Methanogenesis Process Research Articles

The effects of phytochemicals on methanogenesis: insights from ruminant digestion and implications for industrial biogas digesters management

The overarching objective of this review is to compare methanogenesis in ruminant gut systems and industrial digesters that employ plants, crops and their processing residues or by-products as feedstocks. An important consideration is the presence of phytochemicals in these feedstocks, with the understanding that depending on the type and quantities, these phytochemicals affect the activity of biomass degrading microorganisms to varying extents. This review aims to evaluate currently available knowledge on the effects of phytochemicals in ruminants’ methanogenesis as well as industrial anaerobic digestion processes. Technology and scale up in industrial fermentation have always benefited from our understanding of natural biological processes as such, knowledge from the process of methanogenesis in the gastrointestinal tract of ruminants can be used to make inferences about the performance of industrial digester systems for biogas production. This is particularly important when the same feedstock is used in both systems. Thus, in this review, comparisons of the two methane generating systems are discussed with an emphasis on phytochemical inhibitory effects on microorganisms that are involved in anaerobic digestion.

The process of methanogenesis in paddy fields under different elevated CO2 concentrations

Understanding the process of methanogenesis in paddy fields under the scenarios of future climate change is of great significance for reducing greenhouse gas emissions and regulating the soil carbon cycle. Methyl Coenzyme M Reductase subunit A (mcrA) of methanogens is a rate-limiting enzyme that catalyzes the final step of CH4 production. However, the mechanism of methanogenesis change in the paddy fields under different elevated CO2 concentrations (e[CO2]) is rarely explored in earlier studies. In this research, we explored how the methanogens affect CH4 flux in paddy fields under various (e[CO2]). CH4 flux and CH4 production potential (MPP), and mcrA gene abundance were quantitatively analyzed under C (ambient CO2 concentration), C1 (C + 160 ppm CO2), and C2 (C + 200 ppm CO2) treatments. Additionally, the community composition and structure of methanogens were also compared with Illumina MiSeq sequencing. The results showed that C2 treatment significantly increased CH4 flux and MPP at the tillering stage. E[CO2] had a positive effect on the abundance of methanogens, but the effect was insignificant. We detected four known dominant orders of methanogenesis in this study, such as Methanosarcinales, Methanobacteriales, Methanocellales, and Methanomicrobiales. Although e[CO2] did not significantly change the overall community structure and diversity of methanogens, C2 treatment significantly reduced the relative abundance of two uncultured genera compared to C treatment. A linear regression model of DOC, methanogenic abundance, and MPP can explain 67.2% of the variation of CH4 flux under e[CO2]. Overall, our results demonstrated that CH4 flux in paddy fields under e[CO2] was mainly controlled by soil unstable C substrate and the abundance and activity of methanogens in rhizosphere soil.

Вплив віброкавітаційного оброблення суспензії ціанобактерій на інтенсивність синтезу біогазу

Досліджено вплив віброкавітаційного оброблення біомаси суспензії ціанобактерій на інтенсивність та ефективність синтезу біогазу внаслідок реалізації в подальшому процесу метаногенезу. Показано актуальність розроблення технології утилізації надлишкової біомаси ціанобактерій, яка утворюється внаслідок їх неконтрольованого розвитку у мілководних ділянках штучних водойм і в технологіях біологічного очищення забруднених поверхневих та стічних вод. Запропоновано використання для руйнування міжклітинних перегородок ціанобактерій та вивільнення їх біомаси, яка стає доступною біорозкладу та реалізації метаногенезу, віброкавітаційного оброблення. Наведено опис конструкції віброкавітатора, який сприяє ефективній руйнації міжклітинних перегородок біомаси ціанобактерій та може працювати у безперервному режимі. Сконструйовано та використано для попереднього оброблення ціанобактерій лабораторну установку віброкавітатора, який працює у періодичному режимі. Створено дослідну установку для дослідження динаміки метаногенезу обробленої у віброкавітаторі біомаси ціанобактерій у безперервному режимі із використанням відеореєстрації синтезу біогазу та передачі даних щодо динаміки синтезу біогазу на монітор. Отримано експериментальні залежності динаміки синтезу біогазу із біомаси ціанобактерій від періоду їх оброблення у віброкавітаційному полі. Встановлено що оптимальні параметри синтезу біогазу досягаються після попереднього оброблення біомаси ціанобактерій у віброкавітаторові впродовж 15 хв. Встановлено автокаталітичний характер процесу, що дає змогу висунути припущення про підпорядкованість його рівнянню Міхаеліса – Ментена. Отриманий після закінчення процесу метаногенезу дигестат запропоновано використовувати як ефективне біоорганічне або органомінеральне (у разі створення композиції із природними дисперсними сорбентами) добриво.

