Contact Angle Measurement and Cell Hydrophobicity of Granular Sludge from Upflow Anaerobic Sludge Bed Reactors

The contact angle, which is generally used to evaluate the hydrophobicities of pure bacterial strains and solid surfaces, was used to study mixed cell cultures of bacteria involved in anaerobic digestion. Previously published data and data from this study showed that most acidogens are hydrophilic (contact angle, <45(deg)) but most of the acetogens and methanogens isolated from granular sludge are hydrophobic (contact angle, >45(deg)). The hydrophobicities of mixtures of hydrophilic and hydrophobic cells were found to be linearly correlated with the cell mixing ratio. The hydrophobicities of cells present in effluents from upflow anaerobic sludge bed reactors which were treating different types of substrates were different depending on the reactor conditions. When the reactor liquid had a high surface tension, cells sloughing off from sludge granules, as well as cells present on the outer surfaces of the granules, were hydrophobic. Short-term batch enrichment cultures revealed that proteins selected for highly hydrophilic cells. Long-term in-reactor enrichment cultures revealed that sugars selected for hydrophilic acidogens on the surfaces of the granules, while fatty acids tended to enrich for hydrophobic methanogens. When linear alkylbenzenesulfonate was added, the cells on the surfaces of granules became more hydrophilic. Control tests performed with pure cultures revealed that there was no change in the surface properties due to linear alkylbenzenesulfonate; hence, the changes in the wash-out observed probably reflect changes in the species composition of the microbial association. A surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity could be distinguished in the grey granules which we studied.     

Hydrophobicities and Electrophoretic Mobilities of Anaerobic Bacterial Isolates from Methanogenic Granular Sludge

The hydrophobicities and electrophoretic mobilities of isolates from methanogenic anaerobic granular sludge were measured and compared with those of strains from culture collections. All new isolates were highly hydrophobic, indicating that the upflow anaerobic sludge blanket reactor concept selects for hydrophobic bacteria. Methanothrix soehngenii, a methanogen often observed in methanogenic granular sludge, was highly hydrophobic and showed low electrophoretic mobility at pH 7. The role of this strain in the formation of methanogenic granular sludge is discussed.     

Structural analysis of anaerobic granules in a phase separated reactor by electron microscopy

This paper discusses the microbial community structure of anaerobic granules and the effect of phase separation in anaerobic reactor on the characteristics of granules. Electron micrographs revealed that the core of anaerobic granular sludge consists predominantly of Methanosaeta-like cells, a key microorganism in granulation process. Granules in the methanogenic dominant zone of the reactor were stable and densely packed with smooth regular surface. On the other hand, granules subjected to acidogenic activities were less stable structures with broken parts and an irregular fissured surface. Anaerobic granules consisted of a vast diversity of species from the outer surface to the core of the granule and possessed a multi-layered structure. Viruses in the granules suggests the presence of bacteriophage in the granular biomass. These could be responsible for destroying cells and weakening the internal structure of granules, and thus possibly causing the breaking of granules. The observation of protozoa-like microorganism on the exterior zone of granular structure is believed to play an important role as bacterial predator and control the growth of bacterial cells. The images observed in this study shows that anaerobic granule harbour diverse number of microbial species, and act differently in acidogenic and methanogenic microbial zones.     

Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Different types of sludge granules in UASB reactors treating acidified wastewaters

