Rate-limiting Step In Anaerobic Digestion Research Articles

Enhancing waste sludge solubilization through radio frequency treatment perforating bacterial cells

Sludge solubilization is known as a rate-limiting step of anaerobic digestion. Although radio frequency (RF) has been applied for sludge pretreatment due to its similar thermal effect as microwave, the potential non-thermal effects of RF treatment remain controversial. In this study, we demonstrate that RF pretreatment enhances the solubilization and lysis of sludge by 8.02%–19.69% through both thermal and non-thermal mechanisms with less energy input. Scanning electron microscope images provide direct evidence that RF-induced microcurrents penetrated bacterial cells, leading to the release of intracellular substances through formed pores. Additionally, the non-thermal effect of RF treatment which could weaken the cell protection and accelerate the lysis rate involves the disruption of binding forces between extracellular polymeric substances and microbial cells. On average, the utilization of RF at a frequency of 27.12 MHz demonstrates its efficacy as a sludge pretreatment technique, as evidenced by a 13.39% reduction in energy consumption and a 16.9% improvement in treatment performance compared to conductive heating (CH). The findings of this study elucidate the possible mechanism of RF treatment of sludge and could establish a theoretical basis for the practical application of RF treatment in sludge management.

Research progress and perspective on sludge anaerobic digestion technology: A bibliometric analysis.

Rational disposal of sludge is an ongoing concern. This work is the first attempt for in-depth statistical analysis of anaerobic digestion (AD) research in recent three decades (1986-2022) using both quantitative and qualitative approaches in bibliometrics to investigate the research progress, trends and hot spots. All publications in the Web of Science Core Collection database from 1986 to April 4, 2022 were analyzed. Results showed that the research on AD started in 1999 and the number of papers significantly increased since 2012. The research about the disposal of sewage sludge mainly focuses on energy recovery (e.g. methane and short chain volatile organic acids) by AD. Besides, different pretreatment technologies were studied in this study to eliminate the negative effects on the disposal of sludge caused by hydrolysis (rate-limiting step of AD), water content (increasing the costs) and heavy metal (toxic to the environment) of sludge. Of those, the treatment technologies related to direct interspecies electron transfer were worth further studied in the future. Towards that end, iron conductive material, iron-based advanced oxidation and biological treatment were concluded as the prospective technologies and worth to further study.

The microbial and functional reconstruction of instable syntrophic propionate-oxidizing methanogenesis by system recovering and injection modes changing

Microbial enrichment is an effective method to obtain highly abundant syntrophic methanogenic propionate-degrading microbial consortia (syntrophic consortia) in complex environments. Nevertheless, there were few researches studying dynamic microbial community change and syntrophic mechanisms over a long-time bioreactor operation. In this study, the highly efficient syntrophic consortia were enriched in the anaerobic bioreactor, and the effect of propionic acid loading (PAL) on microbial community structure and functional pathways was investigated. By single injection mode (30 mL propionic acid was injected into the reactor once per day), acid accumulation occurred severely at the PAL of 2.7 g/(L·d). After the injection mode was changed to continuous injection mode (a syringe pump was used to inject 30 mL propionic acid evenly in 8 h), the reactor performance was more stable and the volumetric biogas production (VBP) reached 0.96 L/(L·d). 16S rDNA sequencing analysis and gene predictions showed that microbial community structures, functional microorganisms and metabolic pathways varied at different PALs and different injection modes. When the propionic acid shock was great, hydrogenic methanogenesis was the major pathway to relieve the acid accumulation. In continuous injection mode, the bacterial structure was more stable. Syntrophobacter became the dominant propionate oxidation bacterium and acetoclastic methanogenesis conducted by Methanosaeta enhanced. This study provides an effective method for the microbial enrichment in the rate-limiting step of anaerobic digestion and lays a foundation for the development of bio-enhancement with high organic loading.