The Impact of Methane Seepage on the Pore-Water Geochemistry across the East Siberian Arctic Shelf

East Siberian Arctic Shelf, the widest and the shallowest shelf of the World Ocean, covering greater than two million square kilometers, has recently been shown to be a significant modern source of atmospheric methane (CH4). The CH4 emitted to the water column could result from modern methanogenesis processes and/or could originate from seabed deposits (pre-formed CH4 preserved as free gas and/or gas hydrates). This paper focuses primarily on understanding the source and transformation of geofluid in the methane seepage areas using ions/trace elements and element ratios in the sediment pore-water. Six piston cores and totally 42 pore-water samples were collected in the East Siberian Sea and the Laptev Sea at water depths ranging from 22 to 68 m. In the active zones of methane release, concentrations of vanadium, thorium, phosphorus, aluminum are increased, while concentrations of cobalt, iron, manganese, uranium, molybdenum, copper are generally low. The behavior of these elements is determined by biogeochemical processes occurring in the pore-waters at the methane seeps sites (sulfate reduction, anaerobic oxidation of methane, secondary precipitation of carbonates and sulfides). These processes affect the geochemical environment and, consequently, the species of these elements within the pore-waters and the processes of their redistribution in the corresponding water–rock system.

Comparison of methods for calculating biological wastewater treatment

Due to the increase in the discharge of nitrogen and phosphorus compounds into water bodies in many countries, the problem of eutrophication has become the most relevant in the field of biological treatment technologies. To solve this problem, many technological schemes for purification from biogenic elements have been developed, based on different methods. The concept of biological purification, the composition and process of nutrition of heterotrophic microorganisms, as well as the course of the process of methanogenesis are analyzed. The analysis of the methods of the two countries, Russia and Germany. Most of the facilities in Russia are designed in accordance with SNiP 2.04.03.85, while Germany offers a different biological treatment method developed by the German Water Association DWA A131.

CO Biomethanation with Different Anaerobic Granular Sludges

Biomethanation of the syngas from biomass gasification provides an alternative method for production of biofuel and chemicals. CO in syngas plays a key role in biomethanation of syngas, as it is both a substrate and an inhibitor of certain methanogenesis processes. In this study, CO biomethanation by using a mixture of N2 and CO as the gas substrate, was investigated with the help of 5 adapted anaerobic granular sludges under thermophilic and mesophilic conditions. The results show that CO biomethanation by the adapted inocula is omnipresent. The sludge from the juice plant has a methane yield more than 80% of the theoretical value both at 37 °C and 55 °C. Increasing the temperature from 37 °C to 55 °C has a slight effect on the final methane production, but can significantly increase the CO consumption rate and shorten the time for CO biomethanation.

Enhancement of short-chain fatty acids production from microalgae by potassium ferrate addition: Feasibility, mechanisms and implications

Anaerobic fermentation of microalgae was always hindered by its rigid cell wall structure. This paper reports a novel technique, i.e., adding potassium ferrate (K2FeO4) into microalgae fermentation systems to enhance short-chain fatty acids (SCFAs) production. The results showed that the maximum SCFAs production and acetic acid proportion were 732.6 mg COD/g VS and 54.6% at a dosage of 112.8 mg Fe(VI)/g VS, which were 168% and 208% of those in the control, respectively. Mechanism studies revealed that K2FeO4 effectively destroyed surface morphology and cell structure, and thus facilitated microalgae solubilization, providing a large number of biodegradable substrates for subsequent SCFA production. Although K2FeO4 inhibited all the microbial activities relevant to hydrolysis, acidification and methanogenesis processes to some degree, its inhibition to methanogens was much severer than that to other microbes. Illumina MiSeq sequencing analyses revealed that K2FeO4 addition increased the relative abundance (from 9.45% to 50.4%) of hydrolytic and SCFAs-forming bacteria.

