Mechanistic understanding of acclimation and energy metabolism of acetoclastic methanogens under different substrate to microorganism ratios.

Mechanistic understanding of acetoclastic methanogenesis is pivotal for optimizing anaerobic digestion for efficient methane production. In this study, two different operational modes, continuous flow reactor (CFR) and sequencing batch reactor (SBR), accompanied with solids retention times (SRT) of 10 days (SBR10d and CFR10d) and 25 days (SBR25d and CFR25d) were implemented to elucidate their impacts on microbial communities and energy metabolism of methanogens in acetate-fed systems. Microbial community analysis revealed that the relative abundance of Methanosarcina (16.0%-46.0%) surpassed Methanothrix (3.7%-22.9%) in each reactor. SBRs had the potential to enrich both Methanothrix and Methanosarcina. Compared to SBRs, CFRs had lower total relative abundance of methanogens. Methanosarcina exhibited a superior enrichment in reactors with 10-day SRT, while Methanothrix preferred to be acclimated in reactors with 25-day SRT. The operational mode and SRT were also observed to affect the distribution of acetate-utilizing bacteria, including Pseudomonas, Desulfocurvus, Mesotoga, and Thauera. Regarding enzymes involved in energy metabolism, Ech and Vho/Vht demonstrated higher relative abundances at 10-day SRT compared to 25-day SRT, whereas Fpo and MtrA-H showed higher relative abundances in SBRs than those in CFRs. The relative abundance of genes encoding ATPase harbored by Methanothrix was higher than Methanosarcina at 25-day SRT. Additionally, the relative abundance of V/A-type ATPase (typically for methanogens) was observed higher in SBRs compared to CFRs, while the F-type ATPase (typically for bacteria) exhibited higher relative abundance in CFRs than that in SBRs.

Effects of Antibiotics on the DAMO Process and Microbes in Cattle Manure.

Denitrifying anaerobic methane oxidation (DAMO) can mitigate methane emissions; however, this process has not been studied in cattle manure, an important source of methane emissions in animal agriculture. The objective of this study was to investigate the occurrence of DAMO microbes in cattle manure and examine the impacts of veterinary antibiotics on the DAMO process in cattle manure. Results show that DAMO archaea and bacteria consistently occur at high concentrations in beef cattle manure. During the long-term operation of a sequencing batch reactor seeded with beef cattle manure, the DAMO activities intensified, and DAMO microbial biomass increased. Exposure to chlortetracycline at initial concentrations up to 5000 µg L-1 did not inhibit DAMO activities or affect the concentrations of the 16S rRNA gene and functional genes of DAMO microbes. In contrast, exposure to tylosin at initial concentrations of 50 and 500 µg L-1 increased the activities of the DAMO microbes. An initial concentration of 5000 µg L-1 TYL almost entirely halted DAMO activities and reduced the concentrations of DAMO microbes. These results show the occurrence of DAMO microbes in cattle manure and reveal that elevated concentrations of dissolved antibiotics could inhibit the DAMO process, potentially affecting net methane emissions from cattle manure.

Meta-analysis of microbial source tracking for the identification of fecal contamination in aquatic environments based on data-mining.

Microbial source tracking (MST) technology represents an innovative approach employed to trace fecal contamination in environmental water systems. The performance of primers may be affected by amplification techniques, target primer categories, and regional differences. To investigate the influence of these factors on primer recognition performance, a meta-analysis was conducted on the application of MST in water environments using three databases: Web of Science, Scopus, and PubMed (n = 2291). After data screening, 46 studies were included in the final analysis. The investigation encompassed Polymerase Chain Reaction (PCR)/quantitative PCR (qPCR) methodologies, dye-based (SYBR)/probe-based (TaqMan) techniques, and geographical differences of a human host-specific (HF183) primer and other 21 additional primers. The results indicated that the primers analyzed were capable of differentiating host specificity to a certain degree. Nonetheless, by comparing sensitivity and specificity outcomes, it was observed that virus-based primers exhibited superior specificity and recognition capacity, as well as a stronger correlation with human pathogenicity in water environments compared to bacteria-based primers. This finding highlights an important direction for future advancements. Moreover, within the same category, qPCR did not demonstrate significant benefits over conventional PCR amplification methods. In comparing dye-based and probe-based techniques, it was revealed that the probe-based method's advantage lay primarily in specificity, which may be associated with the increased propensity of dye-based methods to produce false positives. Furthermore, the heterogeneity of the HF183 primer was not detected in China, Canada, and Singapore respectively, indicating a low likelihood of regional differences. The variation among the 21 other primers may be attributable to regional differences, sample sources, detection techniques, or alternative factors. Finally, we identified that economic factors, climatic conditions, and geographical distribution significantly influence primer performance.