Impact of thermal pre-treatment on anaerobic co-digestion of sewage sludge and landfill leachate

The complex microbial flocs with rigid cell walls and undigested organic matter in the domestic sewage sludge defy anaerobic digestion, especially hydrolysis, the rate-limiting step of anaerobic digestion. The anaerobic digestion of municipal solid waste landfill leachate is challenging because of its high refractory organics and ammoniacal nitrogen. However, if sewage sludge and landfill leachate are mixed in an optimal ratio and pre-treated, they can be anaerobically co-digested and clean bioenergy can be harnessed. Among the pre-treatment methods, thermal pre-treatment is preferred because it is simple and economical yet effective. In this investigation, sewage sludge from a domestic sewage treatment plant and landfill leachate from an active municipal solid waste landfill were mixed in the ratio of 75:25 and thermally pre-treated at 70 °C for 60 min. The pre-treated substrates were anaerobically digested at 37 ± 1 °C using fresh cow dung slurry as the inocula. The results of this experiment were compared with the control experiment in which substrates without pre-treatment were used. Though biogas production from the fresh substrates was higher during the initial days, it decreased after the seventh day. For the thermal pre-treated substrates, the biogas production was low during the first week, which started to increase from the tenth day until the end of the experiment. The cumulative biogas yields from untreated and thermal pre-treated reactors were 723.5 and 418 mL, respectively.

Methane production improvement in an osmotic membrane bioreactor for sludge anaerobic digestion: pretreatment optimization and long-term performance.

Hydrolysis is the first step and also rate-limiting step of anaerobic digestion which recovers energy from waste sludge. In order to accelerate the reaction rate of the hydrolysis, many pretreatment conditions had been taken into account. In this study, thermal pretreatment and alkaline pretreatment were combined with each other, serving as a thermal-alkaline pretreatment approach. Firstly, an orthogonal designed batch experiment was conducted to evaluate the pretreatment conditions, and then the optimal conditions were applied to an osmotic membrane bioreactor for a long-term investigation. Based on batch experiments, sludge treated by NaOH at pH 9 or 10 showed a better effect in cell solubilization. Sludge treated by Ca(OH)2 at pH 9, and sludge treated by NaOH at pH 9 or 10 showed advantages in methane production. Ultimately, sludge treated by NaOH at pH 9 and then heated at 90 °C for 60 min was selected as the optimal pretreatment condition. During the long-term operation of osmotic membrane bioreactor for sludge anaerobic digestion, the volume methane production of the sludge treated by thermal-alkaline was maintained at around 200-300 mL/L/d, which was 2-3 times of the sludge treated by ultrasound.

Reconsidering hydrolysis kinetics for anaerobic digestion of waste activated sludge applying cascade reactors with ultra-short residence times

Hydrolysis is considered to be the rate-limiting step in anaerobic digestion of waste activated sludge (WAS). In this study, an innovative 4 stages cascade anaerobic digestion system was researched to (1) comprehensively clarify whether cascading configuration enhances WAS hydrolysis, and to (2) better understand the governing hydrolysis kinetics in this system. The cascade system consisted of three 2.2 L ultra-short solids retention times (SRT) continuous stirred tank reactors (CSTRs) and one 15.4 L CSTR. The cascade system was compared with a reference conventional CSTR digester (22 L) in terms of process performance, hydrolytic enzyme activities and microbial community dynamics under mesophilic conditions (35 °C). The results showed that the cascade system achieved a high and stable total chemical oxygen demand (tCOD) reduction efficiency of 40–42%, even at 12 days total SRT that corresponded to only 1.2 days SRT each in the first three reactors of the cascade. The reference-CSTR converted only 31% tCOD into biogas and suffered process deterioration at the applied low SRTs. Calculated specific hydrolysis rates in the first reactors of the cascade system were significantly higher compared to the reference-CSTR, especially at the lowest applied SRTs. The activities of several hydrolytic enzymes produced in the different stages revealed that protease, cellulase, amino peptidases, and most of the tested glycosyl-hydrolases had significantly higher activities in the first three small digesters of the cascade system, compared to the reference-CSTR. This increase in hydrolytic enzyme production by far exceeded the increase in specific hydrolysis rate, indicating that hydrolysis was limited by solids-surface availability for enzymatic attack. Correspondingly, high relative abundances of hydrolytic-fermentative bacteria and hydrogenotrophic methanogens as well as the presence of syntrophic bacteria were found in the first three digesters of the cascade system. However, in the fourth reactor, acetoclastic methanogens dominated, similarly as in the reference-CSTR. Overall, the results concluded that using multiple CSTRs that are operated at low SRTs in a cascade mode of operation significantly improved the enzymatic hydrolysis rate and extend in anaerobic WAS digestion. Moreover, the governing hydrolysis kinetics in the cascading reactors were far more complex than the generally assumed simplified first-order kinetics.