Direct aerobic NSZD of a basalt vadose zone LNAPL source in Hawaii

In recent years, a number of methods have been used to measure the biodegradation of petroleum light non-aqueous phase liquids (LNAPL) at petroleum release sites, a process known as natural source zone depletion (NSZD). Most commonly, NSZD rates have been measured at sites with unconsolidated geology and relatively shallow groundwater (<50 ft. bgs, <15 m bgs). For this study, we have used two methods (1. carbon dioxide flux measured using carbon traps and 2. heat flux based on subsurface temperature gradients) to measure NSZD rates at a petroleum release site in Hawaii with basalt geology and deep groundwater (>300 ft. bgs, >100 m bgs). Both methods documented the occurrence of NSZD at the facility and the two methods yield estimates of the NSZD rate that agreed within a factor of 2 (4600 to 7400 gal/yr; 17,000 to 28,000 L/yr for the flux method and 8600 to 13,000 gal/yr; 33,000 to 49,000 L/yr for the temperature method). Soil gas samples collected directly above the water table and at shallower depths within the vadose zone indicated aerobic conditions throughout the vadose zone (oxygen >13%) and no detectable methane. These results indicate that NSZD occurs at this site through the direct aerobic biodegradation of LNAPL rather than the two-step process of anaerobic methanogenesis followed by methane oxidation at a shallow depth interval documented at other sites.

Anaerobic digestion of food waste at varying operating conditions

Food waste is one of the major problems contributing to the degradation of the environment, and thus needs serious attention. Among different options, anaerobic digestion is possibly the most effective technique for managing degradable waste, and produce renewable energy and fertilizer. Despite multiple-use and benefits of the technology, its application is limited due to a few technical challenges. This study focuses on the anaerobic digestion of food waste with the addition of different percentages of digested cow manure as inoculum to it, at different total solid content in ambient temperature. Anaerobic digestion of food waste in batch and semi-continuous processes were carried out in three different trials at an average temperature range of 20-26℃: Food waste with 20% inoculum, food waste with 50%, 100%, and 200% inoculum and 10% total solid content in batch process and food waste with 20% inoculum with 6% and 10% total solids content in the semi-continuous process. During each trial, some amount of gas production was observed, however, the gas composition showed a negligible amount of methane production (maximum 13% of CH4). There were two common problems detected in each trial: the inability to complete the methanogenesis process, and instability of the overall process due to the high degradability and acidic nature of food waste. Therefore, this study suggests that the mono digestion of food waste is not a suitable option. However, anaerobic co-digestion of food waste with different organic substrate might provide a favorable condition for stable anaerobic digestion as seen from preliminary results.

Development of aerobic methane oxidation, denitrification coupled to methanogenesis (AMODM) in a microaerophilic expanded granular sludge blanket biofilm reactor

The issue of enhancing nitrogen removal and managing dissolved methane emission in anaerobic treatment systems is a major bottleneck in its wider application to treat high-strength organic wastewater with nitrate. Herein, a novel aerobic methane oxidation, denitrification coupled to methanogenesis (AMODM) process was developed in a glucose-fed microaerobic expanded granular sludge blanket biofilm reactor (EGSBBR) through in-situ utilization of produced methane for nitrogen removal. The 162-day operation demonstrated that long-term treatment performance under the decreased COD/NO3−-N (C/N) ratio from 66.7 to 10 and the optimal C/N ratio for completing AMODM was found to be 16.7. Microbial community analysis further evidenced that Methanothrix as key methanogen predominated in the sludge bed, while Methlogaea as aerobic methane oxidizer was mainly detected in the packing bed of the hybrid system. Meanwhile, some facultative heterotrophic and dissimilated nitrate-reduction (DNRA) genera also co-existed. The profiling of key functional genes further proved concurrent occurrence of methanogenesis, aerobic methane oxidation and denitrification. Furthermore, possible microbial mechanism on AMODM process was elucidated from the prospective of targeted species interaction within the reactor. This research provides a robust and environment-friendly alternative process treating nitrate-containing organic wastewater towards efficient nitrogen removal, low resource consumption, bioenergy recovery and greenhouse gas reduction.

Anaerobic co-digestion of waste activated sludge and fish waste: Methane production performance and mechanism analysis

The performances of anaerobic co-digestion (AcD) of waste activated sludge (WAS) and fish waste (FW) were studied through mesophilic biochemical methane potential test. Mixtures of various compositions (0, 1.5, 3, 6 and 10% of FW on the volume basis) were tested as the AcD substrates. Experimental results showed that 1.5% and 3% of FW addition could enhance the cumulative methane production remarkably, increasing from 212.3 mL CH4/g VSfed (100% WAS) to 410.1 mL CH4/g VSfed and 683.8 mL CH4/g VSfed, respectively. However, methane producing was inhibited severely with more than 6% of FW addition, whose cumulative methane production dramatically decreased to approximately 51 mL CH4/g VSfed. Mechanism analysis showed that large amounts of fatty acids accumulation occurred during the AcD with FW addition above 6%, leading to methanogenesis process failure. Microbial community analysis showed that methane producing during AcD was dominated by hydrogenotrophic and methylotrophic methanogenesis pathways led by Methanobacterium and Methanomassiliicoccus.

