Two-stage Anaerobic Digestion Research Articles

Critical Review on Two-Stage Anaerobic Digestion with H2 and CH4 Production from Various Wastes

Anaerobic digestion (AD) is a promising method for resource recovery from various wastes. Compared to the conventional single-stage AD process, a two-stage AD process with separate H2 and CH4 production provides higher energy recovery efficiency and enhanced operation stability. The stage separation makes it possible to apply optimal conditions for different functional microorganisms in their respective stages. This review elaborates the mechanisms of the two-stage AD process and evaluates recent research trends on this topic. A comprehensive comparison between single- and two-stage AD processes is made from the perspective of biogas production, organics degradation, energy recovery, and operation stability. The main influence factors on the two-stage AD process are discussed, including substrates, inoculum, and operation parameters, such as pH, temperature, etc. Upgrading technologies for the two-stage AD process are assessed. The microbial communities in the two-stage AD process for treating different substrates and the influence factors on microbial systems are also summarized. Furthermore, future research opportunities for enhancing the application of this technology are highlighted.

Microbial Characterisation of a Two-Stage Anaerobic Digestion Process for Conversion of Agri-Based Feedstock in Biogas and Long-Chain Fatty Acids in a Circular Economy Framework

Microbial Characterisation of a Two-Stage Anaerobic Digestion Process for Conversion of Agri-Based Feedstock in Biogas and Long-Chain Fatty Acids in a Circular Economy Framework

A robust, low-temperature, closed-loop anaerobic system for high-solid mixed farm wastes: advancing agricultural waste management solutions in Canada.

This study investigates the effectiveness of low-temperature (20 ± 1 °C) anaerobic digestion (AD) for two organic multiple farm substrate combinations: Set 1 comprising chicken manure (CM), dairy manure (DM), and waste corn silage (CS) and Set 2 comprising CM, DM, pig manure (PM), and CS. Inoculum adaptation steps were carried out using CM and CM+DM for Set 1 and Set 2, respectively. Over three consecutive operating cycles spanning 245 days with increasing organic loads, 4.3 and 2.8 g VS L-1 d-1 for Sets 1 and 2 during Cycles 1 to 5.1 and 4.6 g VS L-1 d-1for Sets 1 and 2 during Cycle 3, a closed-loop two-stage liquid-solid AD system was employed, with performance assessed via stability ratios of short-chain volatile fatty acids and alkalinity. Results demonstrate that mono-digestion of CM with adapted inoculum yielded the highest biogas production of 424 ± 4 L over 77 days, indicating superior performance by Set 1 during Phase I, whereas a similar performance was observed during Phase 2, where Sets 1 and 2 exhibited highest specific methane yields of 0.233 ± 0.028 and 0.262 ± 0.004 L g-1 VSfed, respectively, over 68 days. Analysis of heavy metal concentrations in digestates revealed a significant decrease compared to initial raw substrate concentrations, highlighting their role as nutrients for microbial growth. This study, the first of its kind, highlights the potential of low-temperature AD systems to manage diverse organic residues/byproducts and offers insights into effective performance monitoring without compromising system integrity.

Comprehensive insights into the effects of acidogenic off-gas utilization on successive biogas production, microbial community structure and metabolite distribution during two-stage anaerobic digestion

Although two-stage anaerobic digestion (TSAD) technology has been investigated, the mechanisms regarding the impact of acidogenic off-gas (AOG) on successive methane production have not been well addressed. In this study, a novel TSAD system was designed. Food waste, as the main substrate, was co-digested with chicken manure and corn straw. The acidogenic gas beyond atmospheric pressure was introduced into the bottom of the methanogenesis reactor through a stainless steel diffuser. Results showed the addition of AOG increased the methane yield from 435.2 to 597.1 mL/g VSin in successive methanogenesis stage, improved by 37.2 %, and increased the energy yield from 9.0 to 11.3 kJ/g VSsubstrate. However, the theoretical contribution of hydrogenotrophic methanogenesis using H2 contained in AOG was only 15.2 % of the increased methane yield. After the addition of AOG, the decreased levels of ammonia nitrogen and butyrate indicate that the stability of the AD system was improved. The electron transfer system and co-enzyme F420 activity were enhanced; however, the decrease in acetate kinase activity indicates aceticlastic methanogenesis may have been weakened. The microbial diversity and species richness were improved by the added AOG. Methanosarcina was more competitive than Methanothermobacter, enhancing the syntrophic effect. The relative abundance of protein degradation bacteria norank_f_Anaerolineaceae and lipid degradation bacteria Syntrophomonas was increased. Metabolite analysis confirmed that the addition of AOG promoted amino acid metabolism, the biosynthesis of other secondary metabolism and lipid metabolism. The improved degradation of recalcitrant organic components (lipids and proteins) in food waste was responsible for the increased methane yield. This study provides an in-depth understanding of the impact of AOG utilization on successive methane production and has practical implications for the treatment of food waste.

