Stimulating Anaerobic Degradation of Butyrate via Syntrophomonas wolfei and Geobacter sulfurreducens: Characteristics and Mechanism.

Anaerobic digestion (AD) has been widely applied for the degradation of organic wastewater due to its advantages of high-load operation and energy recovery. However, some challenges, such as low treatment capacity and instability caused by the accumulation of volatile fatty acids, limit its further application. Here, S. wolfei and G. sulfurreducens were initially co-cultured in the anaerobic anode of bio-electrochemical system for degrading butyric acid. Butyrate degradation characteristics in different conditions were quantitatively described. Moreover, G. sulfurreducens simultaneously strengthened the consumption of H2 and acetic acid via direct interspecies electron transfer, thereby strengthening the degradation of butyric acid via a co-metabolic process. During butyrate degradation, the co-culture of S. wolfei and G. sulfurreducens showed more advantages than that of S. wolfei and methanogens. This present study provides a new perspective of butyrate metabolism, which was independent of methanogens in an AD process.

Pyrolysis of antibiotic mycelial residue for biochar: Kinetic deconvolution, biochar properties, and heavy metal immobilization.

The safe disposal of antibiotic mycelial residue (AMR), a hazardous waste, is a pressing problem owing to the spread of antibiotic and heavy metal pollution. In this study, AMR pyrolysis at different temperatures and heating rates was investigated to prepare valuable biochar for heavy metal immobilization. The results showed that AMR decomposition mainly involved three pseudo-reactions, with average activation energies of 252.4, 149.8, and 219.7 kJ/mol, that fitted a three-dimensional diffusion model. Increasing the pyrolysis temperature and heating rate decreased the yield and volatile matter content of biochar, but the ash content, fixed carbon content, and aromaticity increased. The AMR-derived biochar had a favorable fuel property (18.1-19.8 MJ/kg) and stability against degradation in soil. Calcium oxalate hydrate, a major mineral in AMR, degraded during biochar formation. Furthermore, high pyrolysis temperature promoted the residual fractions of Cr, Cu, Zn, Cd, and Pb in biochar, more so than did the heating rate, inducing a low potential ecological risk. In particular, the leaching rate of Zn decreased from 46.9% in AMR to 0.3% in biochar obtained at 700 °C with a heating rate of 10 °C/min. This study elucidates the formation process and physicochemical properties of AMR biochar, which helps in the harmless utilization of AMR as a carbon resource.

Inoculating indoleacetic acid bacteria promotes the enrichment of halotolerant bacteria during secondary fermentation of composting.

The secondary fermentation stage is critical for stabilizing composting products and producing various secondary metabolites. However, the low metabolic rate of mesophilic bacteria is regarded as the rate-limiting stage in composting process. In present study, two indoleacetic acid (IAA)-producing bacteria (Bacillus safensis 33C and Corynebacterium stationis subsp. safensis 29B) were inoculated to strengthen the secondary fermentation stage to improve the plant-growth promoting potential of composting products. The results showed that the addition of IAA-producing bacteria promoted the assimilation of soluble salt, the condensation and aromatization of humus, and the accumulation of dissolved organic nitrogen (DON) and dissolved organic carbon (DOC). The bioaugmentation strategy also enabled faster microbial community succession during the medium-late phase of secondary fermentation. However, the colonization of Bacillus and Corynebacterium could not explain the disproportionate increase of IAA yield, which reached up to 5.6 times compared to the control group. Deeper analysis combined with physicochemical properties and microbial community structure suggested that IAA-producing bacteria might induce the increase of salinity, which enriched halotolerant bacteria capable of producing IAA, such as Halomonas, Brachybacterium and Flavobacterium. In addition, the results also proved that it was necessary to shorten secondary fermentation time to avoid IAA degradation without affecting composting maturity. In summary, enhancing secondary fermentation of composting via adding proper IAA-producing bacteria is an efficient strategy for upgrading the quality of organic fertilizer.

Higher Temperatures Do Not Always Achieve Better Antibiotic Resistance Gene Removal in Anaerobic Digestion of Swine Manure.