Inhibition mitigation of methanogenesis processes by conductive materials: A critical review

Methanogenesis can be promoted by the addition of conductive materials. Although stimulating effects of conductive materials on methane (CH4) production has been extensively reported, the crucial roles on recovering methanogenic activities under inhibitory conditions have not been systematically discussed. This critical review presents the current findings on the effects of conductive materials in methanogenic systems under volatile fatty acids (VFAs), ammonia, sulfate, and nano-cytotoxicity stressed conditions. Conductive materials induce fast VFAs degradation, avoiding VFAs accumulation during anaerobic digestion. Under high ammonia concentrations, conductive materials may ensure sufficient energy conservation for methanogens to maintain intracellular pH and proton balance. When encountering the competition of sulfate-reducing bacteria, conductive materials can benefit electron competitive capability of methanogens, recovering CH4 production activity. Conductive nanomaterials stimulate the excretion of extracellular polymeric substances, which can prevent cells from nano-cytotoxicity. Future perspectives about unraveling mitigation mechanisms induced by conductive materials in methanogenesis processes are further discussed.

The effect of microwave pretreatment on anaerobic co-digestion of sludge and food waste: Performance, kinetics and energy recovery

This paper studied the effect of microwave (MW) pretreatment on anaerobic co-digestion of sludge (SS) and food waste (FW). Using SS and FW as digestive substrates, the MW pretreatment method was used to determine the changes in the substrate matrix by means of batch anaerobic digestion at 37 °C. The kinetics of methane production were calculated, and the changes in organic matter during anaerobic co-digestion, the properties of the anaerobic-digested effluent, and the net energy output of the co-digestion system were determinated. The results showed that MW pretreatment was beneficial to the dissolution of organic matter, conversion of protein to NH4+-N, cumulative methane production, unit biomethane yield, and reaction rate of methane production in the SS and FW anaerobic co-digestion system. The highest cumulative methane production in the co-digestion system reached 3446.3 ± 172.3 mL (35 days), which was 19.93% higher than that of the control. Furthermore, MW pretreatment significantly increased the accumulation of VFAs and the content of butyric acid in the anaerobic-digested effluent, which was beneficial to the methanogenesis process. The MW pretreatment of all co-digested substrates produced a greater net energy output than the control, and the MW-SS + MW-FW group yielded the highest net energy output, which was 76.25 kJ/g Fed VS. The results indicated that MW pretreatment prior to SS and FW anaerobic co-digestion is an effective way to improve the anaerobic digestion efficiency and energy recovery rate.

Direct interspecies electron transfer (DIET) can be suppressed under ammonia-stressed condition – Reevaluate the role of conductive materials

Thermal hydrolysis pretreatment (THP) and anaerobic digestion (AD) integrated (THP-AD) process is a promising process for sludge management. However, the high ammonia production during the THP-AD process severely affects system’s stability and performance. Conductive materials are widely reported to stimulate AD, thus they are potentially helpful in alleviating ammonia inhibition. This study investigated the effects of three widely studied conductive materials, i.e. zero-valent iron (ZVI), magnetite nanoparticles (Mag.) and powder activated carbon (PAC), on THP-AD process. Results showed that all the tested materials could effectively stimulate methanogenesis process under non-ammonia inhibition conditions. However, upon ammonia stress, these materials behaved distinctively with the best methanogenic performance in ZVI group followed by Mag. Group, and even worsened inhibition occurred in PAC group. The mechanisms behind were investigated from two levels–the reaction kinetics of each anaerobic digestion step and the responses of intracellular metabolism. It is revealed that ZVI effectively promoted all AD reactions, especially the energy unfavorable propanoate and butanoate metabolism and overall methanogenesis. In addition, ZVI likely acted as intracellular electron shuttles, and the conjunction point of ZVI to electron transfer system was identified as EtfAB: quinone oxidoreductase. On the contrary, the declined methanogenic performance in PAC group was attributed to selectively stimulated the growth of acetoclastic methanogen – Methanosaeta, which is sensitive to ammonia toxicity. The proteomic information further revealed that ammonia stress was unfavorable to the formation of direct interspecies electron transfer between syntrophic anaerobes. Overall, the present study provides fundamental knowledge about the role of different conductive materials in AD systems from intracellular proteomic level.