The influence of a high energy substrate, i.e. sucrose, on the granular sludge yield and the development of different types of granular sludge was investigated by using Upflow Anaerobic Sludge Bed (UASB) reactors fed with synthetic wastewater. The feed COD was a mixture of volatile fatty acids (VFA) i.e., 20, 40, and 40% of the COD as C₂-, C₃-, and C₄-VFA, respectively. Furthermore, experiments were carried out in which 10 and 30% of the VFA COD was substituted with sucrose. The following distinctly different types of granules were observed in each testrun: in the reactor fed with solely VFA, black (B) and white (W) granules developed; in the reactor fed with a mixture of 90% VFA and 10% sucrose, three types of granules i.e., B, W, and grey (G) granules could be seen; in the reactor fed with 70% VFA and 30% sucrose, only W and G granules were found. The granular sludge yield increased proportional to the amount of sucrose COD. At steady-state performance of the reactors, specific acidogenic (SAA) and methanogenic (SMA) activity tests on these granules revealed that B granules had the highest SMA with low SAA. The W granules had very high SMA with low SAA. G granules gave the highest SAA with a considerable SMA. Measurement of coenzyme F₄₂₀ revealed that B granules consist mainly of acetoclastic methanogens. The fore-mentioned tests were supplemented with analyses of the wash-out cells present in the reactor effluent and the results suggested that acidogens, if present, prevail at the granule surface. The B granules were particularly rich in Ca, Mn, and Zn minerals. The size distribution analysis showed that the granule diameter increased in the following order: B相似文献   

Inhibition of Methanogenesis from Acetate in Granular Sludge by Long-Chain Fatty Acids

The effect of four saturated long-chain fatty acids (caprylic, capric, lauric, and myristic) and one unsaturated long-chain fatty acid (oleic) on the microbial formation of methane from acetate was investigated in batch anaerobic toxicity assays. The tests were carried out with granular sludge from an upflow anaerobic sludge bed reactor. In this sludge, Methanothrix spp. are the predominant acetoclastic methanogens. Lauric acid appeared to be the most versatile inhibitor: inhibition started at 1.6 mM, and at 4.3 mM the maximum specific acetoclastic methanogenic activity had been reduced to 50%. Caprylic acid appeared to be only slightly inhibitory. Oleic acid was almost as inhibitory as lauric acid. Although adsorption of the inhibitor on the cell wall might play an important role in the mechanism of inhibition, the inhibition was found to be correlated with concentration rather than with the amount per unit of biomass. In practical situations, as in anaerobic waste treatment processes, synergism can be expected to enhance the inhibition of methanogenesis. In the present research a background concentration of lauric acid below its MIC strongly enhanced the toxicity of capric acid and (to an even greater extent) myristic acid.     

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     

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     

High rate treatment of terephthalic acid production wastewater in a two-stage anaerobic bioreactor

The feasibility was studied of anaerobic treatment of wastewater generated during purified terephthalic acid (PTA) production in two-stage upflow anaerobic sludge blanket (UASB) reactor system. The artificial influent of the system contained the main organic substrates of PTA-wastewater: acetate, benzoate, and terephthalate. Three parallel operated reactors were used for the second stage, and seeded with a suspended terephthalate degrading culture, with and without additional methanogenic granular sludge (two different types). The first stage UASB-reactor was seeded with methanogenic granular sludge. Reactors were operated at 37 degrees C and pH 7. During the first 300 days of operation a clear distinction between the biomass grown in both reactor stages was obtained. In the first stage, acetate and benzoate were degraded at a volumetric loading rate of 40 g-COD/L . day at a COD-removal efficiency of 95% within the first 25 days of operation. No degradation of terephthalate was obtained in the first stage during the first 300 days of operation despite operation of the reactor at a decreased volumetric loading rate with acetate and benzoate of 9 g-COD/L . day from day 150. Batch incubation of biomass from the reactor with terephthalate showed that the lag-phase prior to terephthalate degradation remained largely unchanged, indicating that no net growth of terephthalate degrading biomass occurred in the first stage reactor. From day 300, however, terephthalate degradation was observed in the first stage, and the biomass in this reactor could successfully be enriched with terephthalate degrading biomass, resulting in terephthalate removal capacities of 15 g-COD/L . day. Even though no single reason could be identified why (suddenly) terephthalate degradation was obtained after such a long period of operation, it is suggested that the solid retention time as well the prevailing reactor concentrations acetate and benzoate may have played an important role. From day 1 of operation, terephthalate was degraded in the second stage. In presence of methanogenic granular biomass, high terephthalate removal capacities were obtained in these reactors (15 g-COD/L . day) after approximately 125 days of operation. From the results obtained it is concluded that terephthalate degradation is the bottleneck during anaerobic treatment of PTA-wastewater. Pre-removal of acetate and benzoate in staged bioreactor reduces the lag-phase prior to terephthalate degradation in latter stages, and enables high rate treatment of PTA-wastewater.     

