutrient supplementation, substrate characteristics, and the unique role of microbial consortia proximity in enhancing performance. Reactor configuration modifications, essential nutrient additions, and the importance of close microbial proximity were all found to contribute to improvement in thermophilic anaerobic digestion in all the studies. It was found that, in substrates that shunt significant amounts of the electron donor through propionate, performance was critically related to reactor optimization, with propionate removal efficiency considerably improved using intact upflow anaerobic sludge blanket granules, less so in a homogenized granule slurry blanket, and noticeably reduced even more when the completely stirred reactor configuration of homogenized granules was used. The critical importance of extremely close microbial consortia proximity in maintaining hydrogen intermediates at very low levels to efficiently convert propionate to hydrogen and acetate was demonstrated. Compared to mesophilic digestion, thermophilic digestion manifested elevated levels of propionate, except in the nonmixed reactors, which had close microbial consortia proximity. The reactor configuration with the best results was the anaerobic digestion elutriated phased treatment (ADEPT) scheme, in which the raw sludge was elutriated of its fermenting volatile fatty acids, as they are generated in a short 5- to 8-day solids retention time (SRT) in one reactor and the elutriate then metabolized by passing up through a methanogenic granule or slurry blanket (with its close microbial consortia proximity) in a separate reactor with a 20- to 50-day SRT. Loading rates and performance of the ADEPT reactor configuration were superior to the standard continuously stirred tank reactor, and ADEPT thermophilic temperatures allowed higher organic loading rates without high propionate concentrations in the effluent.

Diversity, Localization, and Physiological Properties of Filamentous Microbes Belonging to Chloroflexi Subphylum I in Mesophilic and Thermophilic Methanogenic Sludge Granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.

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.

Mechanisms and models for anaerobic granulation in upflow anaerobic sludge blanket reactor

Upflow anaerobic sludge blanket (UASB) reactor has been employed in industrial and municipal wastewater treatment for decades. However, the long start-up period required for the development of anaerobic granules seriously limits the application of this technology. In order to develop the strategy for rapid UASB start-up, the mechanisms for anaerobic granulation should be understood. This paper attempts to provide a up-to-date review on the existing mechanisms and models for anaerobic granulation in the UASB reactor, which include inert nuclei model, selection pressure model, multi-valence positive ion-bonding model, synthetic and natural polymer-bonding model, Capetown's model, spaghetti theory, syntrophic microcolony model, multi-layer model, secondary minimum adhesion model, local dehydration and hydrophobic interaction model, surface tension model, proton translocation-dehydration theory, cellular automaton model and cell-to-cell communication model. Based on those previous works, a general model for anaerobic granulation is also proposed. It is expected that this paper would be helpful for researchers to further develop a unified theory for anaerobic granulation and technology for expediting the formation of the UASB granules.

Removal of 3-chlorobenzoate using an upflow anaerobic sludge blanket reactor under light conditions

The possibility of 3-chlorobenzoate removal from water using an upflow anaerobic sludge blanket (UASB) reactor without the addition of any extra dechlorinating culture under light conditions has been studied on a laboratory scale. Benzoate removal was observed in the first three months of operation under light conditions, but the 3-chlorobenzoate removal was not observed. After three months of operation under light conditions, the 3-chlorobenzoate concentration in the UASB reactor effluent gradually decreased to less than 1 mg x h(-1). The 3-chlorobenzoate concentration in the effluent did not increase under dark conditions. The DOC concentration in the effluent decreased according to the removal of the 3-chlorobenzoate by the UASB granules. These results indicated that granules in the UASB reactor provided the 3-chlorobenzoate removability after 80-100 d of adaptation to the 3-chlorobenzoate, and that the UASB reactor is useful for 3-chlorobenzoate removal.

Microbial distribution in UASB granules and its resulting effects

Microscopic (SEM and TEM) examinations of biogranules sampled from various UASB (Upflow Anaerobic Sludge Blanket) reactors indicated that microbes are densely packed. The microbial distribution is strongly dependent upon the degradation thermodynamics and kinetics of individual substrates. Biogranules degrading carbohydrates exhibited typically a layered distribution with a surface layer populated with hydrolytic/fermentative acidogens, a mid-layer comprising syntrophic colonies and an interior comprising acetotrophic methanogens. On the other hand, those substrates having a rate-limiting hydrolytic/fermentative step did not exhibit any layered pattern; instead, bacteria were interwined and distributed evenly. These observations have two implications. Biogranules are developed through evolution instead of random aggregation of suspended microbes. Furthermore, biogranules should be less vulnerable to the changes of mixed liquor condition, because the large majority of microbes inside the biogranules are shielded from the hostile mixed liquor environment. The latter is supported by experimental evidence that biogranules are more resistant than suspended sludge to the toxicity of hydrogen sulfide, heavy metals and aromatic pollutants in wastewater.