Approaches to the mitigation of ammonia inhibition during anaerobic digestion – a review

AbstractThe digestion process of organic waste rich in high ammonia content has always been a gridlock during the methanogenesis process. The free ammonia may increase inhibition/toxicity, which in turn affects the microbial community in the digester and eventually leads to process failures. Substantial methods have been proposed and assessed for curtailing ammonia emissions in anaerobic digesters to attain a safe and steady process so that, along with high methane production, high quality effluents can also be recovered. There are several means for lowering the erratic ammonia in organic wastes that are in use currently, such as decrease of pH, which favours the formation of ammonium over ammonia in the equilibrium; for example, the use of chemical additives that attach ammonium-N. Ammonia can also be removed from nitrogen-rich substrates during anaerobic digestion through other methods such as struvite precipitation, membrane distillation, air stripping, ion exchange, and adsorption. A thorough survey of different articles has shown that ion exchange, adsorption and changing of the C/N ratio through the co-digestion technique are the most commonly studied methods for mitigating ammonia inhibition in wastewater during anaerobic digestion. A detailed review of these methods in the context of nitrogen-rich substrates will be discussed in this paper.

Ruminal Methanogenic Responses to the Thiamine Supplementation in High-Concentrate Diets.

Simple SummaryOptimal methane (CH4) mitigation strategy should decrease carbon footprint and meanwhile avoid causing hazard residues and productive reduction. Thiamine is a critical coenzyme in carbohydrates, and metabolism has been confirmed to effectively attenuate subacute ruminal acidosis (SARA). Whether thiamine supplementation impacts ruminal methanogenesis is still unclear. In the present study, thiamine supplementation in high-concentrate diets (HC) was conducted to investigate the impacts on ruminal methanogenesis process. Results indicated that thiamine supplementation in HC could efficiently reduce CH4 emissions compared with high-forage diets; meanwhile, it did not cause ruminal metabolic disorders compared with HC treatment. The effective decrease in methane production and changes of methanogens after thiamine supplementation in HC compared with high-forage diets contributed a better understanding of thiamine on ruminal carbohydrate metabolism. This finding here provided a brand-new perspective on mitigating methane emission in dairy cattle production, and an optimal strategy to decrease the carbon footprint without compromising the livestock production efficiency.Background: Thiamine supplementation in high-concentrate diets (HC) was confirmed to attenuate ruminal subacute acidosis through promoting carbohydrate metabolism, however, whether thiamine supplementation in HC impacts methane metabolism is still unclear. Therefore, in the present study, thiamine was supplemented in the high-concentrate diets to investigate its effects on ruminal methanogens and methanogenesis process. Methods: an in vitro fermentation experiment which included three treatments: control diet (CON, concentrate/forage = 4:6; DM basis), high-concentrate diet (HC, concentrate/forage = 6:4; DM basis) and high-concentrate diet supplemented with thiamine (HCT, concentrate/forage = 6:4, DM basis; thiamine supplementation content = 180 mg/kg DM) was conducted. Each treatment concluded with four repeats, with three bottles in each repeat. The in vitro fermentation was sustained for 48h each time and repeated three times. At the end of fermentation, fermentable parameters, ruminal bacteria and methanogens community were measured. Results: HC significantly decreased ruminal pH, thiamine and acetate content, while significantly increasing propionate content compared with CON (p < 0.05). Conversely, thiamine supplementation significantly increased ruminal pH, acetate while significantly decreasing propionate content compared with HC treatment (p < 0.05). No significant difference of ruminal methanogens abundances among three treatments was observed. Thiamine supplementation significantly decreased methane production compared with CON, while no significant change was found in HCT compared with HC. Conclusion: thiamine supplementation in the high-concentrate diet (HC) could efficiently reduce CH4 emissions compared with high-forage diets while without causing ruminal metabolic disorders compared with HC treatment. This study demonstrated that supplementation of proper thiamine in concentrate diets could be an effective nutritional strategy to decrease CH4 production in dairy cows.