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.     

The performance of a phase separated granular bed bioreactor treating brewery wastewater

This study presents the performance characteristics of a plug flow phase separated anaerobic granular bed baffled reactor (GRABBR) fed with brewery wastewater at various operating conditions. The reactor achieved chemical oxygen demand (COD) removal of 93-96% with high methane production when operated at organic loading rates (OLRs) of 2.16-13.38kg COD m(-3)d(-1). The reactor configuration and microbial environment encouraged the acidogenic dominant zone to produce intermediate products suitable for degradation in the predominantly methanogenic zone. Noticeable phase separation between acidogenesis and methanogenesis mainly occurred at high OLR, involving a greater number of compartments to contribute to wastewater treatment. The highly active nature and good settling characteristics of methanogenic granular sludge offered high biomass retention and enhanced methanogenic activities within the system. The granular structure in the acidogenic dominant zone of the GRABBR was susceptible to disintegration and flotation. Methanogenic granular sludge was a multi-layered structure with Methanosaeta-like organisms dominant in the core.     

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Methanogenesis in an Upflow Anaerobic Sludge Blanket Reactor at pH 6 on an Acetate-Propionate Mixture

High-rate anaerobic digestion can be applied in upflow anaerobic sludge blanket reactors for the treatment of various wastewaters. In upflow anaerobic sludge blanket reactors, sludge retention time is increased by a natural immobilization mechanism (viz. the formation of a granular type of sludge). When this sludge is cultivated on acid-containing wastewater, the granules mainly consist of an acetoclastic methanogen resembling Methanothrix soehngenii. This organism grows either in rods or in long filaments. Attempts to cultivate a stable sludge consisting predominantly of Methanosarcina sp. on an acetate-propionate mixture as substrate by lowering the pH from 7.5 during the start-up to approximately 6 failed. After 140 days of continuous operation of the reactor a filamentous organism resembling Methanothrix soehngenii prevailed in the sludge. The specific methanogenic activity of this sludge on acetate-propionate was optimal at pH 6.6 to 6.8 and 7.0 to 7.2, respectively.     

Influence of co-substrates on structure of microbial aggregates in long-chain fatty acid-fed anaerobic digesters

AIMS: The purpose of this study was to investigate the influence of co-substrates, such as glucose and cysteine, on the structure of microbial aggregates in anaerobic digesters treating oleate, a long-chain fatty acid (LCFA). METHODS AND RESULTS: Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to examine the structure of microbial aggregates. Fluorescence in situ hybridization (FISH) techniques were also used to characterize and localize the different trophic groups present in the aggregates. Oleate was found to inhibit the methanogenic activity and formation of granular biomass in digesters. The addition of co-substrates, such as glucose and cysteine either singly or in combination, increased the methanogenic activity and formation of granular biomass. Glucose was more effective than cysteine in reducing the inhibition by oleate on the methanogenic bacteria and in enhancing the formation of granules. CONCLUSIONS: The addition of nutrient substrate, such as glucose and cysteine could decrease the toxicity of LCFA on anaerobic granulation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the addition of other substrates might decrease the toxicity of LCFA on the granulation of biomass in anaerobic digesters and enhance methanogenic activity. A combination of TEM, CLSM and FISH techniques provides a better tool for visualizing microbial aggregates and for differentiating and localizing different microbial groups within these aggregates.     