Methane production from sugarcane vinasse: The alkalinizing potential of fermentative-sulfidogenic processes in two-stage anaerobic digestion

The two-stage anaerobic digestion (2st-AD) of sugarcane vinasse is widely studied and well-known for improving the energy recovery potential in sugarcane biorefineries. Maintaining enhanced substrate acidification in a separate (first stage) reactor directly improves the performance of methanogenesis (second stage). However, problems derived from the presence of sulfate (SO42−) and the subsequent sulfide formation in the second stage are not prevented in conventional 2st-AD systems. In addition, high costs related to reactor alkalinization still represent significant drawbacks in that configuration. The energy recovery potential via methanogenesis was assessed from acidified sugarcane vinasse samples collected from different dark fermentative systems, namely: V1 (subjected to NaOH+NaHCO3 dosing), V2 (subjected to NaOH dosing) and V3 (subjected to no pH control). Despite the harmfulness of sulfide, the enhanced production of acetate from the incomplete oxidation of organic matter in sulfidogenesis can benefit methanogens. The highest methane yield (296.3 NmL-CH4 g-COD−1) and global energy recovery potential (354,603 GWh per season) were obtained from the lactate and SO42−rich vinasse (V2). Nevertheless, from a technological perspective, the methanogenesis of vinasses subjected to the fermentative-sulfidogenic process (V1) provided a higher quality biogas due to a higher calorific power (26.4-27.0 MJ Nm−3) and decreased H2S content in the biogas. Finally, the fermentative-sulfidogenic process as an alkalinizing strategy was demonstrated to be the best economic approach for scaling up the 2st-AD of sugarcane vinasse, overcoming the main economic drawback of this configuration.

Co-digestion of filter cake, biogas effluent, and anaerobic sludge for hydrogen and methane production: Optimizing energy recovery through two-stage anaerobic digestion

This study assesses a two-stage anaerobic digestion process designed to efficiently recover energy through hydrogen and methane production by co-digesting filter cake (FC), biogas effluent (BE), and anaerobic sludge (AS) from the sugar and ethanol industry. The optimal proportions of FC, BE, and AS were determined in batch fermentation experiments using Design Expert software, which identified a suitable ratio of 31.05:28.95:0.00 (g VS/L), respectively. The results indicated that hydrogen production in the first stage could occur solely through the hydrogenic bacteria present in the BE and FC mixture, without the need for AS as an inoculum. This optimized FC:BE ratio was then applied in a semi-continuous fermentation system, achieving a hydrogen production rate of 193.6 mL H2/L.d and a hydrogen yield of 9.8 mL H2/g VS at an optimal hydraulic retention time (HRT) of 3 d. In the subsequent second stage, the effluent from the hydrogen reactor was used for methane production. This stage achieved a methane production rate of 422.0 mL CH4/L·d and a methane yield of 140.2 mL CH4/g VS, with an HRT of 20 d. Overall, the two-stage process exhibited an impressive energy output, peaking at 5.17 kJ/g VS. This suggests the potential for an annual electricity generation of 194,655 MWh and an estimated reduction of 85,668 tCO2eq/year in greenhouse gas emissions. This study highlights the efficiency of the two-stage anaerobic digestion process for harnessing energy through the co-digestion of FC and BE, without the need for additional AS inoculum. The findings demonstrate the potential for sustainable energy recovery from industrial waste streams while mitigating environmental impacts.