Revealing the mechanisms of Triclosan affecting of methane production from waste activated sludge

Triclosan (TCS), as an antimicrobial agent, is considered as a representative emerging contaminant and was frequently detected in excess sludge. This study investigated the effect of TCS on activate wastewater sludge (WAS) digestion through laboratory methane production experiment. It was concluded that TCS had a tendency to restrain methane production from sludge with its exposure level increasing. The results displayed that the yields of final maximum cumulative methane production were similar about 108.4 mL/g VSS at TCS level lower 200 mg TCS/kg TSS, while the values were approximately 95.2 mL/g VSS with TCS level over 550 mg TCS/kg TSS. Although TCS could be degraded, its intermediates in this study had no effect on sludge digestion. In addition, TCS at higher levels had seriously negative effect on the solubilization, hydrolysis, acidification, and methanogenesis processes. Microbial community was further analyzed to understand the TCS’s effect on digestion system from a micro perspective.

Octylphenol facilitates fermentative volatile fatty acids recovery from waste activated sludge

The presence of endocrine disruptor compounds (EDCs) in wastewater treatment plants has attracted widespread attention, but their potential impact on anaerobic fermentation of waste activated sludge (WAS) remains unclear. Therefore, this study aims to reveal the effect of typical EDC octylphenol (OP) on the recovery of volatile fatty acids (VFAs) in anaerobic fermentation. The results show that OP has a positive effect on the recovery of VFAs from WAS. The presence of 200 mg/kg dry sludge of OP increased the cumulative amount of VFAs from 3245 in the control (without OP) to 6828 mg COD/L. The increase in VFA production was mainly attributed to the accumulation of acetic acid, which rose from 1511 to 4425 mg COD/L, almost tripled. Further research found that OP promoted solubilization and hydrolysis by improving the biodegradability of WAS, and severely inhibited the methanogenesis process by inhibiting the activity of coenzyme F420, thereby significantly increasing the accumulation of acetic acid. These findings are of great significance to clarify the role of OP in anaerobic fermentation, and provide theoretical basis and guidance for the selection of target products in anaerobic fermentation of WAS containing OP.

Zero-valent iron enhanced anaerobic digestion of pre-concentrated domestic wastewater for bioenergy recovery: Characteristics and mechanisms

Pre-concentrated domestic wastewater (PDWW) rich in organic matters can be a suitable substrate for anaerobic digestion (AD) towards holistic resource and bioenergy recovery. Micron zero-valent iron (ZVI) was applied in designed batch experiments during anaerobic treatment of PDWW to verify its roles in performance enhancement and associated mechanisms. In the selected range of food to microorganism (F/M) ratio, 0.5 gCOD/gMLVSS was most appropriate as biomethane production potential (BMP) of 0.275 L CH4/gCOD was obtained. The optimal ZVI dosage at fixed F/M of 0.5 was 6 g/L, further enhancing the BMP by 15.2%. Furthermore, ZVI improved the hydrolysis process (producing more soluble organics) and regulated acidification process (affecting volatile fatty acids distribution). No obvious impact on acetoclastic and hydrogenotrophic methanogenesis processes was noted with ZVI addition. ZVI based AD of the PDWW is promising for promoting the practical application of advanced domestic wastewater treatment strategy (pre-concentration plus anaerobic digestion).

Effect of Aso limonite on anaerobic digestion of waste sewage sludge

The effect of Aso volcanic limonite was explored in anaerobic digestion using waste sewage sludge (WSS). In this study, methane and hydrogen sulfide were remarkably inhibited when Aso limonite was mixed with WSS as well as a significant reduction of ammonia. Although pH was lowered after adding Aso limonite, methane was still inhibited in neutralized pH condition at 7.0. Hydrolysis stage was not influenced by Aso limonite as supported by the result that a high protease activity was still detected in the presence of the material. However, acidogenesis stage was affected by Aso limonite as indicated by the different productions of organic acids. Acetic acid, was accumulated in the presence of Aso limonite due to the inhibition of methane production, except in the highest concentration of Aso limonite which the production of acetate may be inhibited. Besides, the production of propionate and butyrate reduced in accordance to the increased concentration of Aso limonite. In addition, Archaeal activity (methanogens) in WSS with Aso limonite was low in agreement with the low methane production. Thus, these results indicate that Aso limonite influences the acidogenesis and methanogenesis processes, by which the productions of methane and ammonia were inhibited. On the other hand, in the contactless of Aso limonite during the anaerobic digestion of WSS (Aso limonite was placed in the area of headspace in the vial), Aso limonite had the adsorptive ability for hydrogen sulfide from WSS, but not for methane. This contactless system of Aso limonite may be a practical means to remove hydrogen sulfide without inhibiting methane production as an important bioenergy source.