Enhanced Methane Recovery from Waste-Activated Sludge by Alginate-Degrading Consortia: The Overlooked Role of Alginate in Extracellular Polymeric Substances

The hydrolysis of extracellular polymeric substances (EPS) in waste-activated sludge (WAS) is considered as the rate-limiting step in anaerobic digestion. Uronic acids such as alginate are one of m...

Direct interspecies electron transfer in anaerobic digestion: Research and technological application

Anaerobic digestion (AD), an economical and eco-friendly strategy for renewable energy recovery, has been widely investigated and applied in the stabilization of various organic wastes. However, two major factors involved in the slow fermentation rate and low methane conversion efficiency still limit its large-scale applications around the world. Methane formation via conventional methanogenic pathway in AD undergoes the three stages: Hydrolysis/acidogenesis, acetogenesis and methanogenesis. The structure of some saccharide- and protein-like organic compounds contained in complex organic waste is relatively complex and stable, limiting the rate of converting them into amino acids, fatty acids and alcohols by fermentative bacteria. Therefore, the hydrolysis/acidogenesis is widely recognized as a rate-limiting step of AD in practical applications. On the other hand, the obligate anaerobic methanogens can only utilize methanol, methylamine, formate, acetate and hydrogen/carbon dioxide as substrates. Methane formation with amino acids, fatty acids and alcohols as substrates requires the essential conversion of them into acetate and hydrogen/carbon dioxide by acetogenic bacteria, and then depends on acetate- and hydrogen-utilizing methanogens to metabolize acetate and hydrogen/carbon dioxide into methane, respectively. This working mode of syntrophic metabolism of organic compounds into methane between acetogenic bacteria and methanogens that depends on hydrogen as an interspecies electron carrier has been widely described as Interspecies Hydrogen Transfer (IHT). Formate may be also served as a substitute for hydrogen as an interspecies electron carrier (Interspecies Formate Transfer, IFT). Syntrophic metabolism of organic compounds is strictly limited by the hydrogen partial pressure, since the oxidation of organic compounds into acetate with H+ as an electron acceptor is not feasible in thermodynamics ( E H + / H 2 = −0.414 V, pH 7). Syntrophic metabolism via IHT functions well as long as the hydrogen-utilizing methanogens continuously maintain the hydrogen partial pressure low enough (

Integrative Effects of Sonication and Particle Size on Biomethanation of Tropical Grass Pennisetum purpureum Using Superior Diverse Inocula Cultures

Biogas from the fast growing crop, Pennisetum purpureum, has received considerable attention in Southeast Asia since wastewater and bio-waste materials are almost completely utilized. To overcome slow hydrolysis, a rate-limiting step in anaerobic digestion of lignocellulosic biomass, superior microorganism culture, size reduction, and sonication pretreatment were co-applied. In the first experiment, the selection of anaerobic microbial culture to be used in digestion, so-called inoculum, was carried out. Specific anaerobic activities for hydrolysis and methanogenesis of sludge from different sources, a slurry digester of cattle farm (CF) and a wastewater digester of rubber latex factory (RL) were assessed. Results revealed a remarkable synergistic capability in the combined sludge, adding 10% and 49% to the overall biomethanation efficiency over the individual CF and RL sludges. In the second part, interactive effects of size reduction and sonication intensity were studied. Biomethanation efficiency as methane yield increased by 62% by size and 115% by sonication variation, but when optimally combined an additional gain of 40% was recorded. The regression model generated could estimate the energy yield increase as a function of size and sonication intensity with a satisfactory statistical precision R2 of 0.945.

Mediative mechanism of bicarbonate on anaerobic propionate degradation revealed by microbial community and thermodynamics.