This study employed high-throughput quantitative PCR and 16S rRNA sequencing to evaluate the effect of temperature and residual antibiotics on the dynamics of antibiotic resistance genes (ARGs) and microbial communities during anaerobic digestion of swine manure. The abundances of total ARGs and 16S rRNA genes significantly decreased in all of four treatments (25°C, 37°C, and 37°C with 50 mg of wet weight antibiotics of body weight, and 55°C). The abundances of most ARG types were significantly correlated with those of the 16S rRNA gene and transposase gene (P < 0.01). However, the abundances of total ARGs at 55°C were much higher than those of other treatments. Meanwhile, the microbial communities at 55°C, where the Streptococcus pathogen remained at a relatively high abundance and cellulose degraders and hydrogen producers, such as Ethanoligenens and Coprococcus bacteria, increased, were markedly different from those of other treatments. Redundancy analysis indicates that temperature, pH, and the genus Streptococcus had the highest explanation for ARG variation among experimental factors, chemical properties, and representative genera, respectively. Network analysis further showed that the genus Streptococcus contributed greatly to the higher ARG abundance at 55°C. The moderate antibiotic residue only caused a slight and transitory inhibition for microbially diverse populations and promotion for ARG abundance, probably due to the degradation of antibiotics and microbial adaptability. Our results clarify the cooperativity of gene transfer-related items on ARG variation and intensively prove that higher temperature cannot always achieve better ARG removal in anaerobic digestion unless pathogens and gene transfer elements are more efficiently inhibited.IMPORTANCE Antibiotic resistance genes (ARGs) are frequently detected with high abundance in manure-applied soils. Anaerobic digestion is one of widely used processes for animal waste treatment. Thus, it is critical to understand the potential of anaerobic digestion to attenuate ARGs. Although some previous studies recommended thermophilic digestion for ARG removal, they did not get sufficient evidence to support this view. The antibiotics applied to animals are mostly excreted through feces and urine because of incomplete metabolism. It is indispensable to know whether residual antibiotics in manure will hinder ARG attenuation in anaerobic digesters. The significance of our research is in comprehensively understanding the evolution and mechanism of ARGs in anaerobic digestion of swine manure affected by temperature and residual antibiotics, which will allow the development of an ARG elimination strategy before their release into the environment.

Syntrophic methane production from volatile fatty acids: Focus on interspecies electron transfer.

Methane is a renewable biomass energy source produced via anaerobic digestion (AD). Interspecies electron transfer (IET) between methanogens and syntrophic bacteria is crucial for mitigating energy barriers in this process. Understanding IET is essential for enhancing the efficiency of syntrophic methanogenesis in anaerobic digestion. Interspecies electron transfer mechanisms include interspecies H2/formate transfer, direct interspecies electron transfer (DIET), and electron-shuttle-mediated transfer. This review summarizes the mechanisms, developments, and research gaps in IET pathways. Interspecies H2/formate transfer requires strict control of low H2 partial pressure and involves complex enzymatic reactions. In contrast, DIET enhances the electron transfer efficiency and process stability. Conductive materials and key microorganisms can be modulated to stimulate the DIET. Electron shuttles (ES) allow microorganisms to interact with extracellular electron acceptors without direct contact; however, their efficiency depends on various factors. Future studies should elucidate the key functional groups, metabolic pathways, and regulatory mechanisms of IET to guide the optimization of AD processes for efficient renewable energy production.

Study of oyster shell as a potential substrate for constructed wetlands.

We tested the suitability of oyster shell (OS) as a substrate for phosphorus removal in constructed wetlands (CWs) treating swine wastewater. OS is proven to have a significant phosphorus adsorption capacity; significant phosphorus removal was achieved in vertical subsurface flow constructed wetlands (VSSFs) that were filled with OS and used to treat swine wastewater. In the VSSF system, OS adsorption and precipitation played the greatest role in phosphorus removal, and the phosphorus distribution in the substrate layers was attributed to the vertical flow state of wastewater in the system. Ca-P was the predominant form of phosphorus in the system. Overall, the study results showed that OS could be used for phosphorus removal in CWs. OS also allowed for reuse of a waste substance, making the overall system more environmentally friendly.

Iron-modified biochar boosts anaerobic digestion of sulfamethoxazole pharmaceutical wastewater: Performance and microbial mechanism.

