Anaerobic Digestion Reactor Research Articles

Filamentous foam disintegration with free nitrous acid: Effect on anaerobic digestion

Sludge foaming is a common problem in wastewater treatment plants negatively affecting operation of anaerobic digestion reactors. Therefore, in common practice, foam is removed from reactors without being fermented, leading to increase in sludge mass for disposal. However, foam is rich in lipids and can be a good source of methane if operational problems can be overcome. In this paper, in a two-stage experiment, we show that foam disintegration with free nitrous acid (FNA) can boost methane production and decrease foaming potential. In the first stage, the biochemical methane potential (BMP) of foam was evaluated to be higher by 19–63% (191-263NmL/gVS) than the BMP of waste activated sludge (WAS) (161 ± 1NmL/gVS) confirming previous assumptions. The main findings of the second stage (continuous experiments) are: (1) foam and WAS co-digestion leads to sludge stratification and thickened biomass accumulation in the upper part of the reactor, (2) FNA disintegration destroyed foam structure, resulting in lower biomass stratification and 14% higher methane production (134 mL/gVS) than observed in the reference reactor, (3) FNA disintegration of both substrates (foam and WAS) does not provide noticeable benefits in terms of biomass stratification. However, it does enhance methane production to 140 mL/gVS and sludge mineralization efficiency. A significantly higher impact of FNA on methane yield from foam than WAS was attributed to the high content of M.parvicella and the ability of these bacteria to adsorb and accumulate lipids. Anaerobic digestion of FNA disintegrated foam leads to substantial benefits in terms of methane production, reactor volume, and reagents consumption.

Carbon-based conductive materials enhance biomethane recovery from organic wastes: A review of the impacts on anaerobic treatment

Amongst the most important sustainable waste management strategies, anaerobic biotechnology has had a central role over the past century in the management of high-pollution load sources, such as food, agricultural and municipal wastes. During anaerobic digestion (AD), valuable by-products such as digestate and biogas are produced. Biogas (mainly composed of methane) is generated through a series of reactions between bacteria and archaea. Enhancement of AD process with higher methane yield, accelerated methane production rate, and shorter start-up time is possible via tapping into a novel methanogenic pathway discovered a decade ago. This fundamentally new concept that is a substitute to interspecies hydrogen transfer is called direct interspecies electron transfer (DIET). DIET, a thermodynamically more feasible way of electron transfer, has been proven to occur between bacteria and methanogens. It is well-documented that amendment of carbon-based conductive materials (CCMs) can stimulate DIET via serving as an electrical conduit between microorganisms. Therefore, different types of CCMs such as biochar and activated carbon have been amended to a variety of AD reactors and enhancement of process performance was reported. In this review, a comparative analysis is presented for enhancement of AD performance in relation to major CCM related factors; electrical conductivity, redox properties, particle size and dosage. Additionally, the impacts of AD operational conditions such as organic loading rate and temperature on CCM amended reactors were discussed. Further, the changes in microbial communities of CCM amended reactors were reviewed and future perspectives along with challenges for CCM application in AD have been provided.

Treatment of fracturing wastewater by anaerobic granular sludge: The short-term effect of salinity and its mechanism

The effects of salinity shock on the anaerobic treatment of fracturing wastewater regarding chemical oxygen demand (COD) removal performance, sludge characteristics and microbial community were investigated. Results showed COD removal efficiency decreased from 76.0% to 69.1%, 65.6%, 33.7% and 21.9% with the increase of salinity from 2.5 g/L to 10, 15, 25 and 45 g/L, respectively. The cumulative biogas production decreased by 13.8%–81.1% when salinity increased to 15–85 g/L. The increase of salinity led to the decline in particle size of granular sludge, and the activity of granular sludge, including SMA, coenzyme F420 and dehydrogenase, was inhibited significantly. Flow cytometry indicated the percentage of damaged cells in granular sludge gradually increased with the increase of salinity. Sequence analysis illustrated that microbial community structure in anaerobic digestion reactor was influenced by the salinity, high salinity reduced the diversity of archaea and decreased the abundance of methanogens, especially Methanosaeta.