Syntrophic acetogenesis of volatile fatty acids (VFAs) such as propionate and butyrate is considered as the rate-limiting step of anaerobic digestion. Though being extensively researched, the mechanism is not well understood as the main constraint on developing effective solutions to the practical problem. In the present research work, the mediation of methanogenic propionate degradation by exogenous bicarbonate was evaluated, while the mechanism was revealed by microbial community and thermodynamics. It was found that the exogenous bicarbonate not more than 0.10mol/L acted as a mediative role to enrich syntrophic acetogenic bacteria and decrease the actual Gibbs free energy change (ΔG) of syntrophic acetogenesis reaction, resulted in the increased degradation rate and methane production rate of propionate. The remarkably increased ΔG of methanogenic propionate degradation by the exogenous bicarbonate more than 0.15mol/L decreased the degradation rate and methane production rate of propionate, though the ΔG of syntrophic acetogenesis reaction was also decreased by the exogenous bicarbonate. This research work provided a control strategy to enhance syntrophic acetogenesis, as well as the methanogenic VFAs degradation.

Mesophilic and thermophilic anaerobic digestion of lipid-extracted microalgae N. gaditana for methane production

In the last years, a huge effort has been made to make biodiesel production from microalgae a feasible option. Besides the potential of biodiesel for replacing fossil fuels as a cleaner alternative, some limitations have still to be overcome. Among them, the low energy yields of the process and the high-energy requirements of the harvesting and drying steps lead to a high cost per litre of fuel. In this context, anaerobic digestion of the microalgal biomass after lipid extraction can improve the energy balance of the process, by producing methane and revalorizing a waste generated during biodiesel production. In this study, the production of biogas by anaerobic digestion of the marine microalgae Nannochloropsis gaditana after oil extraction was studied. As the hydrolysis is known to be the rate-limiting step of anaerobic digestion of solid substrates, the influence of the process temperature on this step was assessed. For this purpose, two different anaerobic sludges, i.e. mesophilic (35 °C) and thermophilic (55 °C), were used as inocula for different batch tests and for two continuous anaerobic bioreactors. The influence of the oil extraction process on the structural integrity of the microalgae was also studied. The results obtained from scanning electron microscopy and flow cytometry showed that the lipid extraction did not cause cell lysis, but the structure of their surface was affected by the process. Batch assays showed that thermophilic conditions did not improve the biomethane potentials when compared to mesophilic conditions. Two continuous anaerobic bioreactors operated for 120 days confirmed the batch results. However, measurements of the chemical oxygen demand showed that the soluble fraction in the thermophilic reactor was higher than under mesophilic conditions, indicating an improved hydrolysis step. This was confirmed by the results of scanning electron microscopy and flow cytometry, which suggested a more intense disintegration of microalgae in the thermophilic reactor, indicating a greater degree of hydrolysis. Nevertheless, this advantage of thermophilic temperatures over mesophilic conditions did not improve the methane productivity.

Effect of different pH-values on process parameters in two-phase anaerobic digestion of high-solid substrates

In many publications, primary fermentation is described as a limiting step in the anaerobic digestion of fibre-rich biomass [Eastman JA, Ferguson JF. Solubilization of particulacte carbon during the anaerobic digeston. J WPCF. 1981;53:352–366; Noike T, Endo G, Chang J, Yaguchi J, Matsumoto J. Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion. Biotechnol Bioeng. 1985;27:1482–1489; Arntz HJ, Stoppok E, Buchholz K. Anaerobic hydroysis of beet pulp-discontiniuous experiments. Biotechnol Lett. 1985;7:113–118]. The microorganisms of the primary fermentation process differ widely from the methanogenic microorganisms [Pohland FG, Ghosh S. Developments in anaerobic stabilization of organic wastes-the two-phase concept. Environ Lett. 1971;1:255–266]. To optimize the biogas process, a separation in two phases is suggested by many authors [Fox P, Pohland GK. Anaerobic treatment applications and fundamentals: substrate specificity during phase separation. Water Environ Res. 1994;66:716–724; Cohen A, Zoetemeyer RJ, van Deursen A, van Andel JG. Anaerobic digestion of glucose with separated acid production and methane formation. Water Res. 1979;13:571–580]. To carry out the examination, a two-phase laboratory-scale biogas plant was established, with a physical phase separation. In previous studies, the regulation of the pH-value during the acid formation was usually carried out by the addition of sodium hydroxide [Cohen A, Zoetemeyer RJ, van Deursen A, van Andel JG. Anaerobic digestion of glucose with separated acid production and methane formation. Water Res. 1979;13:571–580; Ueno Y, Tatara M, Fukui H, Makiuchi T, Goto M, Sode K. Production of hydrogen and methane from organic solid wastes by phase separation of anaerobic process. Bioresour Technol. 2007;98:1861–1865; Zoetemeyer RJ, van den Heuvel JC, Cohen A. pH influence on acidogenic dissimilation of glucose in an anaerobic digestor. Water Res. 1982;16:303–311]. A new technology without the use of additives was developed in which the pH-regulation is executed by the pH-dependent recycling of effluent from the anaerobic filter into the acidification reactor. During this investigation, the influence of the different target pH-values (5.5, 6.0, 7.0 and 7.5) on the degradation rate, the gas composition and the methane yield of the substrate maize silage was determined. With an increase in the target pH-value from 5.5 to 7.5, the acetic acid equivalent decreased by 88.1% and the chemical oxygen demand-concentration by 18.3% in the hydrolysate. In response, there was a 58% increase in the specific methane yield of the overall system. Contrary to earlier studies, a marked increase in biogas production and in substrate degradation was determined with increasing pH-values. However, these led to a successive approximation of a single-phase process. Based on these results, pH-values above 7.0 seem to be favourable for the digestion of fibre-rich substrates.