The accumulation of volatile fatty acids (VFAs) caused by antibiotic inhibition significantly reduces the treatment efficiency of sulfamethoxazole (SMX) wastewater. Few studies have been conducted to study the VFAs gradient metabolism of extracellular respiratory bacteria (ERB) and hydrogenotrophic methanogen (HM) under high-concentration sulfonamide antibiotics (SAs). And the effects of iron-modified biochar on antibiotics are unknown. Here, the iron-modified biochar was added to an anaerobic baffled reactor (ABR) to intensify the anaerobic digestion of SMX pharmaceutical wastewater. The results demonstrated that ERB and HM were developed after adding iron-modified biochar, promoting the degradation of butyric, propionic and acetic acids. The content of VFAs reduced from 1166.0 mg L-1 to 291.5 mg L-1. Therefore, chemical oxygen demand (COD) and SMX removal efficiency were improved by 22.76% and 36.51%, and methane production was enhanced by 6.19 times. Furthermore, the antibiotic resistance genes (ARGs) such as sul1, sul2, intl1 in effluent were decreased by 39.31%, 43.33%, 44.11%. AUTHM297 (18.07%), Methanobacterium (16.05%), Geobacter (6.05%) were enriched after enhancement. The net energy after enhancement was 0.7122 kWh m-3. These results confirmed that ERB and HM were enriched via iron-modified biochar to achieve high efficiency of SMX wastewater treatment.

pH and hydraulic retention time regulation for anaerobic fermentation: Focus on volatile fatty acids production/distribution, microbial community succession and interactive correlation.

Enriching suitable fermentative products by optimizing operation conditions could effectively improve the efficiency of anaerobic digestion. In the present study, pH (5.0-6.0) and hydraulic retention time (HRT) (2 h-12 h) were regulated for volatile fatty acids (VFAs) production during glucose fermentation in acidogenic continuous stirred tank reactor (CSTR). Results showed that acetate and butyrate dominated during pH regulation. HRT reduction favored butyrate production and formate retainment. Maximum total VFAs production with highest acetate content was achieved at pH of 6.0 and HRT of 6 h. Microbial analysis revealed that Clostridium_sensu_stricto_1 was predominant butyrate producer during pH regulation, and Bacteroides was main contributor when HRT shorter than 6 h. In addition to acetyl-CoA pathway, acetate could also be produced via homoacetogenesis by Parabacteroides, UCG-004 and norank_f__Acidaminococcaceae. These results would give guidance for enhancing targeted VFAs products by optimizing operational parameters or bio-augmentation with specific bacteria.

Two-stage hybrid microalgal electroactive wetland-coupled anaerobic digestion for swine wastewater treatment in South China: Full-scale verification.

Constructed wetlands have been widely used for organic wastewater treatment owing to low operating costs and simple maintenance. However, there are some disadvantages such as unstable efficiency in winter. In this study, a microalgal electroactive biofilm-constructed wetland was coupled with anaerobic digestion for full-scale treatment of swine wastewater. In a 12-month outdoor trial, the overall removal efficiencies of chemical oxygen demand, ammonium, nitrate, total nitrogen, total phosphorus, and nitrite reached 98.26%/95.14%, 97.96%/92.07%, 85.45%/66.04%, 95.07%/91.48%, 91.44%/91.52%, and 85.45%/84.67% in summer/winter, respectively. Hydrolytic bacteria were dominant in the anaerobic digestion part, and Cyanobium, Shewanella, and Azoarcus were enriched in the microalgal electroactive biofilm. The operating cost of the entire system was approximately 0.118 $/m3 of wastewater. These results confirm that the microalgal electroactive biofilm significantly enhances the efficiency and stability of constructed wetlands. In conclusion, the anaerobic digestion-microalgal electroactive biofilm-constructed wetland is technically and economically feasible for the treatment of swine wastewater.

Compost-derived indole-3-acetic-acid-producing bacteria and their effects on enhancing the secondary fermentation of a swine manure-corn stalk composting.

Composting, as an effectively bio-oxidative process, has been widely used for converting organic waste to organic fertilizer. However, the low fertilizer efficiency of composting product limited its application in agriculture. To improve the growth-promoting effect of composting product, the present study investigated the bioaugmentation strategy of inoculating indole-3-acetic-acid (IAA)-producing bacteria. Firstly, two IAA-producing bacteria (Bacillus safensis 33C and Rhodococcus rhodochrous YZ) were isolated from composting products with high IAA yields of 39.18 and 16.32 µg mL-1, respectively. Secondly, the microbial inoculants were prepared with 33C, YZ and a previously isolated IAA-producing strain Corynebacterium stationis 29B. To increase the accumulation of microbial secondary metabolites, microbial inoculants were amended at the secondary fermentation stage of composting. Physicochemical characterization showed that the maturity of composting product was significantly promoted by inoculating microbial inoculants prepared with 33C and 29B (single and combined inoculants). Finally, bioaugmentation with 33C and 29B increased the IAA contents of composting products by 2.9-5.2 times, which benefited the germination and early vegetative growth of plants. In summary, inoculating proper IAA-producing bacteria during secondary fermentation of composting could improve the quality of composting product and expand its application.