Anaerobic Digestion of Cigarette Butts: Microbial Community Analysis and Energy Production Estimation

Anaerobic digestion using cigarette butts, one of most littered items, was studied not only as a waste treatment, but also as an energy production method. Methane production from cigarette butts was measured through the biochemical methane potential (BMP) test and it was evaluated whether it is possible to produce electrical energy. Intact cigarettes or individual components (filter, paper, and leaf) were supplied as the sole carbon source (substrate) for the BMP test. The tendency of methane production indicated biodegradation in the order of paper, filter, and leaves; however, the filter of cigarettes was the substrate produced the highest amount of methane per total solid. The microbial community was also analyzed in each anaerobic digestion reactor, and substrate-specific microorganisms were identified, such as Proteiniphilum strain (filter) and Methanobacterium formicicum (paper). In intact cigarettes, the related microbial community became dominant over time in the order of paper, filter, and leaf. The conversion of cigarette butts to methane, a renewable energy source, can be proposed as a sustainable route for energy demand, for example, in a smoking room.

Vane geometry for measurement of influent rheological behaviour in dry anaerobic digestion

The knowledge of the rheology of influents involved in dry anaerobic digestion is an essential factor for process optimization. Despite this fact, it is difficult to characterize the rheological behaviour of these influents with high total solid (TS) content. Indeed, standard methods of rheological measurements are poorly adapted to these influents because of the sliding effects observed during the measurements. In this study, we used a non-standard method, the Vane geometry, for its particularity to limit the sliding effects. Two types of measurements were carried out on potato influents of 18–35% TS, extracted from the digesters. The first measurement was to submit to the sample a rising shear velocity ramp from 0 to 200 s−1 at 37 °C and 55 °C. The results of these measurements show a good correlation between the rheograms and Herschel-Bulkley model and we deduced a pseudoplastic threshold behaviour. The second measurement was performed in oscillations at 10 Hz at 25 °C, 37 °C and 55 °C. The results show different evolutions of the elastic G′ and viscous G″ modulus as a function of temperature. There is a crossing of G′ and G″ at 25 °C, G′ greater than G″ at 37 °C, and finally G′ and G″ forming a plateau at 55 °C, characteristic of a gel. The knowledge of these rheological behaviour is crucial for the design and operating instructions of anaerobic digestion reactors for influents with high TS content. The Vane geometry would, therefore, be an effective tool for characterizing the rheological behaviour of influents in dry anaerobic digestion.

Preliminary determination of antibacterial substances during anaerobic preservation of food waste and their effects on methanogenesis

Food waste can easily breed bacteria and produce odour during production, collection, transportation, preservation and disposal. This work studied the effects of aerobic preservation and anaerobic preservation on the antibacterial and antiseptic properties of food waste and subsequent methane production by anaerobic fermentation. Compared with aerobic preservation, the concentration of lactic acid produced on the 5th day of anaerobic preservation increased by 2.01 times, the pH decreased rapidly and the number of Escherichia coli was reduced by 99.96% on the 7th day; all these changes could effectively inhibit the activity of spoilage bacteria. The results of the biological toxicity test showed that lactic acid and acetic acid were the main antibacterial substances in anaerobic preservation. The biological toxicity of lactate or acetate was significantly lower than that of the corresponding organic acid molecules. When food waste enters the anaerobic digestion reactor after preservation, materials with high lactic acid concentration are greatly diluted, which will not affect the methane fermentation process, but can lead to elevate biogas production. These observations are consistent with the conclusion obtained by kinetic analysis that anaerobic preservation has a faster hydrolysis rate and a shorter system stagnation period than aerobic preservation. Therefore, anaerobic preservation is a suitable way of storing food waste, and it can promote methane fermentation.