Revealing impacts of operational modes on anaerobic digestion systems coupling with sulfate reduction.

Anaerobic digestion (AD) is promising for treating high-strength wastewater. However, the effect of operational parameters on microbial communities of AD with sulfate is not yet fully understood. To explore this, four reactors were operated under rapid- and slow-filling modes with different organic carbons. Reactors in the rapid-filling mode generally exhibited a fast kinetic property. For example, the degradation of ethanol was 4.6 times faster in ASBRER than in ASBRES, and the degradation of acetate was 11.2 times faster in ASBRAR than in ASBRAS. Nevertheless, reactors in the slow-filling mode could mitigate propionate accumulation when using ethanol as organic carbon. Taxonomic and functional analysis further supported that rapid- and slow-filling modes were suitable for the growth of r-strategists (e.g., Desulfomicrobium) and K-strategists (e.g., Geobacter), respectively. Overall, this study provides valuable insights into microbial interactions of AD processes with sulfate through the application of the r/K selection theory.

Distribution of extracellular amino acids and their potential functions in microbial cross-feeding in anaerobic digestion systems.

Anaerobic digestion is a prevalent bioenergy production process relying on a complex network of symbiotic interactions, where the nutrient based cross-feeding is an essential microbial mechanism. Here, the cross-feeding function was assessed by analyzing extracellular polymeric substances-associated amino acids in microbial aggregates collected from 14 lab-scale anaerobic digesters, as well as deciphering their genetically biosynthetic potential by syntrophic bacteria and methanogens. The total concentration of essential amino acids ranged from 1.2 mg/g VSS to 174.0 mg/g VSS. The percentages of glutamic acid (8.5 âˆ¼ 37.6%), lysine (2.7 âˆ¼ 22.6%), alanine (5.6 âˆ¼ 13.2%), and valine (3.0 âˆ¼ 10.4%) to the total amount of detected amino acids were the highest in most samples. Through metagenomics analysis, several investigated syntrophs (i.e., Smithella, Syntrophobacter, Syntrophomonas, and Mesotoga) and methanogens (i.e., Methanothrix and Methanosarcina) were auxotrophies, but the genetic ability of syntrophs and methanogens to synthesize some essential amino acids could be complementary, implying potential cross-feeding partnership.

The r/K selection theory and its application in biological wastewater treatment processes.

Understanding the characteristics of functional organisms is the key to managing and updating biological processes for wastewater treatment. This review, for the first time, systematically characterized two typical types of strategists in wastewater treatment ecosystems via the r/K selection theory and provided novel strategies for selectively enriching microbial community. Functional organisms involved in nitrification (e.g., Nitrosomonas and Nitrosococcus), anammox (Candidatus Brocadia), and methanogenesis (Methanosarcinaceae) are identified as r-strategists with fast growth capacities and low substrate affinities. These r-strategists can achieve high pollutant removal loading rates. On the other hand, other organisms such as Nitrosospira spp., Candidatus Kuenenia, and Methanosaetaceae, are characterized as K-strategists with slow growth rates but high substrate affinities, which can decrease the pollutant concentration to low levels. More importantly, K-strategists may play crucial roles in the biodegradation of recalcitrant organic pollutants. The food-to-microorganism ratio, mass transfer, cell size, and biomass morphology are the key factors determining the selection of r-/K-strategists. These factors can be related with operating parameters (e.g., solids and hydraulic retention time), biomass morphology (biofilm or granules), and operating modes (continuous-flow or sequencing batch), etc., to achieve the efficient acclimation of targeted r-/K-strategists. For practical applications, the concept of substrate flux was put forward to further benefit the selective enrichment of r-/K-strategists, fulfilling effective management and improvement of engineered pollution control bioprocesses. Finally, the future perspectives regarding the development of the r/K selection theory in wastewater treatment processes were discussed.