Deeper insights into effect of activated carbon and nano-zero-valent iron addition on acidogenesis and whole anaerobic digestion.

Conductive materials presented promising advantages for enhancing anaerobic digestion (AD) performance. This study evaluated the effects of activated carbon (AC) and nano-zero-valent iron (nZVI) on the acidogenesis and whole AD to explore their potential mechanisms. AC increased the content of lactic and propionic acids in acidogenesis. nZVI increased the production of formic acid, acetic acid and H2 in acidogenesis, thus significantly promoted the methane yield in the whole AD. Mechanism exploration proved that AC enriched Trichococcus, and norank_f__Bacteroidetes_vadinHA17, and then improved the activity of enzymes involved in the production of lactic and propionic acids. nZVI buffered the pH to increase the activity of pyruvate formate-lyase (PFL) in formic acid production. Furthermore, nZVI enriched the Methanobacterium which use H2 and formic acid as substrate. The research paves pathway for the efficient enhancement of conductive materials added novel AD process.

Novel insights into the anaerobic digestion of propionate via Syntrophobacter fumaroxidans and Geobacter sulfurreducens: Process and mechanism.

The accumulation of volatile fatty acids, particularly propionic acid, significantly inhibits the efficiency of the anaerobic digestion system. In propionate degradation metabolism, the unfavorable thermodynamics of syntrophic reactions, strict ecological niche of syntrophic priopionate oxidizing bacteria, and slow metabolic rate of methanogens are regarded as major limitations. In this study, Geobacter sulfurreducens was co-cultured with Syntrophobacter fumaroxidans in bioelelectrochemical cells to analyze the propionate degradation process, impact factor, mechanism metabolic pathways, and electron transfer comprehensively. The results revealed that the syntroph S. fumaroxidans and syntrophic partner G. sulfurreducens achieved more efficient propionate degradation than the control group, comprising S. fumaroxidans and methanogens. Moreover, the carbon resource concentration and pH were both significantly correlated with propionate degradation (P < 0.01). The results further confirmed that G. sulfurreducen strengthened the consumption of H2 and acetate via direct interspecific electron transfer in propionate degradation. These findings indicate that G. sulfurreducens plays an unidentified functional role in propionate degradation.

The application status, development and future trend of nano-iron materials in anaerobic digestion system.

Growing environment problem and emphasis of environmental protection motivate intense research efforts in exploring technology to improve treatment efficiency on refractory organic pollutants. Hence, finding a method to make up for the deficiency of anaerobic digestion (AD) is very attractive and challenging tasks. The recent spark in the interest for the usage of some nanomaterials as an additive to strengthen AD system. The adoption of iron compounds can influence the performance and stability in AD system. However, different iron species and compounds can influence AD system in significantly different ways, both positive and negative. Therefore, strengthening mechanism, treatment efficiency, microbial community changes in Nanoscale Zero Valent Iron (nZVI) and Fe3O4 nanoparticles (Fe3O4 NPs) added AD systems were summarized by this review. The strengthening effects of nZVI and Fe3O4 NPs in different pollutants treatment system were analyzed. Previous study on the effects of nZVI and Fe3O4 NPs addition on AD have reported the concentration of nZVI and Fe3O4 NPs, and the types and biodegradability of pollutants might be the key factors that determine the direction and extent of effect in AD system. This review provides a summary on the nZVI and Fe3O4 NPs added AD system to establish experiment systems and conduct follow-up experiments in future study.

Novel strategy for enhancing acetic and formic acids generation in acidogenesis of anaerobic digestion via targeted adjusting environmental niches.

Optimization of acetic acid and formic acid production efficient methanogenesis is always the research hot spot in anaerobic digestion. It is a promising approach to adjust the operation parameters to influence the functional microorganisms for better acetic acid and formic acid production in acidogenesis. Herein, the effects of pH, oxidation-reduction potential (ORP) and carbon-nitrogen (C/N) ratio were determined in batch experiments to probe acetic and formic acids production, and were further verified in continuous stirred tank reactor (CSTR). The results revealed that the content of volatile fatty acids (VFAs) reached to maximum at pH 6.0 or ORP -350 mV, while the production of acetic and formic acids was the highest at pH 7.0 or ORP -450 mV in 9 h fermentation. Also, fermentation products dominated by acetic and formic acids were adjusted in the CSTR under the operating conditions of pH 7.0 and ORP -450 mV. Microbiological analysis from batch test showed that fermentation at pH value of 7.0 enriched the diversity of microorganism, and provided a niche for microbes (Petrimonas, norank_f__Synergistaceae, vadinBC27_wastewater-sludge_group, and Trichococcus) to produce acetic and formic acids. Correspondingly, 78.70% of the carbon was converted to acetic and formic acids in pH 7.0. This study provides a promising strategy for the targeted regulation of acetic and formic acids production in acidogenesis of anaerobic digestion.