Microbial community assembly and dynamics in Granular, Fixed-Biofilm and planktonic microbiomes valorizing Long-Chain fatty acids at 20 °C

Distinct microbial assemblages evolve in anaerobic digestion (AD) reactors to drive sequential conversions of organics to methane. The spatio-temporal development of three such assemblages (granules, biofilms, planktonic) derived from the same inoculum was studied in replicated bioreactors treating long-chain fatty acids (LCFA)-rich wastewater at 20 °C at hydraulic retention times (HRTs) of 12–72 h. We found granular, biofilm and planktonic assemblages differentiated by diversity, structure, and assembly mechanisms; demonstrating a spatial compartmentalisation of the microbiomes from the initial community reservoir. Our analysis linked abundant Methanosaeta and Syntrophaceae-affiliated taxa (Syntrophus and uncultured) to their putative, active roles in syntrophic LCFA bioconversion. LCFA loading rates (stearate, palmitate), and HRT, were significant drivers shaping microbial community dynamics and assembly. This study of the archaea and syntrophic bacteria actively valorising LCFAs at short HRTs and 20 °C will help uncover the microbiology underpinning anaerobic bioconversions of fats, oil and grease.

Enhancing anaerobic digestion using free nitrous acid: Identifying the optimal pre-treatment condition in continuous operation

Free Nitrous Acid (FNA) pre-treatment is a promising technology demonstrated effective in improving waste activated sludge degradability and anaerobic digestion (AD) performance. Pre-treatment conditions including FNA concentration and treatment duration determine operational and capital cost of full-scale implementation, which have not been studied in long-term experiments. The knowledge of FNA pre-treatment conditions improving the AD performance is urgently required to determine suitable conditions for the technology implementation. In this work, five different FNA concentrations (2.2, 4.4, 7.2, 12 mgN/L and nitrite only without pH adjustment) and three treatment durations (8, 24 and 48 h) were studied in four lab-scale semi-continuous AD reactors for over 300 days. FNA pre-treatment was shown under all tested conditions effective in enhancing AD performances, while its effectiveness and resulted benefits varied substantially amongst different pre-treatment conditions. The long-term experiment demonstrated that the methane production, sludge reduction and digested sludge viscosity of AD are positively correlated with the FNA concentration and durations, until an optimal condition is reached, which was identified in this work to be FNA concentration of 7.2 mgN/L and treatment duration of 24 h. Microbial community changes supported the apparent observation of enhanced sludge degradation at elevating FNA concentrations applied during pre-treatment. The short-term sludge solubilization results were inconsistent with the long-term AD performance, which was potentially caused by inhibitions from stringent FNA pre-treatment conditions applied (FNA = 12 mgN/L with 24-hour treatment & FNA = 7.2 mgN/L with 48-hour treatment). Overall, results suggested FNA pre-treatment at the optimized condition is highly beneficial to WWTPs and competitive with other pre-treatment technologies, e.g., thermal hydrolysis pre-treatment. This work comprehensively evaluated the key design parameters of FNA pre-treatment process, reached a major milestone in the development and applications of FNA technologies.

Microbial community redundance in biomethanation systems lead to faster recovery of methane production rates after starvation