Buswell’s Equation for Quantifying Biohydrogen

Biohydrogen is widely generated by dark fermentation and two-stage anaerobic digestion. Anaerobic digestion can be chemically represented by the Buswell’s equation, which is based on elemental composition of organic matter. When compared to Buswell’s equation for biomethane, its equation for biohydrogen has been given less attention. In this article, the nature of Buswell’s equation for biohydrogen is introduced by using the H-atom method. The mean oxidation number of organic carbons is employed as a metric for counting theoretical quantity of biohydrogen and parameters of Buswell’s equation for biohydrogen. Based on an empirical formula and mean oxidation number of organic carbons, the general Buswell’s equation for biohydrogen is developed. The mathematical relationships among mean oxidation number of organic carbons, empirical formula, quantity of biohydrogen, theoretical biohydrogen potential, and biohydrogen percent yield are also established. Biowastes and bio-substrates are chosen to demonstrate this notion.

An integrated energy recovery approach of biohythane-biocrude production from microalgae-sludge through co-digestion and co-liquefaction

Higher energy recovery from wet-waste is a challenging task while considering the waste-to-energy approach to tackle the huge wet-waste biomass generated around the globe. This study took the opportunity to explore the combined approach of biohythane (e.g., bioH2 and bioCH4) and biocrude generation using two-stage anaerobic digestion coupled with hydrothermal liquefaction (TSAD-HTL). The finding of this study suggests that the highest biohythane productivity of 268.7 mL/g-VS with energy recovery of 9.95 MJ/kg-VS was produced from sludge-microalgae co-digestion, which was 17% and 205% higher than mono-digestion of microalgae and sludge, respectively. In addition, the highest biocrude yield of 45.4% was obtained from sludge-microalgae-derived digestate. The lighter fraction (<C20) of biocrude was observed at 91.8 % (20 % naphtha, 58 % jet fuel, and 13 % diesel) with an HHV of 35.85 MJ/kg. The higher energy recovery of 81 % and lower energy consumption ratio of 0.25 suggests that the integrated TSAD-HTL is an innovative energy-positive approach for producing biohythane-biocrude from wet wastes as a sustainable concept of zero waste.

The Advantage of Citrus Residues as Feedstock for Biogas Production: A Two-Stage Anaerobic Digestion System

Anaerobic digestion (AD) is an important step in waste recovery. In Colombia, the production of citrus food significantly contributes to environmental impact via waste generation. In 2021, the waste produced, specifically citrus rind, amounted to 725,035 tons/year. During degradation, wastes generate leachate and greenhouse gases (GHGs), which negatively impact water sources (leachate), soil, and human and animal health. This article describes the design of a two-phase biodigestion system for the degradation of organic matter and biogas production. The system uses citrus waste to produce biogas with neutral emissions. The biodigestion process begins with the stabilization of the methanogenesis reactor (UASB), which takes approximately 19 days. During this period, the biogas produced contains approximately 60% methane by volume. Subsequently, the packed bed reactor operates for 7 days, where hydrolytic and acetogenic bacteria decompose the citrus waste, leading to the production and accumulation of volatile fatty acids. The final step involves combining the two phases for 5 days, resulting in a daily biogas production ranging from 700 to 1100 mL. Of this biogas, 54.90% is methane (CH4) with a yield of 0.51 LCH4gSV−1. This study assesses the methane production capacity of citrus waste, with the process benefiting from the pH value of the leachate, enhancing its degradability. Consequently, this approach leads to a notable 27.30% reduction in solids within the digestion system. The two-phase anaerobic biodigestion system described in this article demonstrates a promising method to mitigate the environmental impact of citrus waste while concurrently producing a renewable source of energy.

Distinct Mechanisms on Accelerating Electron Transfer to Facilitate Two-Stage Anaerobic Digestion Modulated by Various Microalgae Biochar

Distinct Mechanisms on Accelerating Electron Transfer to Facilitate Two-Stage Anaerobic Digestion Modulated by Various Microalgae Biochar

ADM1-Based Modeling of Biohydrogen Production through Anaerobic Co-Digestion of Agro-Industrial Wastes in a Continuous-Flow Stirred-Tank Reactor System