Improvement of methane production from waste paper by pretreatment with rumen fluid

Cellulose hydrolysis is the rate-limiting step in anaerobic digestion. In the present study, waste paper was used as a model of cellulosic biomass and was pretreated with rumen fluid prior to methane production. To achieve a high methane yield, the reaction time of the pretreatment was examined. Waste paper was soaked with rumen fluid for 6 and 24h at 37°C. Various volatile fatty acids, especially acetate, were produced by the pretreatment. Semicontinuous methane production was carried out over a 20-day period. The best daily methane yield was obtained by the 6-h pretreatment. The amount was 2.6 times higher than that of untreated paper, which resulted in 73.4% of the theoretical methane yield. During methane production, the cellulose, hemicellulose and lignin degradabilities were improved by the pretreatment. Pretreatment by rumen fluid is therefore a powerful method to accelerate the methane yield from a cellulosic biomass.

Enhancement of solubilization rate of cellulose in anaerobic digestion and its drawbacks

Hydrolysis is widely acknowledged as the rate-limiting step in anaerobic digestion of solid cellulose to biogas (methane), and pretreatment is generally considered to facilitate the process. However, few studies have investigated how such pretreatment may affect the rest of this complex process. The present study compared the solubilization rate in anaerobic digestion of cotton linter (high crystalline cellulose), with that of regenerated cellulose (amorphous cellulose), using pretreatment with NMMO. Batch digestions were performed, with the initial cellulose concentrations ranging between 5 and 40 g/l, and during 30 days of incubation, biogas and VFAs production as well as pH and COD changes were measured. The lag time before digestion started was longer for the high crystalline cellulose than for the amorphous one. The maximum solubilization rates of treated cellulose were 842 and 517 mg sCOD/g cCOD/day at the initial cellulose concentration of 5 and 30 g/l, respectively, while the solubilization rate of untreated cellulose never exceeded 417 mg sCOD/g cCOD/day. The difference between the two cellulose types was a direct result of the high rate of hydrolysis inhibiting the acetogenesis/methanogenesis microorganisms, a drawback to the rest of the process.

Extracellular enzyme activities during regulated hydrolysis of high-solid organic wastes

The hydrolysis process, where the complex insoluble organic materials are hydrolyzed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Recirculating the leachate from hydrolysis reactor and recycling the effluent from methanogenic reactor to hydrolysis reactor in the two-stage solid–liquid anaerobic digestion process could accelerate degradation of organic solid wastes. To justify the influencing mechanism of recirculation and recycling on hydrolysis, the relationship of hydrolysis to the synthesis and locations of extracellular enzymes was evaluated by regulating the dilution rate of the methanogenic effluent recycle. The results showed that the hydrolysis could be enhanced by increasing the dilution rate, resulting from improved total extracellular enzyme activities. About 15%, 25%, 37%, 56% and 92% of carbon, and about 9%, 18%, 27%, 45% and 80% of nitrogen were converted from the solid phase to the liquid phase at dilution rates of 0.09, 0.25, 0.5, 0.9 and 1.8 d −1, respectively. The hydrolysis of organic wastes was mainly attributable to cell-free enzyme, followed by biofilm-associated enzyme. Increasing the dilution rate afforded cell-free extracellular enzymes with more opportunity to access the surface of organic solid waste, which ensured a faster renewal of niche where extracellular enzymes functioned actively. Meanwhile, the increment of biofilm-associated enzyme was promoted concomitantly, and therefore improved the hydrolysis of organic solid wastes.