Synergetic effect of nano zero-valent iron and activated carbon on high-level ciprofloxacin removal in hydrolysis-acidogenesis of anaerobic digestion.

Ciprofloxacin is the most commonly prescribed antibiotic, and its widespread use poses threat to environmental safety. The removal of ciprofloxacin from contaminated water has remained a major challenge. The present study investigated adding nanoscale zero-valent iron (NZVI) and activated carbon (AC) on high-level ciprofloxacin removal in hydrolysis-acidogenesis stage of anaerobic digestion. The results showed that the degradation rate of ciprofloxacin increased from 22.61% (Blank group) to 72.41% after adding NZVI/AC with concentration of ciprofloxacin in effluent decreasing from 8.25 mg L-1 to 3.48 mg L-1. The volatile fatty acids (VFAs) yield increased by 173.7% compared with the Blank group. In addition, the NZVI/AC group achieved the highest chemical oxygen demand (COD) removal rate and acidogenesis rate. The microbial community analysis presented that hydrolytic and acidogenic bacteria and microorganisms related to degrading ciprofloxacin were obviously improved in the NZVI/AC group. Moreover, eleven transformation products and the main degradation pathways were proposed based on mass spectrometry information. In summary, the NZVI/AC addition supplied promising approach for ciprofloxacin wastewater treatment.

Deep insights into the network of acetate metabolism in anaerobic digestion: focusing on syntrophic acetate oxidation and homoacetogenesis.

Acetate is a pivotal intermediate product during anaerobic decomposition of organic matter. Its generation and consumption network is quite complex, which almost covers the most steps in anaerobic digestion (AD) process. Besides acidogenesis, acetogenesis and methanogenesis, syntrophic acetate oxidation (SAO) replaced acetoclastic methanogenesis to release the inhibition of AD at some special conditions, and the importance of considering homoacetogenesis had also been proved when analysing anaerobic fermentations. Syntrophic acetate-oxidizing bacteria (SAOB), with function of SAO, can survive under high temperature and ammonia/ volatile fatty acids (VFAs) concentrations, while, homoacetogens, performed homoacetogenesis, are more active under acidic, alkaline and low temperature (10°C-20°C) conditions, This review summarized the roles of SAO and homoacetogenesis in AD process, which contains the biochemical reactions, metabolism pathways, physiological characteristics and energy conservation of functional bacteria. The specific roles of these two processes in the subprocess of AD (i.e., acidogenesis, acetogenesis and methanogenesis) were also analyzed in detail. A two phases anaerobic digester is proposed for protein-rich waste(water) treatment by enhancing the functions of homoacetogens and SAOB compared to the traditional two-phases anaerobic digesters, in which the first phase is fermentation phase including acidogens and homoacetogens for acetate production, and second phase is a mixed culture coupling syntrophic fatty acids bacteria, SAOB and hydrogenotrophic methanogens for methane production. This review provides a new insight into the network on production and consumption of acetate in AD process.

Carbon- and metal-based mediators modulate anaerobic methanogenesis and phenol removal: Focusing on stimulatory and inhibitory mechanism.

In this study, anaerobic batch experiments were conducted to investigate the effect of carbon-based (biochar) and metal-based (nanoscale zero-valent iron, NZVI and zero valent iron, ZVI) mediators on the AD process treating phenolic wastewater. Fresh apricot shell- and wood-derived biochar (BiocharA, BiocharB) could remove the phenol efficiently (77.1% and 86.2%), suggesting that biodegradation cooperated with adsorption had advantage in phenol removal. BiocharB, NZVI and ZVI enhanced the methane production by 17.6%, 23.7% and 23.2%, respectively. Apart from serving as carrier for microbial growth, BiocharB might promote the direct interspecies electron transfer (DIET) since the Anaerolineaceae/Clostridium sensu stricto, which have potential for DIET, were enriched. NZVI and ZVI added systems mainly enhanced the abundance of Clostridium sensu stricto (24.5%, 37.6%) and Methanosaeta. Interestingly, BiocharA inhibited the methanogenesis completely. An inhibitory mechanism was proposed: the exposure of absorbed microbes on the BiocharA to the highly concentrated phenol in biochar' pores resulted in the inhibition of methanogens, especially for Methanosarcina. In conclusion, this study showed that suitable biochar (BiocharB) could serve as an alternative redox mediator for realizing simultaneously the efficient phenol removal and methane production.