Biological treatment is a promising alternative for waste management considering the environmentally sustainable concept that the European Union demands. In this direction, anaerobic digestion comprises a viable waste treatment process, producing high energy-carrier gases such as biomethane and biohydrogen under certain operating conditions. The mathematical modeling of this bioprocess can be used as a valuable tool for process scale-up with cost-effective implications. The scope of this work was the evaluation of the well-established Anaerobic Digestion Model 1 (ADM1) for use in two-stage anaerobic digestion of agro-industrial waste. Certain equations for the description of the metabolic pathways for lactate and bioethanol accumulation were implemented in the existing mechanistic model in order to enhance the model’s accuracy. The model presents a high estimation ability regarding the final product (H2 and biogas) reaching the same maximum value for the theoretical as the experimental data of these products (0.0012 and 0.0036 m3/d, respectively). The adapted ADM1 emerges as a useful instrument for designing anaerobic co-digestion processes with the goal of achieving high yields in fermentative hydrogen production, considering mixed biomass growth mechanisms.

Impact of Waste Cooking Oils Addition on Thermophilic Dry Co-Digestion of Wheat Straw and Horse Manure for Renewable Energy Production in Two Stages.

Anaerobic co-digestion of waste wheat straw and horse manure in two steps was revealed as a promising option for renewable energy production in the form of hydrogen and methane. Addition of waste cooking oils, disposal of which could cause damage to health or the environment, as a third substrate for digestion, is suggested as an approach not only to help handle the increasing volume of food waste worldwide but also to improve process performance. In the present study, waste cooking oil, in a concentration of 5%, appeared to be a positive modulator of anaerobic digestion with the production of hydrogen and did not lead to inhibition of the hydrolysis phase. The overall efficiency of the two-stage anaerobic digestion of the mixture, which contains mainly lignocellulose waste, is positively dependent on thermochemical pretreatment with the alkali reagent (Ca(OH)2), but elevated temperature (55 °C) and cooking oil addition revealed the opportunity to omit the pre-treatment step. Nevertheless, the overall energy production was lower due to the methane production step. However, the addition of waste cooking oils to the process in which lig-nocellulose is not pretreated (V3) led to an increase in the methane production and energy yield compared to V1. The anaerobic digestion of lignocellulosic waste is a complex process and comprises successive degradation pathways and syntrophic microbial associations' activities, so the division in two reactors ensured suitable conditions for the microorganisms residing in each of them. In this study, along with the production of hydrogen and methane and the separation of the hydrolysis and methanogenesis stages, utilization of agriculture- and kitchen-generated wastes was realized in the context of waste-to-energy sustainable production methods.

Enhancing Single- and Two-Stage Anaerobic Digestion of Thickened Waste-Activated Sludge through FNA-Heat Pretreatment

This study aimed to investigate the effect of combined Free Nitrous Acid (FNA)-Heat (i.e., FNH) pretreatment on single- and two-stage anaerobic digestion (AD) of thickened waste-activated sludge (TWAS). Single-stage AD was conducted in batches, while two-stage AD involved acidogenic fermentation under semi-continuous flow followed by batch methanogenesis. FNH pretreatment was applied before the acidogenic stage, using 1.4 mg HNO2-N/L FNA concentration at 25 °C, 37 °C, and 60 °C for 24 h. Among the scenarios, the most promising results were observed with two-stage AD fed with FNH-pretreated TWAS at 60 °C, showing higher COD solubilization and a reduction in volatile solids. Combined FNA-Heat pretreatment in two-stage AD yielded elevated methane production (363–415 mL CH4/g VS added) compared to single-stage digestion. Methane yields from FNA-Heat pretreated single-stage ranged from 332 to 347 mL CH4/g VS added, contrasting with 212 mL CH4/g VS added for untreated TWAS. Methane generation commenced early in both untreated and pretreated samples, attributed to soluble substrate abundance.

Effects of food wastes based on different components on digestibility and energy recovery in hydrogen and methane co-production

This study was conducted for four organic fractions (carbohydrates, proteins, cellulose, lipids) at an inoculum concentration of 30 % and a total solid (TS) of 8 % to investigate the effect of the main components of food waste on the performance of the two-stage anaerobic digestion. The results showed that the gas phase products were closely related to the composition of the substrate, with the carbohydrate and lipid groups showing the best hydrogen (154.91 ± 2.39mL/gVS) and methane (381.83 ± 12.691mL/gVS) production performance, respectively. However, the increased protein content predisposes the system to inhibition of gas production, which is mutually supported by changes in the activity of dehydrogenase and coenzyme F420. Butyric acid (53.19 %) dominated the liquid phase products in both stages, indicating that all four organic fractions were butyric acid-based fermentation and that the final soluble chemical oxygen demand degradation reached 72.97 %–82.86 %. The carbohydrate and cellulose groups achieved the best energy recovery performance, with conversion rates exceeding 65 %. The above results can provide a useful reference for the resource utilization of food waste.

