A half-submerged integrated two-phase anaerobic reactor for agricultural solid waste codigestion

Anaerobic digestion is widely used in bioenergy recovery from waste. In this study, a half-submerged, integrated, two-phase anaerobic reactor consisting of a top roller acting as an acidogenic unit and a recycling bottom reactor acting as a methanogenic unit was developed for the codigestion of wheat straw (WS) and fruit/vegetable waste (FVW). The reactor was operated for 21 batches (nearly 300 d). Anaerobic granular sludge was inoculated into the methanogenic unit. The residence time for the mixed waste was maintained as 10 d when the operation stabilized, and the temperature was kept at 35 °C. The highest organic loading rate was 1.37 kg VS/(m³ d), and the maximum daily biogas production was 328 L/d. Volatile solid removal efficiencies exceeded 85%. WS digestion could be confirmed, and efficiency was affected by both the ratio of WS to FVW and the loading rate. The dominant bacteria were Bacteroides-like species, which are involved in glycan and cellulose decomposition. Methanogenic community structures, pH levels, and volatile fatty acid concentrations in the acidogenic and methanogenic units differed, indicating successful phase separation. This novel reactor can improve the mass transfer and microbial cooperation between acidogenic and methanogenic units and can efficiently and steady codigest solid waste.     

Extraction of extracellular polymeric substances (EPS) of sludges

The efficacies of extracting extracellular polymeric substances (EPS) from aerobic, acidogenic and methanogenic sludges using EDTA, cation exchange resin and formaldehyde under various conditions were compared. Results show that formaldehye plus NaOH was most effective in extracting EPS for all sludges; only 1.1-1.2% of DNA in the sludge samples were detected, suggesting the EPS extracted were not contaminated by intracellular substances. For each gram of volatile solids, formaldehyde-NaOH extracted 165, 179 and 102 mg of EPS from aerobic, acidogenic and methanogenic sludges, respectively. All EPS were mainly composed of carbohydrate, protein and humic substance, plus small quantities of uronic acid and DNA. Carbohydrate was predominant in the acidogenic sludge (62% in the EPS extracted by formaldehyde-NaOH), whereas protein was predominant in the methanogenic sludge (41%). Humic substance, which has often been overlooked, accounted for 30.6, 8.4 and 22.8% of the extracted EPS from aerobic, acidogenic and methanogenic sludges, respectively. However, judging from EPS quantities estimated from confocal laser scanning microscopic observations, formaldehyde-NaOH extracted only a limited portion of EPS. Optimization of extraction procedures and/or development of a more effective extraction method are warranted.     

The effect of temperature and retention time on methane production and microbial community composition in staged anaerobic digesters fed with food waste

Background

Food waste is a large bio-resource that may be converted to biogas that can be used for heat and power production, or as transport fuel. We studied the anaerobic digestion of food waste in a staged digestion system consisting of separate acidogenic and methanogenic reactor vessels. Two anaerobic digestion parameters were investigated. First, we tested the effect of 55 vs. 65 °C acidogenic reactor temperature, and second, we examined the effect of reducing the hydraulic retention time (HRT) from 17 to 10 days in the methanogenic reactor. Process parameters including biogas production were monitored, and the microbial community composition was characterized by 16S amplicon sequencing.

Results

Neither organic matter removal nor methane production were significantly different for the 55 and 65 °C systems, despite the higher acetate and butyrate concentrations observed in the 65 °C acidogenic reactor. Ammonium levels in the methanogenic reactors were about 950 mg/L NH₄ ⁺ when HRT was 17 days but were reduced to 550 mg/L NH₄ ⁺ at 10 days HRT. Methane production increased from ~ 3600 mL/day to ~ 7800 when the HRT was decreased. Each reactor had unique environmental parameters and a correspondingly unique microbial community. In fact, the distinct values in each reactor for just two parameters, pH and ammonium concentration, recapitulate the separation seen in microbial community composition. The thermophilic and mesophilic digesters were particularly distinct from one another. The 55 °C acidogenic reactor was mainly dominated by Thermoanaerobacterium and Ruminococcus, whereas the 65 °C acidogenic reactor was initially dominated by Thermoanaerobacterium but later was overtaken by Coprothermobacter. The acidogenic reactors were lower in diversity (34–101 observed OTU_0.97, 1.3–2.5 Shannon) compared to the methanogenic reactors (472–513 observed OTU_0.97, 5.1–5.6 Shannon). The microbial communities in the acidogenic reactors were > 90% Firmicutes, and the Euryarchaeota were higher in relative abundance in the methanogenic reactors.