Wastewater treatment and poly-β-hydroxybutyrate production using lighted upflow anaerobic sludge blanket method

We investigated the performance of a lighted upflow anaerobic sludge blanket (LUASB) reactor for wastewater treatment and poly-β-hydroxybutyrate (PHB) production. Phototrophic bacteria were induced from UASB (upflow anaerobic sludge blanket) granules under light conditions (100 μE•m −2•s −1). The ammonium and phosphate ion removal efficiencies of the LUASB reactor were higher than those of the UASB reactor. The difference in the results from runs under light and dark conditions suggested that the ammonium and phosphate ion removal efficiencies were improved by increasing the amount of phototrophic bacteria in the LUASB reactor. The average production rate of PHB from the biomass in the effluent from the LUASB reactor was 6.6–14.0mg•l −1-reactor•d −1 using acetate-based media and the average PHB content based on the dry bacterial biomass was 15.1–25.3%. The PHB concentration increased by reincubation of the effluent from the LUASB reactor with sodium acetate under light conditions. The UASB granular sludge can decompose a variety of organic substances and in addition the LUASB method can remove ammonium and phosphate ions. The LUASB method thus appears to be appropriate for wastewater treatment and production of phototrophic bacteria and PHB from various wastewaters.

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 μl 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.

Startup of thermophilic (55°C) UASB reactors using different mesophilic seed sludges

Performances during startup of three 2.8-litre UASB (upflow anaerobic sludge blanket) reactors operated under thermophilic condition were investigated. All reactors were seeded with mesophilic sludges: one with flocculent digester sludge (Reactor-F), another with UASB granules (Reactor-G), and the third with disintegrated granules (Reactor-D). The reactors were operated in parallel at 55°C and 24 hours of retention time, using sucrose and milk as substrate at COD (chemical oxygen demand) loadings up to 10 g-COD/l·day. Immediately after temperature was step-increased from 37°C to 55°C, all reactors encountered sludge washout and deterioration of COD removal efficiency; however, the impact of temperature increase was more severe on Reactor-F. Sludge granulation took place in all reactors; first granules became noticeable after 45 days in Reactor-D, and after 90 days in Reactor-F. Reactor-G and Reactor-D were capable of removing 95% of soluble COD after 75 days, while Reactor-F after 110 days. Throughout this study, there was little difference in performance between Reactors G and D. The thermophilic granule were estimated to have a yield of 0.099 g-VSS/g-COD, and a methanogenic activity of 0.71-1.55 g-methane-COD/g-VSS·day, comparable to that of mesophilic granules.

Startup of thermophilic (55°C) UASB reactors using different mesophilic seed sludges

Performances during startup of three 2.8-litre UASB (upflow anaerobic sludge blanket) reactors operated under thermophilic condition were investigated. All reactors were seeded with mesophilic sludges: one with flocculent digester sludge (Reactor-F), another with UASB granules (Reactor-G), and the third with disintegrated granules (Reactor-D). The reactors were operated in parallel at 55°C and 24 hours of retention time, using sucrose and milk as substrate at COD (chemical oxygen demand) loadings up to 10 g-COD/l·day. Immediately after temperature was step-increased from 37°C to 55°C, all reactors encountered sludge washout and deterioration of COD removal efficiency; however, the impact of temperature increase was more severe on Reactor-F. Sludge granulation took place in all reactors; first granules became noticeable after 45 days in Reactor-D, and after 90 days in Reactor-F. Reactor-G and Reactor-D were capable of removing 95% of soluble COD after 75 days, while Reactor-F after 110 days. Throughout this study, there was little difference in performance between Reactors O and D. The thermophilic granule were estimated to have a yield of 0.099 g-VSS/g-COD, and a methanogenic activity of 0.71–1.55 g-methane-COD/g-VSS·day, comparable to that of mesophilic granules.

Evaluation of Two Upflow Anaerobic Digesters Purifying Industrial Wastewaters High in Organic Matter

Two full-scale anaerobic digesters, one a clarigester purifying a maize processing wastewater and the other with an upflow anaerobic sludge blanket (UASB) configuration treating brewery effluent, contained well settling, granular sludges efficient in pollutant removal. Due to differences in both digester design and feed composition, the sludges differed in activity and microbial population. The clarigester granules contained a diverse population with a multiformity of hydrolytic, acidogenic and acetogenic bacteria while the predominant methanogens, in order of significance, were Methanothrix and Methanosarcina. These granules did not reconstitute on re-start up following digester shutdown and possible reasons for this are discussed. The UASB granules contained a more uniform population with three major microbial morphotypes, the predominant methanogens being Methanothrix and, possibly, Methanobacterium. In this paper the differences in digester design, feed composition, sludge microbiology and process performance are discussed.

Qualitative Changes in Upflow Anaerobic Sludge Blanket Granules during Extracellular Polymer Extraction by Autoclaving.

Extracellular or surface polymers play a major role as a matrix keeping the cells together and mediating the adhesion of bacteria in natural or man-made eco-systems. It is presumptive that the bacterial aggregation in upflow anaerobic sludge blanket reactors is brought about in the form of granules by the combination of extracellular polymeric material and inorganic ash. The extracellular polymeric material in the granules of upflow anaerobic sludge blanket reactor was extracted by the autoclaving technique and qualitative changes in the granules before and after autoclaving were investigated with the help of scanning electron microscopy. Autoclaving for thirty, sixty and ninety minutes intervals was carried out and the effect of autoclaving time on the extraction of extracellular polymeric material was also studied. Most of the extracellular polymeric material could be extracted at 30 min autoclaving time without significant damage to the bacterial cells. 90 min of autoclaving resulted in the release of slightly more extracellular material but caused damage to the cells. 30 min autoclaving time seems to be suitable for the extraction of extracellular polymeric material from the upflow anaerobic sludge blanket granular sludges.