Methanogenic Granules Research Articles

Effect of storage on the performance of methanogenic granules

Anaerobic methanogenic granules were stored under anaerobic condition at 4° and 22°C for 1- to 18-month period to evaluate the effect of storage on degradation of volatile fatty acids (VFAs), including acetate, propionate and butyrate, and on methane production. The length of storage period affected the activities of different microbial trophic groups. During storage at 22°C, the degradation rates for all the three acids decreased gradually. At low temperature (4°C), reduction in degradation rates of acetate and propionate was relatively slower than that at 22°C. Reduction in butyrate degradation rate was faster (by 45%) during the first month of storage at 4°C, but the rate declined afterwards. Nevertheless, the granules maintained, although at reduced level, their metabolic activities for all three VFAs even after storage for 18 months. Higher decay coefficients were obtained at 22°C than those at 4°C. For a relatively short period (1–5 months), granules can be stored at ambient temperature (approx. 20–22°C) with limited loss in their VFA degradation rates. However, granules can maintain higher levels of VFA degradation rates when they are stored at low (4°C) rather than at ambient temperature. Reactor studies indicated that the granules can completely recover their original VFA degradation rates in three days when stored for 31 d at 22°C. The granules stored at 22°C for 9 months were used successfully as inoculum to start a laboratory-scale reactor. The original VFA degradation rates of the granules were achieved after 15–20 d of reactor operation at 35°C.

Acceleration of mass transfer in methane-producing loop reactors.

Gas bubbles entrapped in methanogenic granules subjected to hydrostatic pressure oscillations during recirculation in loop reactors will induce intraparticle liquid flows, and thereby enhance mass transfer in excess of diffusion. This 'breathing particle' concept was clearly demonstrated in a well defined inorganic model system. The experimental results could be described satisfactory with a structured mathematical model, while a 30% improvement is predicted for methanogenic loop reactors as compared to constant pressure systems. It is concluded that acceleration of mass transfer in gas-producing systems offers challenging perspectives for both heterogeneous catalysis and biological fermentations.

Size distribution model for methanogenic granules from full scale UASB and IC reactors

The size distribution of methanogenic granules from ten full scale UASB and Internal Circulation (IC) reactors were studied at regular time intervals. A size distribution model was developed based on these data and additional lab-scale experiments. Full scale data were used, since the hydrodynamic conditions in full scale reactors influencing the size distribution cannot be simulated properly in lab scale reactors. It was concluded that breaking and/or disintegration did not significantly contribute to the size distribution. Washout of granules is under normal conditions negligible, since the settling velocity is much higher than the liquid upflow velocity. Gradient measurements showed that the sludge bed is well mixed. Thus, sluicing will not systematically remove large granules. The most significant process limiting the maximum granule size in normal operation is the regular sluicing of surplus biomass. Shear forces appear to have no influence on the size distribution. High numbers of precursors in the influent result in short size distributions, while in wastewaters with little or no suspended solids wide size distributions can be found. The developed size distribution model accurately described the wider size distributions found in IC-reactors. IC-granules are larger and less strong as compared to UASB granules. Furthermore, it was concluded that operating the systems at high loadings results in granules with a lower strength. The density of methanogenic granules is strongly correlated with the ash content.

Methanogenic granule development in full scale internal circulation reactors

Internal Circulation (IC) reactors can be operated at higher reactor volume loading rates than Upflow Anaerobic Sludge Blanket (UASB) reactors. This results in increased gas production rates and subsequently higher average shear rates in IC-reactors. Furthermore, the liquid upflow velocity is 8-20 times higher, still granules develop successfully in IC-reactors. To investigate the granule development in IC-reactors and elucidate the process limitations with respect to granule development and biomass retention, granule samples from three full scale IC-reactors are characterized. Characterization included size distribution, strength, settling velocity, density, ash content and methanogenic activity. Granules were compared with samples from UASB reactors treating similar types of wastewaters. A hydrodynamic model was developed to describe the liquid circulation in IC reactors. The average shear rate in IC reactors is approximately twice as high compared to UASB-reactors. The two stage design of the IC-reactor allows 3-6 times higher loading rate. The experimental results showed that IC-granules are larger than UASB-granules grown on similar wastewater, while the strength of IC-granules is lower as a result of the higher sludge loading rate. Although wash-out is slightly enhanced in IC-reactors, the conditions in the second stage are tranquil enough to ensure adequate biomass retention in IC-reactors. The development of characteristic IC-granules after seeding proceeds within a few months. Physical characteristics of granules are determined mainly by biological factors.