New insights into the r/K selection theory achieved in methanogenic systems through continuous-flow and sequencing batch operational modes.

Anaerobic digestion is achieved through cooperation among various types of microorganisms, and the regulation of microbial communities is key to achieving stable system operation. In this study, the r/K selection theory was adopted to examine the system performance and microbial characteristics in anaerobic reactors with different operating modes (continuous-flow reactors, CFRs; sequencing batch reactors, SBRs) and sludge retention times (25 and 10 days). Four lab-scale reactors (CFR25d, CFR10d, SBR25d, and SBR10d) were operated. In the cycle reaction, CFR25d achieved the highest methane yield (678.0 mL/L) and methane production rate (140.8 mL/(L·h)); while those in CFR10d were the lowest, which could have been due to an accumulation of volatile fatty acids. CFR could wash out r-strategists efficiently, such as Methanosarcina. CFR25d and CFR10d significantly enriched the K-strategist Geobacter, with the relative abundances of 34.0% and 72.6%, respectively. In addition, the hydrogenotrophic methanogens of Methanolinea and Methanospirillum (K-strategists) dominated in CFR25d and CFR10d. Methanobacterium adapted to the diverse operational conditions, but the slow grower Methanosaeta only accounted for 0.9% in CFR10d. Failure to enrich propionate oxidizers resulted in a functional absence of propionate degradation in the CFRs.

New insights into the effect of ethanol and volatile fatty acids proportions on methanogenic activities and pathways.

During anaerobic digestion, methanogenic activities and pathways can be affected by intermediates. Here, the effects of intermediates acetate, propionate, and ethanol on methanogenesis were investigated. Four anaerobic sequencing batch reactors were acclimated with propionate (ASBR_P), ethanol/propionate (ASBR_EP), acetate/propionate (ASBR_AP), and ethanol/acetate/propionate (ASBR_EAP). Ethanol was the easiest one to be biodegraded, thereby enhancing the maximum methane production rate and shortening the lag phase, while the longest acclimation time and lowest methane production rate were observed in ASBR_P. Different microbial communities and syntrophic patterns existed in four reactors. Desulfovibrio and Geobacter were the dominant ethanol-oxidizing bacteria in ASBR_EP and ASBR_EAP, respectively. Both Desulfovibrio and Geobacter possessed the potential of extracellular electron transfer, which might be the advantage of ethanol dosage for enhancing methanogenesis through direct interspecies electron transfer. Methanosarcina was enriched in ASBR_P and ASBR_AP, while Methanosaeta in ASBR_EP and ASBR_EAP. Genes responsible for acetoclastic methanogenesis were significantly enriched in ASBR_EAP, possibly resulting in the highest methanogenic activity from acetate. Results from this study will advance the optimization of practical anaerobic systems, which can be achieved by regulating the intermediates with different fermenting pathways.

Temporary addition of carbon fibers facilitates methanogenic degradation of ethanol during anaerobic treatment.

Syntrophic methanogenesis can be improved by the addition of conductive materials. In this study, conductive carbon fibers (CFs) were applied to efficiently enrich syntrophic microorganisms with potential direct interspecies electron transfer (DIET) ability and promote methanogenic activity. With ethanol as the substrate, CFs shortened the acclimation time remarkably. The maximum methane production rate and the ethanol degradation rate of suspended biomass were increased by 40% and 68%, respectively, even when CFs were subsequently removed. However, with acetate and propionate as the mixed substrate, CFs decreased the methanogenic activity. In the reactor fed with ethanol, CFs increased the relative abundance of Geobacter, Desulfovibrio, and methanogens by 57%, 39%, and 63%, respectively. Methanosaeta possessed most methane production genes and might involve in DIET. Furthermore, CFs increased the relative abundance of ethanol-degradation genes assigned to Geobacter, Desulfovibrio and Pelobacter, suggesting the promoted ethanol-degradation. The triggered electron transport system activity and acetoclastic methanogenesis also explained the accelerated effects on ethanol-degradation by long-term acclimation with CFs. Notably, the dominance of Geobacter and Methanosaeta combined with the increased electron transfer constant in the CFs-amended ethanol reactor indicated the potential role of DIET after the removal of CFs, which deserved further clarification.

Microbial physiology and interactions in anammox systems with the intermittent addition of organic carbons.