The Power-to-Gas concept corresponds to the use of the electric energy surplus to produce H2 by water electrolysis, that can be further converted to methane by biomethanation. However, the fluctuant production of renewable energy sources can lead to discontinuous H2 injections into the reactors, that may interfere with the adaptation of the microbial community to high H2 partial pressures. In this study, the response of the microbial community to H2 and organic feed starvation was evaluated in in-situ and ex-situ biomethanation. The fed-batch reactors were fed with acetate or glucose and H2, and one or four weeks of starvation periods were investigated. Methane productivity was mostly affected by the four-week starvation period. However, both in-situ and ex-situ biomethanation reactors recovered their methane production rate after starvation within approximately one-week of normal operation, while the anaerobic digestion (AD) reactors did not recover their performances even after 3 weeks of normal operation. The recovery failure of the AD reactors was probably related to a slow growth of the syntrophic and methanogen microorganisms, that led to a VFA accumulation. On the contrary, the faster recovery of both biomethanation reactors was related to the replacement of Methanoculleus sp. by Methanobacterium sp., restoring the methane production in the in-situ and ex-situ biomethanation reactors. This study has shown that biomethanation processes can respond favourably to the intermittent H2 addition without compromising their CH4 production performance.

Complete Genome Sequence of Gelria sp. Strain Kuro-4, a Thermophilic Anaerobe Isolated from a Thermophilic Anaerobic Digestion Reactor Treating Poly(L-Lactic Acid).

ABSTRACTStrain Kuro-4 was isolated as a novel member of the genus Gelria from a thermophilic anaerobic digestion reactor treating poly(l-lactic acid). Here, we report a 2,880,462-bp complete circular genome sequence of Kuro-4, with a G+C content of 61.9%. The chromosome harbors 2,831 protein-coding genes and 62 RNA-coding genes.

Specialized trace elements and volatile fatty acids interactions for enhanced methane production and biomethanization process stability during high organic loading rate

Volatile fatty acids (VFAs) and trace elements (TEs) interactions (VFAs*TEs) during biomethanization have effects that could be exploited to enhance anaerobic digestion (AD) of biomass. The goal of this study was to validate biocatalytic effects of specialized VFAs*TEs identified from a batch-derived Optimum TEs Configuration (or simply ‘Optimum’) on high organic loading rate (OLR) involving mixed fruit residue (MFR) fed in semi-continuous AD operation. The specialized VFAs*TEs were formulated as Variants of the Optimum and included Optimum –Cobalt (Co) for specialized VFAs*Co effects, and Optimum +Selenium (Se) for specialized VFAs*Se effects. Four duplicate AD reactors were treated with formulations reflecting the Optimum and the Variants. Each duplicate reactor was semi-continuously fed with MFR at varying OLR until instability occurred. Methane production, total volatile organic acidity (FOS) / total alkalinity (TAC) and VFAs fingerprints were measured as main responses. The results showed that reactors of the Optimum and its Variants were unstable at OLR of 8g oDM/L/d, but stability was restored in the Optimum –Co (FOS/TAC values of 0.6 compared to 1.51 and 1.67 for Optimum and Optimum +Se respectively). The average specific CH4 production (Nml/g oDM) of the Optimum and its Variants were Control: 431±36; Optimum: 553±16; Optimum –Co: 580±12; and Optimum +Se: 545±13. Optimum –Co also had the lowest acetic acid and butyric acid accumulation, but had higher propionic acid concentration (0.7 g/L) compared to the Optimum (0.3 g/L) and Optimum +Se (0.4 g/L).

Wastewater treatment and resource recovery technologies in the brewery industry: Current trends and emerging practices

Brewery is a water intensive industry where the waste stream consists mostly of wastewater originating from the processing and cleaning of equipment. A large amount of this wastewater is directly discharged into water bodies causing surface water contamination, which in turn is disastrous for human health and environment. This review intended to recommend waste effluent treatment and residual management for a brewery industry through a systematic literature review regarding the different industrial unit operations and processes, and waste streams, by focusing mainly on the current trends and emerging practices. In the malting process, it was proposed to treat the steeping water and condensate from the kilning stages with an anaerobic digestion reactor to remove organics while producing methane, then reusing the treated water to lower the amount of fresh steeping water required. In the brewing process, it was proposed to treat the wastewater with an upflow anaerobic sludge blanket (UASB) system to produce biogas, followed by an aerobic membrane bioreactor (MBR) to polish the effluent and possibly remove nutrients such as nitrogen and phosphorus. The effluent from MBR can then be passed through a reverse osmosis (RO) system to reuse the water. During the sludge handling process, it was proposed to collect the water from the thickening, dewatering and drying stages, and precipitate out struvite as a product and reduce the nutrient load going back to the wastewater treatment system. Alternatively, after struvite precipitation, the water can be passed through a RO system to reclaim some water for reuse. Future studies should be focused on emerging resource recovery technologies applied at a semi industrial level with detailed technological and economical assessments. In addition, sustainable actions should be promoted at various levels of the industry in order to suggest policies and technologies that will prevent further deterioration of the environment.