Co-digestion approach for enhancement of biogas production by mixture of untreated napier grass and industrial hydrolyzed food waste.

The co-digestion of untreated Napier grass (NG) and industrial hydrolyzed food waste (FW) was carried out in the batch reactor to investigate the effect of substrate ratios on biogas production performance. Two-stage anaerobic digestion was performed with an initial substrate concentration of 5g VSadded/L and a Food to Microorganism Ratio (F/M) of 0.84. The 1:1 ratio of the NG and FW showed the optimum performances on biogas production yield with a value of 1,161.33mL/g VSadded after 60 days of digestion. This was followed by the data on methane yield and concentration were 614.37mL/g VSadded and 67.29%, respectively. The results were similar to the simulation results using a modified Gompertz model, which had a higher potential methane production and maximum production rate, as well as a shorter lag phase and a coefficient of determination of 0.9945. These findings indicated that the co-digestion of Napier grass and hydrolyzed food waste can enhance biogas production in two-stage anaerobic digestion.

Evaluating the role of salinity in enhanced biogas production from two-stage anaerobic digestion of food waste by zero-valent iron

Salts including NaCl are the most common food flavoring agents so they are often accumulated in food waste (FW) and have potential impact on anaerobic digestion (AD) of FW. In this study, the enhanced biogas production from two-stage anaerobic digestion (TSAD) of FW by microscale zero-valent iron (ZVI) under different salinity (3, 6, 9, and 15 g NaCl/L) was evaluated. Under salinity stress, ZVI becomes a continue-release electron donor due to the enhanced corrosion and dissolution effect and the slow-down surface passivation, further improving the performance of TSAD. Experimental results revealed that the biogas production including H2 and CH4 from TSAD with 10 g/L ZVI addition was promoted under salinity stress. The maximum H2 and CH4 yield (303.38 mL H2/g-VS and 253.84 mL CH4/g-VS) were observed at the salinity 9 g NaCl/L. Compared with that of zero salinity, they increased by 40.94% and 318.46%, respectively. Additionally, Sedimentibacter, an exoelectrogen that can participate in the direct interspecies electron transfer, also exhibited the highest relative abundance (34.96%) at the salinity 9 g NaCl/L. These findings obtained in this study might be of great importance for understanding the influence of salinity on the enhanced AD by ZVI.

Breakdown of hardly degradable carbohydrates (lignocellulose) in a two-stage anaerobic digestion plant is favored in the main fermenter

The yield and productivity of biogas plants depend on the degradation performance of their microbiomes. The spatial separation of the anaerobic digestion (AD) process into a separate hydrolysis and a main fermenter should improve cultivation conditions of the microorganisms involved in the degradation of complex substrates like lignocellulosic biomass (LCB) and, thus, the performance of anaerobic digesters. However, relatively little is known about such two-stage processes. Here, we investigated the process performance of a two-stage agricultural AD over one year, focusing on chemical and technical process parameters and metagenome-centric metaproteomics.Technical and chemical parameters indicated stable operation of the main fermenter but varying conditions for the open hydrolysis fermenter. Matching this, the microbiome in the hydrolysis fermenter has a higher dynamic than in the main fermenter. Metaproteomics-based microbiome analysis revealed a partial separation between early and common steps in carbohydrate degradation and primary fermentation in the hydrolysis fermenter but complex carbohydrate degradation, secondary fermentation, and methanogenesis in the main fermenter. Detailed metagenomics and metaproteomics characterization of the single metagenome-assembled genomes showed that the species focus on specific substrate niches and do not utilize their full genetic potential to degrade, for example, LCB.Overall, it seems that a separation of AD in a hydrolysis and a main fermenter does not improve the cleavage of complex substrates but significantly improves the overall process performance. In contrast, the remaining methanogenic activity in the hydrolysis fermenter may cause methane losses.