The rate-limiting step in anaerobic digestion in the presence of phosphine

Methanogenesis is the most important anaerobic biodegradation process in nature, which is accomplished by three different kinds of bacteria - hydrolytic, acetogenic and methanogenic bacteria (MB). An experiment was performed to determine the rate-limiting step of methanogenesis under the influence of various phosphine concentrations (100, 300, 500, 700 and 1000 ppm). It was found that the growth of fermentative bacteria (FB) was severely affected by higher concentrations of phosphine (700 and 971 ppm), while the growth of hydrogen-producing acetogenic bacteria (HPAB) and MB was not affected severely at higher phosphine concentrations. Thus, HPAB and MB are less sensitive to phosphine compared with FB, which means that hydrolysis, and fermentation step is the rate-limiting step during methanogenesis under the influence of phosphine. It is recommended that special attention be paid to the first stage of methanogenesis under high concentrations of phosphine during anaerobic wastewater treatment.

Hydrolytic activity of alpha-amylase in anaerobic digested sludge

Hydrolysis is usually considered to be a rate-limiting step in anaerobic digestion. For improving anaerobic solid waste treatments, it is essential to elucidate the mechanism of hydrolysis. In this study, alpha-amylase, one of the hydrolytic enzymes, was investigated for the elucidation of more precise mechanism of hydrolysis. Alpha-amylase activity of solid starch-degrading bacteria (SDB) was estimated through batch experiments with several different substrates and with distinction between cell-bound and cell-free alpha-amylase. Monitoring of newly isolated strains of SDB was done by fluorescence in situ hybridization. Results indicated that cell-bound alpha-amylase is chiefly responsible for the hydrolysis in the digested sludge, providing very useful information that the contact between microbial cells and solids is significantly important. The activity of alpha-amylase of the digested sludge remained quite low when not required, but increased as they recognized appropriate substrates. Several-fold higher activity was obtained for starch or maltose as compared to glucose only.

Enhanced anaerobic gas production of waste activated sludge pretreated by pulse power technique

An electric pulse-power reactor consisting of one coaxial electrode and multiple ring electrodes was developed to solubilize waste activated sludge (WAS) prior to anaerobic digestion. By pretreatment of WAS, the soluble chemical oxygen demand (SCOD)/total chemical oxygen demand (TCOD) ratio and exocelluar polymers (ECP) content of WAS increased 4.5 times and 6.5 times, respectively. SEM images clearly showed that pulse-power pretreatment of WAS was found to result in destruction of sludge cells. Batch-anaerobic digestion of pulse-power treated sludge showed 2.5 times higher gas production than that of untreated sludge. Solubilized sludge cells by pulse-power pretreatment would be readily utilized for anaerobic microorganisms to produce anaerobically-digested gas. Slow or lagged gas production in the initial anaerobic digestion stage of pulse-power pretreated sludge implied that the methane-forming stage of anaerobic digestion would be the rate-limiting step for anaerobic digestion of pulse-power pretreated sludge.

Influence of the size reduction of organic waste on their anaerobic digestion

The rate-limiting step in anaerobic digestion of organic solid waste is generally their hydrolysis. A size reduction of the particles and the resulting enlargement of the available specific surface can support the biological process in two ways. Firstly, in case of substrates with a high content of fibres and a low xegradability, their comminution yields to an improved digester gas production. This leads to a decreased amount of residues to be disposed of and to an increased quantity of useful digester gas. The second effect of the particle size reduction observed with all the substrates but particularly with those of low degradability is a reduction of the technical digestion time. Furthermore, the particle size of organic waste has an influence on the dewaterability after codigestion with sewage sludge. The presence of organic waste residues improves the dewaterability measured as specific resistance to filtration but this positive effect is attenuated if the particle size of the solids is reduced.

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.