Conclusions

The digestion systems had similar biogas production and COD removal rates, and hence differences in temperature, NH₄ ⁺ concentration, and pH in the reactors resulted in distinct but similarly functioning microbial communities over this range of operating parameters. Consequently, one could reduce operational costs by lowering both the hydrolysis temperature from 65 to 55 °C and the HRT from 17 to 10 days.

     

Influence of the starting microbial nucleus type on the anaerobic granulation dynamics

 The influence of four different granulation precursors, syntroph-enriched methanogenic consortia, Methanosaeta-enriched, Methanosarcina-enriched nuclei and acidogenic flocs, on the time course of complex granule development and the lag time for start-up was investigated in four upflow anaerobic sludge-bed and filter reactors. Although the operational conditions allowed the maintenance of the same specific growth rate of biomass in the four reactors, granulation proceeded rapidly with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei. However, granulation was significantly retarded when acidogenic flocs were used as precursors. The granule mean Sauter diameter increased rapidly in the reactor inoculated with syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei and reached, at the end of the experiment, 3.1, 2.7 and 2.4 mm compared to 1.1 mm in that inoculated with acidogenic flocs. This corresponded to a rate of granule size increase of 31, 21, 18 μm/day in syntroph/methanogenic consortia, Methanosaeta and Methanosarcina nuclei, respectively, compared to 7 μm/day in acidogenic flocs. Biomass specific activities (i.e. acidogenic, syntrophic and methanogenic activities) increased stepwise in all reactors with time, especially in those inoculated with syntroph/methanogenic consortia and Methanosaeta nuclei. From these results it appears that syntrophs and Methanosaeta spp. play an important role in the anaerobic granulation process. Received: 25 January 1996 / Received revision: 3 September 1996 / Accepted: 13 September 1996     

Anaerobic digestion of cheese whey using an upflow anaerobic sludge blanket reactor: III. Sludge and substrate profiles

Anaerobic treatment of cheese whey using a 17·5 litre upflow anaerobic sludge blanket reactor was investigated in the laboratory over a range of influent concentration from 4·5 to 38·1 g COD litre⁻¹ at a constant hydraulic retention time of 5 days. The results indicated that two sludge distribution regions, a sludge bed and a sludge blanket, as well as two distinct reaction phases, acidogenic and methanogenic, were formed. However, as the substrate loading was increased, the acidogenic region extended into the methanogenic region in the upper portion of the reactor until the whole region was acidogenic, leading to the failure of the reactor.     

Determination of the Microbial Population in Thermophilic Anaerobic Reactor: Comparative Analysis by Different Counting Methods

This paper describes the determination of the microbial population, in terms of the number, biomass and composition, of single and two-phase, laboratory-scale thermophilic (55°C) anaerobic reactors, under steady-state conditions. Epifluorescence microscopy with DAPI (4′,6-diamidine-2-phenylindole) as fluorochrome was used to determine the total number of micro-organisms in the reactors, and autofluorescence microscopy for the number of the autofluroescent methanogenic populations. The results obtained by the direct count methods were compared to the quantity of biomass contained in the system, determined by volatile suspended solids. The viable bacterial population was determined by plating techniques using an anaerobic chamber. The total bacterial and F420 autofluorescent populations of single-stage digesters increase when the hydraulic retention time decreases; nevertheless, the percentages of the autofluorescent methanogens remain constant at 13%. In the two-stage reactors, the percentages of this group are 99% and 26% of the total population in the acidogenic and methanogenic factors, respectively. In the single-stage reactors, biomass determinations can be used to estimate microbial concentrations, and vice versa, as there is a high positive correlation between microorganism concentration and biomass. It was obtained a high correlation between direct counts by epifluorescence microscopy and viable plate counts for the combined system studied.     