Assessment of energy efficiency and performance in a two-phase anaerobic process for organic matter removal.

Energy efficiency (EE) depends mainly on the lower heating values (LHVs) of hydrogen and methane selected from the thermodynamics tables under ideal conditions. However, for practical applications, the heating value should be calculated by considering some environmental factors under real conditions. Accordingly, this study compared EE using the ideal LHV with the EE using the real LHV in a two-phase anaerobic digestion reactor treating synthetic wastewater. Additionally, the process performance and the stability were studied. The results showed that the EE value calculated using LHVideal was, on average, 35% higher than that evaluated using LHVreal; these differences are relevant to the estimation of real energy and also for practical applications. At the same time, the index buffer intermediate alkalinity/partial alkalinity was shown to be more accurate than the pH value to analyze the stability of the process. With regards to chemical oxygen demand, the removal efficiency in the methanogenic phase decreased drastically when utilizing 100% of the acidogenic phase. Future considerations for the optimization of each phase are highlighted.

Retraining prior state performances of anaerobic digestion improves prediction accuracy of methane yield in various machine learning models

The prediction of anaerobic digestion (AD) performance using numerical models, which are based on mathematics and kinetics, is being challenged by poor mechanistic understanding and the non-linear relationships between performance and operational parameters. This study demonstrated that various machine learning (ML) models using the 1-step ahead with the retraining method, which utilized AD performance data from prior states, can improve the prediction accuracy of ML models. For the four types of ML models studied, the 1-step ahead with the retraining method could improve the root mean square errors by 32–49% compared to the conventional multi-step ahead method, which was particularly noteworthy during the transition period when AD reactors were faced with loading shocks and showed inhibited methane yields. Moreover, the 1-step ahead with the retraining method showed the potential of achieving accurate predictions using a single input parameter, pH, which was considerably less labor-intensive to monitor than the other parameters often required in AD models (e.g., VSS). As such, the 1-step ahead with retraining method is suitable for efficient real-time prediction of AD performance in real-world operations.

Performance and microbial community analysis of anaerobic sludge digestion enhanced by in-situ microaeration

The effectiveness of in-situ microaeration was investigated with varied air dosages [0–25 mL/(L·d)] to enhance mesophilic anaerobic digestion (AD) of sludge in semi-continuous systems in terms of specific biogas production and volatile solids (VS) removal rate, focusing on performance evaluation, microbial community analysis, and underlying mechanisms. The optimal dosage of 12.5 mL/(L·d) increased specific biogas production by 15.8 ± 7.3% and enhanced VS removal by 18.3 ± 1.6%. In-situ microaeration stimulated activity of hydrolytic enzymes, enlarged sludge particle size, and improved sludge dewaterability. MiSeq sequencing analysis showed that dominant bacterial and archaeal communities were remarkably different between in-situ microaeration and obligate AD process. The presence of oxygen enriched relative abundances of Deltaproteobacteria, Anaerolineae, Clostridia, Synergistia and Caldilineae, enabling the acid-producing process to metabolize more types of substrates. Due to the selective enrichment, in-situ microaeration significantly enriched aceticlastic methanogens rather than hydrogenotrophic methanogens in the AD reactor. Analysis of coupling microaeration pretreatment and in-situ microaeration for anaerobic digestion indicated that excessive microaeration decreased VS removal rate and methane yield owing to substrates consumption by facultative bacteria. In-situ microaeration-based AD could be a promising process for sludge treatment and bioenergy recovery.