Effects of organic loading rate on hydrogen and methane production in a novel two-stage reactor system: performance, enzyme activity and microbial structure

Two-stage anaerobic digestion (AD) system is an efficient technology; however, organic loading rate (OLR), a critical design and process parameter, influences the performance and capacity of digesters. This study investigates the impact of various OLRs on biogas production during co-digestion of food waste, chicken manure and corn straw in an innovatively designed two-stage reactor system, including variations in the enzyme and electron transfer system (ETS) activities and microbial community. The production of hydrogen and methane increased gradually with increasing OLR, with maximum production at 8 g VS/L d (690 mLH2/L d and 690 mLCH4/L d), which is 30–97 % higher than OLR of 1.5 to 6 g VS/L d. However, the maximum methane yield of 535 mL/g VSadded was attained at OLR of 4 g VS/L d consistent with the highest coenzyme F420 activity. Although no substantial ETS activity was seen during the acidogenic stage, the ETS activity decreased with increasing OLR in the methanogenesis stage, indicating that high substrate loading results in low syntrophic methanogenesis. Microbial analysis revealed a more diverse enrichment of acidogens and acetogens at high OLR with a wider range of substrate consumption, resulting in balanced acetoclastic and hydrogenotrophic methanogens (Methanosarcina and Methanothermobacter) in the biomethane process. These findings suggest the strengthening of syntrophic methanogenesis in high substrate-loading AD systems.

Effects of sudden shock load on simultaneous biohythane production in two-stage anerobic digestion of high-strength organic wastewater

The two-stage anaerobic digestion (AD) for biohythane production is a sustainable solution, but it is sensitive to organic shock load that disrupts reactors and inhibits biohythane production. This study investigated biohythane production, reactor performance, and the possibility of post-failure restoration in a two-stage AD system designed for treating high-strength organic wastewater. Sudden shock load was applied by increasing the OLR threefold higher after reaching steady state phase. During shock load phase, hydrogen content, hydrogen yield and methane production rate (MPR) reached its peak values of 62.61 %, 1.641 mol H2/mol glucose, and 1.003 L CH4/L⋅d respectively before declining significantly. Interestingly, during the restorative phase, hydrogen production sharply declined to nearly zero, while methane production exhibited a resilience and reached its peak methane content of 52.2 %. The study successfully demonstrated the system’s resilience to sudden shock load, ensuring stable methane production, while hydrogen production did not exhibit the same capability.

Effect of reactor operation modes on mitigating antibiotic resistance genes (ARGs) and methane production from hydrothermally-pretreated pig manure

Numerous efforts have been made to enhance the performance of anaerobic digestion (AD) for accelerating renewable energy generation, however, it remains unclear whether the intensified measures could enhance the proliferation and transmissions of antibiotic resistance genes (ARGs) in the system. This study assessed the impact of an innovative pig manure AD process, which includes hydrothermal pretreatment (HTP) and a two-stage configuration with separated acidogenic and methanogenic phases, on biomethane (CH4) production and ARGs dynamics. Results showed that HTP significantly increase CH4 production from 0.65 to 0.75 L/L/d in conventional single-stage AD to 0.82 and 0.91 L/L/d in two-stage AD. This improvement correlated with a rise in the relative abundance of Methanosarcina, a key methanogenesis microorganism. In the two-stage AD, the methanogenic stage offered an ideal environment for methanogens growth, resulting in substantially faster and higher CH4 production by about 10% compared to single-stage AD. Overall, the combined use of HTP and the two-stage AD configuration enhanced CH4 production by 40% compared to traditional single-stage AD. The abundance and diversity of ARGs were significantly reduced in the acidogenic reactors after HTP. However, the ARGs levels increased by about two times in the following methanogenesis stage and reached similar or higher levels than in single stage AD. The erm(F), erm(G), ant(6)-Ia, tet(W), mef(A) and erm(B) were the six main ARGs with significant differences in relative abundances in various treatments. The two-stage AD mode could better remove sul2, but it also had a rebound which elevated the risk of ARGs to the environment and human health. Network analysis identified pH and TVFAs as critical factors driving microbial communities and ARG proliferation in the new AD process. With the results, this study offers valuable insights into the trade-offs between AD performance enhancement and ARG-related risks, pinpointing essential areas for future research and practical improvements.