Effect of pH on phase separated anaerobic digestion

A pilot scale experiment was performed for a year to develop a two-phase anaerobic process for piggery wastewater treatment (COD: 6,000 mg/L, BOD: 4,000 mg/L, SS: 500 gm/L, pH 8.4, alkalinity 6,000 mg/L). The acidogenic reactor had a total volume of 3 m³, and the methanogenic reactor, an, anaerobic up-flow sludge filter, combining a filter and a sludge bed, was also of total volume 3 m³ (1.5 m³ of upper packing material). Temperatures of the acidogenic and methanogenic reactors kept at 20°C and 35°C., respectively. When the pH of the acidogenic reactor was controlled at 6.0–7.0 with HCl, the COD removal efficiency increased from 50 to 80% over a period of six months, and as a result, the COD of the final effluent fell in the range of 1,000–1,500 mg/L. BOD removal efficiency over the same period was above 90%, and 300 to 400 mg/L was maintained in the final effluent. The average SS in the final effluent was 270 mg/L. The methane production was 0.32 m³ CH₄/kg COD_removed and methane content of the methanogenic reactor was high value at 80–90%., When the pH of the acidogenic reactor was not controlled over the final two months, the pH reached 8.2 and acid conversion decreased compared with that of pH controlled, while COD removal was similar to the pH controlled operation. Without pH control, the methane content in the gas from methanogenic reactor improved to 90%, compared to 80% with pH control.     

Composting potential of different inoculum sources in the modified SEBAC system treatment of municipal solid wastes

The aim of the work described here was to analyse the biomethanization process for three types of organic fraction of municipal solid waste (OFMSW) using two different inoculum sources in a sequential leach-bed anaerobic reactor under thermophilic (55 degrees C) and dry conditions (20%TS). The OFMSWs studied were: separately collected food waste (SC_OFMSW) from restaurants, synthetic waste (ST_OFMSW) and mechanically selected municipal waste (MS_OFMSW). The inoculum studied were digested mesophilic sludge (SLUDGE) and digested SC_OFMSW. The results show that SLUDGE gave the best performance and this was therefore selected for the rest of the experiments. Three assays were performed to analyse the biomethanization processes of three types of organic waste. The results suggest that all three wastes give rise to an acclimation stage with acidogenic/acetogenic activity between days 5 and 20-30 and a stabilization phase associated with methanogenic activity. In conclusion, a mixture of OFMSW (regardless of its origin) and SWINE arranged in layers in the reactor, as well as SLUDGE used with an inoculum source, enhanced the fast start up phase of a modified sequential leach-bed system under dry thermophilic conditions.     

Production of hydrogen and methane from organic solid wastes by phase-separation of anaerobic process

Phase-separated two-stage anaerobic process was examined and evaluated using artificial organic solid waste in laboratory scale. Acidogenic process, which was combined with subsequent methanogenic process using packed-bed reactor, was operated emphasizing on either hydrogen production, or solublizing efficiency of solid materials. In either effluent from hydrogenogenic, or solublizing operation, maximum allowable OLR achieved at methanogenesis was higher than the single methanogenic process. Hydrogenogenic operation was more suitable to combine methanogenic process than solublizing operation, since retention time of hydrogenogenic operation was much shorter than the solublizing operation, obtaining almost the same levels of overall removal efficiency in both COD and VSS. The combination of hydrogenogenic operation in acidogenic process and methanogenic process produced approximately 442mmoll-reactor(1)days(-1) of methane and 199mmoll-reactor(1)days(-1) of hydrogen at 25h of total retention time indicating 82% of COD removal with 96% of VSS decomposition.     

Effects of the acidogenic biomass on the performance of an anaerobic membrane bioreactor for wastewater treatment

Continuous flow experiments were performed to study the effects of acidogenic biomass development, induced by feeding with non-acidified substrate, on the operation and performance of an anaerobic membrane bioreactor (AnMBR). The AnMBR was operated at cross-flow velocities up to 1.5 m/s and fed with a gelatine–starch–ethanol mixture. A significant fraction of acidogenic biomass developed during reactor operation, which fully determined the sludge rheology, and influenced the particle size distribution. As a result, flux levels of only 6.5 l/m² h were achieved, at a liquid superficial velocity of 1.5 m/s. Even though the soluble microbial products levels in the AMBR were as high as 14 g COD/l, the observed hydraulic flux was not limited by irreversible pore fouling, but by reversible cake layer formation. Propionate oxidation was the limiting step for the applied organic loading rate. The assessed specific methanogenic activity (SMA) with propionate as substrate was, however, similar to the values found by others during thermophilic treatment of non or partially acidified substrates in granular sludge bed reactors, indicating an appropriate level of the propionate oxidation capacity.     

Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion

The characteristics of the degradation of cellulose, soluble starch, and glucose in the acidogenic phase and the effects of the substrate loading rate and biological solids retention time on the methanogenic phase of anaerobic digestion were investigated. The results obtained from continuous experiments using laboratory-scale anaerobic chemostat reactors elucidated the true rate-limiting step of anaerobic digestion. The specific rate of substrate utilization decreased in the following order: glucose, soluble starch, acetic acid, and cellulose. The rate of the hydrolysis of cellulose was so low that this was shown to be the rate-limiting step in overall anaerobic digestion. Among methanogenic bacteria Methanosarcina would provide a higher substrate utilization rate than Methanothrix, and the maximum allowable substrate loading rate in the methanogenic phase was 11.2 g acetic acid/L day.     

Anaerobic digestion of the vegetable fraction of municipal refuses: mesophilic versus thermophilic conditions

The phenomena limiting the anaerobic digestion of vegetable refuses are studied through batch tests carried out using anaerobic sludge previously selected under either mesophilic (37v°C) or thermophilic (55v°C) conditions. The compositions of the hydrolysed cellulosic and hemicellulosic fractions of these materials are simulated by starch and hemicellulose hydrolysates, respectively. Non-hydrolysed mixtures of vegetable waste with sewage sludge are used to ascertain whether the hydrolysis of these polymeric materials is the limiting step of the digestion process or not. The experimental data of methane production are then worked out by a first-order equation derived from the Monod's model to estimate the kinetic rate constant and methane production yield for each material. Comparison of these results shows that passing from mesophilic to thermophilic conditions is responsible for a slight deceleration of methane production but remarkably enhances both methanation yield and methane content of biogas. The final part of the study deals with the fed-batch digestion of the same residues in static digester. Working under thermophilic conditions at a loading rate threshold of 6.0 g_COD/l · d, the hemicellulose hydrolysate ensures the highest methane productivity (60 mmol_CH4/l · d) and methane content of biogas (60%), while unbalance towards the acidogenic phase takes place under the same conditions for the starch hydrolysate. The intermediate behaviour of the non-hydrolysed mixture of vegetable waste with sewage sludge demonstrates that hemicellulose hydrolysis is the limiting step of digestion and suggests the occurrence of ligninic by products inhibition on methane productivity.     

Analysis of the methane production in thermophilic anaerobic reactors: use of autofluorescence microscopy

Methanogenic activity in thermophilic, anaerobic reactors was determined by comparing the amount of methane generated in single- and two-stage systems with the size of the methanogenic population, as determined by microscopy. The methanogenic activities were 2.71 × 10^–9 ml methane cell^–1 d^–1 and 1.10 × 10^–9 ml methane cell^–1 d^–1 for 10 and 4 days of the hydraulic retention time (HRT), in the single-stage system. In the two-stage system, 7.49 × 10^–9 ml methane cell^–1 d^–1 in the acidogenic reactor and 1.56 × 10^–9 ml methane cell^–1 d^–1 in the methanogenic reactor for 4 days of the HRT. A high correlation was evident between the methane production and methanogenic population [0.1354 ln(x) – 2.1375](R ² 0.8619).     

Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester

After immobilization of anaerobes on polyurethane foam in a thermophilic, fixed-bed, anaerobic digester supplied with acetate, the results of real-time PCR analysis indicated that the major immobilized methanogenic archaea were Methanosarcina spp., and that the major free-living methanogenic archaea were Methanosarcina and Methanobacterium spp. 16S rRNA gene densities of Methanosarcina spp. and Methanobacterium spp. immobilized on the polyurethane foam were 7.6x10(9) and 2.6x10(8) copies/cm3, respectively. Immobilized methanogenic archaea could be concentrated 1000 times relative to those in the original anaerobically digested sludge from a completely mixed thermophilic digester supplied with cattle waste. On the other hand, immobilized bacteria could be concentrated only 10 times. The cell densities of the immobilized methanogenic archaea and bacteria were higher than those of the free-living methanogenic archaea and bacteria in the reactor. The results of clone analysis indicate that the major methanogenic archaea of the original thermophilic sludge are members of the order Methanomicrobiales, and that the major methanogenic archaea immobilized on the polyurethane foam are Methanosarcina spp., and those of the liquid phase are Methanobacterium spp. The results of the real time PCR analysis approximately agree with those of the clone analysis. These results indicate that real-time PCR analysis is useful for quantitatively describing methanogenic communities.     