Tuning microbial community in non-conventional two-stage anaerobic bioprocess for microalgae biomass valorization into targeted bioproducts

Unspecific microorganisms consortia are normally used in anaerobic biodegradation of solid wastes. However, these consortia can be tuned to optimally obtain determined bioproducts. In this study, high value-added products and biogas were obtained via an innovative two-stage anaerobic bioprocess from microalgae biomass. The anaerobic fermentation (AF) entailed the production of short-chain fatty acids (SCFAs) and subsequently, only the solid spent of AF effluent was valorized for methane production via conventional anaerobic digestion (AD). Applied conditions in AF (25 °C, HRT 8 days) favored Firmicutes predominance (64%) enabling a conversion efficiency of 32.1% g SCFAs-COD/g CODin. Opposite, a wider microbial biodiversity was determined in the AD reactor (35 °C, HRT 20 days), being mainly composed by Firmicutes (28.6%), Euryarchaeota (17.7%) and Proteobacteria (15.3%). AD of the AF-solid spent reached 168.9 mL CH4 /g CODin. Strikingly, operational conditions imposed mediated a microbial specialization that maximized product output.

Enhanced in-situ biomethanation of food waste by sequential inoculum acclimation: Energy efficiency and carbon savings analysis.

The increasing rate of food waste (FW) generation globally, makes it an attractive resource for renewable energy through anaerobic digestion (AD). The biogas recovered from AD can be upgraded by the methanation of internally produced carbon dioxide, CO2 with externally sourced hydrogen gas, H2 (biomethanation). In this work, H2 was added to AD reactors processing FW in three successive phases, with digestate from preceding phases recycled in succession with the addition of fresh inoculum to enhance acclimation. The concentration of H2 was increased for succeeding phases: 5%, 10% and 15% of the reactor headspace in Phase 1 (EH1), Phase 2 (EH2) and Phase 3 (EH3), respectively. The H2 utilisation rate and biomethane yields increased as acclimation progressed from EH1 through EH3. Biomethane yield from the controls: EH1_Control, EH2_Control and EH3_Control were 417.6, 435.4 and 453.3 NmL-CH4/gVSadded accounting for 64.8, 73.9 and 77.8% of the biogas respectively. And the biomethane yield from the test reactors EH1_Test, EH2_Test and EH3_Test were 468.3, 483.6, and 499.0 NmL-CH4/gVSadded, accounting for 77.2, 78.1 and 81.0% of the biogas respectively. A progressive in-situ biomethanation could lead to biomethane production that meets higher fuel standards for gas-to-grid (GtG) injections and vehicle fuel - i.e. >95% CH4. This would increase the energy yield and carbon savings compared to conventional biogas upgrade methods. For example, biogas upgrade for GtG by in-situ biomethanation could yield 7.3 MWh/tFW energy and 1343kg-CO2e carbon savings, which is better than physicochemical upgrade options (i.e., 4.6-4.8 MWh/tFW energy yield and 846-883kg-CO2e carbon savings).

Enhancement of Thermophilic Biogas Production from Palm Oil Mill Effluent by pH Adjustment and Effluent Recycling