Improved Dechlorinating Performance of Upflow Anaerobic Sludge Blanket Reactors by Incorporation of Dehalospirillum multivorans into Granular Sludge

Dechlorination of tetrachloroethene, also known as perchloroethylene (PCE), was investigated in an upflow anaerobic sludge blanket (UASB) reactor after incorporation of the strictly anaerobic, reductively dechlorinating bacterium Dehalospirillum multivorans into granular sludge. This reactor was compared to the reference 1 (R1) reactor, where the granules were autoclaved to remove all dechlorinating abilities before inoculation, and to the reference 2 (R2) reactor, containing only living granular sludge. All three reactors were fed mineral medium containing 3 to 57 μM PCE, 2 mM formate, and 0.5 mM acetate and were operated under sterile conditions. In the test reactor, an average of 93% (mole/mole) of the effluent chloroethenes was dichloroethene (DCE), compared to 99% (mole/mole) in the R1 reactor. The R2 reactor, with no inoculation, produced only trichloroethene (TCE), averaging 43% (mole/mole) of the effluent chloroethenes. No dechlorination of PCE was observed in an abiotic control consisting of sterile granules without inoculum. During continuous operation with stepwise-reduced hydraulic retention times (HRTs), both the test reactor and the R1 reactor showed conversion of PCE to DCE, even at HRTs much lower than the reciprocal maximum specific growth rate of D. multivorans, indicating that this bacterium was immobilized in the living and autoclaved granular sludge. In contrast, the R2 reactor, with no inoculation of D. multivorans, only converted PCE to TCE under the same conditions. Immobilization could be confirmed by using fluorescein-labeled antibody probes raised against D. multivorans. In granules obtained from the R1 reactor, D. multivorans grew mainly in microcolonies located in the centers of the granules, while in the test reactor, the bacterium mainly covered the surfaces of granules.     

Heterogeneous Distribution of Microbial Activity in Methanogenic Aggregates: pH and Glucose Microprofiles

Methanogenic aggregates, harvested from an upflow anaerobic sludge blanket reactor treating potato starch wastewater, were acclimatized to either glucose or a mixture of sugars and organic nitrogen compounds (i.e., diluted molasses). Both types of granules exhibited internal pH and substrate concentration gradients in mineral medium (pH 7.0, 30°C) as was measured with microelectrodes. Glucose-acclimatized granules suspended in a mineral medium lacking glucose exhibited a distinct internal pH decrease of about 1 U within the granule, suggesting strong metabolism by the acidogenic bacteria. Molasses-acclimatized and aged granules suspended in mineral medium did not exhibit such a pH decrease, suggesting the importance of the metabolic state of these acidogens. The pH gradient did not occur in deactivated granules and was not observable in strongly buffered media (mineral medium containing 33 mM phosphate or reactor liquid). When glucose (0.5 to 5.0 mM) was added to the mineral medium, granules exhibited a convex pH profile. Glucose consumption was located exclusively in the outer 200 to 300 μm of the aggregates (mean diameter = 1.5 mm). The addition of 20 mM 2-bromoethanesulfonic acid to the mineral medium indicated that the higher pH levels in the centre of the granule appeared to be related to the activity of methanogens. It is suggested that acidogenic activity occurs predominantly in the outer 200 to 300 μm of the aggregate and methanogenic activity occurs predominantly in the center of the investigated granules.     

Degradation of Methanethiol by Methylotrophic Methanogenic Archaea in a Lab-Scale Upflow Anaerobic Sludge Blanket Reactor

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30°C to H₂S, CO₂, and CH₄. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter⁻¹ day⁻¹. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.     

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     

Cultivation of low-temperature (15°C), anaerobic, wastewater treatment granules

Aims: Anaerobic sludge granules underpin high‐rate waste‐to‐energy bioreactors. Granulation is a microbiological phenomenon involving the self‐immobilization of several trophic groups. Low‐temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low‐temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15°C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. Methods and Results: One mesophilic (R1; 37°C) and two low‐temperature (R2 and R3, 15°C) laboratory‐scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0·4 mm) granules and were fed a glucose‐based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low‐temperature (LT) bioreactors. Conclusions: Granulation was observed at 15°C in anaerobic bioreactors and was associated with H₂/CO₂‐mediated methanogenesis and distinct community structure development. Significance and Impact of the Study: Granulation underpins high‐rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20°C was not previously documented.