Metabolic properties and kinetics of methanogenic granules

Two types of mesophilic methanogenic granules (R- and F-granules) were developed on different synthetic feeds containing acetate, propionate and butyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H2-CO2 were related to the feed composition used for their development. These granules performed a reversible reaction between H2 production from formate and formate synthesis from H2 plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and butyrate during butyrate or isobutyrate degradation. Valerate and 2-methylbutyrate were produced and consumed during propionate-butyrate degradation. The respective apparent Km (mm) for various substrates in disrupted R- and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an optimum temperature range from 40 to 50° C for H2-CO2 and formate utilization and 40° C for acetate, propionate and butyrate utilization and a similar optimum pH.

Comparison of rod- versus filament-type methanogenic granules: microbial population and reactor performance

Two types of methanogenic granules capable of high chemical oxygen demand removal rates were developed in laboratory-scale upflow reactors at 35° C. One granule type (R-granules) had a rod-type Methanothrix-like species as the predominant species whereas the other (F-granules) had a filament-type M. soehngenii-like acetate-utilizer as the predominant species. These two types of granules were compared in terms of operational performance, physical-chemical characteristics and microbial population. The R-granules had a higher density [65–70 vs 39–43 g suspended solids (SS)/l], specific gravity (1.03 vs 1.01) and specific volumetric methane production rate (180 vs 120 l CH4/l granules per day) than the F-granules. Acetate, propionate and butyrate degraders in both types of granules had similar specific growth rates. The most probable number enumeration indicated that both types of granule had the same population levels (cells/g SS) in terms of methanogens (H2-CO2-, formate- and acetate-utilizing) and syntrophic acetogens. Hydrolytic-fermentative bacteria were present in greater number in the F-granules than in the R-granules. The R-granules had a higher cell density than the F-granules. The differences in operational performance were due mainly to their different microbial composition, especially the predominant acetate-utilizing methanogens in the granules. The long-filamentous M. soehngenii-like rods in the F-granules appeared to be responsible for their lower density and large-sized granules.

Microbial composition and characterization of prevalent methanogens and acetogens isolated from syntrophic methanogenic granules

The microbial species composition of methanogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (1012/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (1011/g dry weight), but low numbers of hydrolytic-fermentative bacteria (109/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospirillum hungatei strain BD, and a non-filamentous, bamboo-shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading sporeforming oval-shaped species, strain PT, and a propionate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophically with an H2-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, acetate and butyrate but they were involved in propionate degradation.

Anaerobic Digestion of Distillery Wastewater in a Two-Phase UASB System

A two-phase UASB system was operated for high-rate treatment of concentrated distillery wastewater. The phase separation was obtained by adjusting pH in each reactor. When influent SS concentration was 4.1 g/l, the first phase UASB reactor was effectively operated up to the loading rate of 16.5 kg COD/m3.day, producing 3.9 g HAc/l.day. In the methanogenic UASB reactor, loading rates up to 44 kg COD/m3.day could be applied while removing 80 % of influent COD with a specific gas production of 16.5 l/l.day. After the formation of granular sludge in both reactors, it was possible to maintain the appropriate pH in the first phase only by recirculating the effluent from the methanogenic phase without the addition of alkaline chemicals. The morphological study on the granular sludge consortia was made with both scanning and transmission electron microscopes. It was interesting to find the granules of different color and shape in the acidogenic phase in 90 days and in the methanogenic phase in 120 days after start-up. The acidogenic granular sludge mainly consisted of long chains of large rods, short plump rods, and various size of cocci, which have a different shape and cytoplasmic appearance from common methanogenic bacteria. Whereas the long multicellular filaments of Methanothrix spp. were prevalent in the methanogenic granule, diverse bacteria were entrapped therein.

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.

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/cm 3 (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 × 10 8, 1.6 × 10 10, and 2 × 10 9 (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.

Chemical composition and kinetic properties of granular methanogenic sludge grown on propionate

The chemical composition and kinetic properties of propionate-grown methanogenic granules were analyzed as a function of the diameter of the granules (range: 0.1–3.0 mm in diameter). No significant variation in the chemical composition of the granules was observed. All granules contained large amounts of ash (between 53 and 59%). Extracellular polymers extracted from the granules were equivalent to 86–99 mg/g mixed-liquor volatile suspended solids (MLVSS), in which proteins, carbohydrates, and nucleic acids were the main constituents. The apparent substrate saturation constant (K s) value for propionate utilization was clearly influenced by the diameter of granules, although the maximum rate of propionate utilization was virtually constant, irrespective of the diameter. This indicates that the influence of mass transfer limitation was more pronounced in larger granules, especially at lower propionate concentrations.