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.     

Anaerobic digestion of food waste in a hybrid anaerobic solid–liquid system with leachate recirculation in an acidogenic reactor

Recirculation of the leachate in the acidogenic reactor was proposed to enhance anaerobic digestion of food waste in the hybrid anaerobic solid–liquid (HASL) system. Recirculation of the leachate in the acidogenic reactor provided better conditions for extraction of organic matter from the treated food waste and buffering capacity to prevent excessive acidification in the acidogenic reactor. It ensured faster supply of nutrients in the methanogenic reactor in experiment. The highest dissolved COD and VFA concentrations in the leachate from the acidogenic reactor were reached for shorter time and were 16,670 mg/l and 9450 mg/l in control and 18,614 mg/l and 11,094 mg/l in experiment, respectively. Recycling of the leachate in the acidogenic reactor intensified anaerobic digestion of food waste and diminished time needed to produce the same quantity of methane by 40% in comparison with anaerobic digestion of food waste without recirculation.     

Methane fermentation of coastal mud sediment by a two-stage upflow anaerobic sludge blanket (UASB) reactor system

The removal of organic matter from a coastal mud sediment was carried out by a methane fermentation process under anaerobic conditions. In a batch acidogenic fermentation, the addition of vitamins containing thiamine, nicotinic acid and biotin dramatically enhanced acetate production from the mud sediment (200 g wet wt l(-1) artificial sea water), yielding 77 mM acetate after 6 days, which corresponded to 77% of the organic matter in the mud sediment, measured on the basis of chemical oxygen demand. Thereafter, the two-fold diluted, post-acidogenic fermentation liquor (PAF liquor) was continuously treated at 2.4x original dilution rate day(-1) for 30 days, using an upflow anaerobic sludge blanket methanogenic reactor containing the acclimated methanogenic sludge from the mud sediment. Acetate, 42 mM in the PAF liquor, was converted to methane at a maximum methane production rate of 96 mmol l(-1) day(-1); and 87.5% of the acetate and 88.7% of the total organic carbon in the PAF liquor were removed. Moreover, an efficient treatment of the mud sediment was carried out by a semi-continuous, two-stage reactor system, where the culture broth was circulated between acidogenic and methanogenic reactors. This two-stage reactor system gave a stable operation at 4-day intervals for one treatment period, yielding 112 mmol methane from the wet mud in the PAF liquor (278 g l(-1)).     

Influence of sulfates and operational parameters on volatile fatty acids concentration profile in acidogenic phase

Anaerobic treatment of distillery wastewaters containing high sulfate concentrations was carried out on a two-phase process. The acidogenic phase was operated so as to produce the more favourable intermediates for methanogenic bacteria coupled with maximum sulfate removal. Sulfate removal was directly affected by pH and dilution rate (D). The maximum sulfate removal and acetic acid production was achieved at pH 6.6 and D=0.035 h^–1. A linear relationship between acetic acid produced and sulfate removal was observed, indicating that acetic acid was mainly produced by sulfate reducing bacteria with important operational advantages. Higher concentrations of butyric acid were obtained at low pH values and high dilution rates.     

Anaerobic membrane reactor with phase separation for the treatment of cheese whey

Two-phase anaerobic digestion of cheese whey was investigated in a system consisting of a stirred acidogenic reactor followed by a stirred methanogenic reactor, the latter being coupled to a membrane filtration system to enable removal of soluble effluent whilst retaining solids. The acidogenic reactor was operated at a hydraulic retention time (HRT) of one day, giving maximum acidification of 52.25% with up to 5 g/l volatile fatty acids, of which 63.7% was acetic acid and 24.7% was propionic acid. The methanogenic reactor received an organic load up to 19.78 g COD/ld, corresponding to a HRT of 4 days, at which 79% CODs and 83% BOD(5) removal efficiencies were obtained. Average removals of COD, BOD(5) and TSS in the two-phase anaerobic digestion process were 98.5%, 99% and 100%, respectively. The daily biogas production exceeded 10 times reactor volume and biogas methane content was greater than 70%.