A sudden pH drops always inhibits the anaerobic digestion (AD) reactor for biogas production from palm oil mill effluent (POME). The pH adjustment of POME by oil palm ash addition and the biogas effluent recycling effect on the preventing of pH drop and change of the archaea community was investigated. The pH adjustment of POME to 7.5 increased the methane yield two times more than raw POME (pH 4.3). The optimal dose for pH adjustment by oil palm ash addition was 5% w/v with a methane yield of 440 mL-CH4/gVS. The optimal dose for pH adjustment by biogas effluent recycling was 20% v/v with a methane yield of 351 mL-CH4/gVS. Methane production from POME in a continuous reactor with pH adjustment by 5% w/v oil palm ash and 20% v/v biogas effluent recycling was 19.1 ± 0.25 and 13.8 ± 0.3 m3 CH4/m3-POME, respectively. The pH adjustment by oil palm ash enhanced methane production for the long-term operation with the stability of pH, alkalinity, and archaea community. Oil palm ash increased the number of Methanosarcina mazei and Methanothermobacter defluvii. Oil palm ash is a cost-effective alkali material as a source of buffer and trace metals for preventing the pH drop and the increased methanogen population in the AD process.

The impact of different types of high surface area brush fibers with different electrical conductivity and biocompatibility on the rates of methane generation in anaerobic digestion

The addition of electrically conductive materials may enhance anaerobic digestion (AD) efficiency by promoting direct interspecies electron transfer (DIET) between electroactive microorganisms, but an equivalent enhancement can also be achieved using non-conductive materials. Four high surface area brush materials were added to AD reactors: non-conductive horsehair (HB) and polyester (PB), and conductive carbon fiber (CB) and stainless steel (SB) brushes. Reactors with the polyester material showed lower methane production (68 ± 5 mL/g CODfed) than the other non-conductive material (horsehair) and the conductive (graphite or stainless steel) materials (83 ± 3 mL/g CODfed) (p < 0.05). This difference was due in part to the higher biomass concentrations using horsehair or carbon (135 ± 43 mg) than polyester or stainless steel or materials (26 ± 1 mg). A microbial community analysis indicated that the relative abundance of electroactive microorganisms was not directly related to enhanced AD performance. These results show that non-conductive materials such as horsehair can produce the same AD enhancement as conductive materials (carbon or stainless steel). However, if the material, such as polyester, does not have good biomass retention, it will not enhance methane production. Thus, electrical conductivity alone was not responsible for enhancing AD performance. Polyester, which has been frequently used as a non-conductive control material in DIET studies, should not be used for this purpose due to its poor biocompatibility as shown by low biomass retention in AD tests.

Enhancing bioenergy production from food waste by in situ biomethanation: Effect of the hydrogen injection point

AbstractThe increasing rate of food waste (FW) generation around the world is a growing environmental concern, notwithstanding, its valorisation through anaerobic digestion (AD) makes it a potential resource. Moreover, there is a growing demand to optimise the biomethane from AD for gas‐to‐grid (GtG) and vehicular applications. This has spurred researches on hydrogen gas (H2) injection into AD systems to enhance the biological conversion of H2 and carbon dioxide (CO2) to methane (CH4), a process known as biomethanation. A simplistic approach for biomethanation is to add H2 directly into working AD reactors (in situ biomethanation). However, a competition for the injected H2 towards other biological reactions besides H2/CO2 conversion to CH4 could follow, thus, reducing the efficiency of the system. Hence, this study was conducted to understand how different H2 injection points would affect H2/CO2 conversion to CH4 during FW in situ biomethanation, to identify an optimal injection point. Experiments were designed using H2 equivalent to 5% of the head‐space of the AD reactor at three injection points representing different stages of AD: before volatile fatty acids (VFA) accumulation, during VFA accumulation and at depleted VFA intermediates. Lower potential for competitive H2 consumption before the accumulation of VFA enabled a high H2/CO2 conversion to CH4. However, enhanced competition for soluble substrates during VFA accumulation reduced the efficiency of H2/CO2 conversion to CH4 when H2 was added at this stage. In general, 12%, 4% and 10% CH4 increases as well as 39%, 25% and 34% CO2 removal were obtained for H2 added before VFA accumulation, during VFA accumulation and at depleted VFA intermediates, respectively. For immediate integration of biomethanation with existing AD facilities, it is suggested that the required H2 be obtained biologically by dark fermentation.