Syntrophic butyrate-oxidizing methanogenesis promoted by anthraquinone-2-sulfonate and cysteine: Distinct tendencies towards the enrichment of methanogens and syntrophic fatty-acid oxidizing bacteria.

Interspecies electron transfer (IET) between syntrophic fatty-acid oxidizing bacteria (SFOBs) and methanogens decided the performance of anaerobic digestion. Electron shuttles, as potential IET accelerators, were controversial concerning their influences on methanogenesis. In this study, concentration-dependent effects of anthraquinone-2-sulfonate (AQS) and cysteine on glucose digestion were firstly demonstrated: low dosage of AQS and cysteine (50 and 100 µM, respectively) had highest methane yield (133.5% and 148.6%, respectively). Using butyrate as substrate, distinct tendencies towards the enrichment of methanogenic community were further revealed. Cysteine just acted as a reductant which lowered ORP quickly and enriched most methanogens. It benefited methanogenesis right until methanogenic substrates accumulated. AQS, however, showed characteristic features of electron shuttles: it was firstly oxidized by SFOBs and then reduced by hydrogenotrophic methanogens, which accelerated methanogenic butyrate degradation. This study showed wide spectrum of SFOBs and methanogens benefited from the addition of electron shuttles, which laid foundation for future application.

Deeper investigation on methane generation from synthetic wastewater containing oxytetracycline in a scale up acidic anaerobic baffled reactor.

Acidic anaerobic digestion attracted much attention and interest due to its significant advantage in wastewater treatment. In the present study, methanogenic fermentation was successfully operated under acidic condition during treating wastewater containing oxytetracycline (OTC) in a scale up anaerobic baffled reactor (ABR). After start-up process, the pH value in the first compartment was 4.60 with high activity of methanogenesis. After stabilization, different OTC loading of 1.0, 3.3 and 5.0 g/m3/d was added in the influent for OTC removal. The resulted showed that OTC addition had little impact on the methane generation with whole COD and OTC removal rate of 95% and 60%, respectively. The microbial analysis, OTC addition could significantly influence the bacteria and archaea communities. To be more specific, Methanosaeta showed the highest relative abundance and tolerance to OTC under acidic condition. The present work supplied deeper insights into methane generation from acidic condition during wastewater containing OTC treatment.

Variation of volatile fatty acid oxidation and methane production during the bioaugmentation of anaerobic digestion system: Microbial community analysis revealing the influence of microbial interactions on metabolic pathways.

Anaerobic digestion (AD) is widely used on waste treatment for its great capability of organic degradation and energy recovery. Accumulation of volatile fatty acids (VFAs) caused by impact loadings often leads to the acidification and failure of AD systems. Bioaugmentation is a promising way to accelerate VFA degradation but the succession of microbial communities usually caused unpredictable consequences. In this study, we used the sludge previously acclimated with VFAs for the bioaugmentation of an acidified anaerobic digestion system and increased the methane yield by 8.03-9.59 times. To see how the succession of microbial communities affected bioaugmentation, dual-chamber devices separated by membrane filters were used to control the interactions between the acidified and acclimated sludges. The experimental group with separated sludges showed significant advantages of VFA consumption (5.5 times less final VFA residue than the control), while the group with mixed sludge produced more methane (4.0 times higher final methane yield than the control). Microbial community analysis further highlighted the great influences of microbial interaction on the differentiation of metabolic pathways. Acetoclastic methanogens from the acclimated sludge acted as the main contributors to pH neutralization and methane production during the early phase of bioaugmentation, and maintained active in the mixed sludge but degenerated in the separated sludges where interactions between sludge microbiotas were limited. Instead, syntrophic butyrate and acetate oxidation coupled with nitrate and sulfate reduction was enriched in the separated sludges, which lowered the methane conversion rate and would cause the failure of bioaugmentation. Our study revealed the importance of microbial interactions and the functionality of enriched microbes, as well as the potential strategies to optimize the durability and efficiency of bioaugmentation.