Organic carbon can affect nitrogen removal in the anaerobic ammonia oxidation (anammox) process. Two continuous up-flow anaerobic sludge blanket (UASB) reactors were operated under autotrophic (UASBN, without organic carbon) and mixotrophic (UASBCN, with the intermittent addition of acetate and propionate) conditions. Stable operation of anammox systems was achieved, with the nitrogen removal rate and percentage of 2.12 g/(L·d) and 86.4% in UASBN, and 2.09 g/(L·d) and 85.0% in UASBCN, respectively. The network of Candidatus Kuenenia, Thauera, and Nitrosomanas contributed to both nitrogen and carbon metabolisms, and the intermittent addition of acetate and propionate strengthened Ca. Kuenenia's ability to utilize several types of carbon sources. Anammox bacteria showed activity in the presence of organic carbon and without inorganic carbon, confirming the mixotrophic characteristic of Ca. Kuenenia. Cross-feeding of amino acids and vitamins existed among functional microorganisms, with extracellular polymeric substances acting as the media for microbial interactions.

Review of characteristics of anammox bacteria and strategies for anammox start-up for sustainable wastewater resource management.

Wastewater management has experienced different stages, including pollutant removal, resource recovery, and water nexus. Within these stages, anaerobic ammonia oxidation-based biotechnology can be incorporated for nitrogen removal, which can help achieve sustainable wastewater management, such as reclamation and ecologization of wastewater. Here, the physiology, metabolism, reaction kinetics and microbial interactions of anammox bacteria are discussed, and strategies to start-up the anammox system are presented. Anammox bacteria are slow growers with a high doubling time and a low reaction rate. Although most anammox bacteria grow autotrophically, some types can grow mixotrophically. The reaction stoichiometric coefficients can be affected by loading rates and other biological reactions. Microbial interactions also contribute to enhanced biological nitrogen removal and promote activities of anammox bacteria. The start-up of the anammox process is the key aspect for its practical application, which can be realized through seed selection, system stimulation, and biomass concentration enhancement.

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.

Potential interactions between syntrophic bacteria and methanogens via type IV pili and quorum-sensing systems.

Interspecies electron transfer plays an important role in syntrophic methanogenesis. Direct interspecies electron transfer (DIET) between syntrophic oxidizers and methanogens via conductive pili has been only confirmed in some specific co-cultures. This study examined potential syntrophic cooperation via type IV pili and quorum sensing between widespread syntrophic bacteria and methanogens through a metagenomic analysis of 12 anaerobic sludge samples. We found that Methanosaeta and Methanosarcina, which are reported to have DIET ability, were dominant in most methanogenic samples. Putative conductive pili genes were found in some typical syntrophic bacteria, which has rarely been reported previously. The existence of diverse quorum-sensing genes suggested that various quorum-sensing systems might participate in the communication of anaerobic microorganisms. Specifically, the diffusible signal factor and 3'-5' cyclic diguanosine monophosphate related genes were mainly assigned to syntrophic bacteria. These results suggest that the combined regulation of these signals might be responsible for the biosynthesis of type IV pili and affect syntrophic interaction during methanogenesis. These novel results provide fresh evidence to support the widespread existence of DIET in anaerobic methanogenic systems; therefore, regulating the quorum-sensing system may promote syntrophic interaction.

Coupled effects of ferroferric oxide supplement and ethanol co-metabolism on the methanogenic oxidation of propionate.

Direct interspecies electron transfer (DIET) is a new electron-transfer strategy for enhanced propionate degradation. Ethanol can enrich the DIET species of Geobacter and conductive ferroferric oxide (Fe3O4) can promote DIET. Therefore, coupled effects of ethanol and Fe3O4 on propionate degradation were investigated. The maximum CH4 production rate was increased by 81.4% by adding Fe3O4 when simultaneously fed with ethanol and propionate, while the improvement could not be observed without ethanol. The sludge conductivity and the electron transfer system activity by adding Fe3O4 were increased by 2.66 and 2.73 times, respectively. Besides, the relative abundance of functional microbes such as Geobacter, Syntrophobacter, Smithella, and Methanosaeta, and their functional genes were increased by the supplement of Fe3O4. The improvement of propionate degradation by adding Fe3O4 was largely attributed to the co-existence of ethanol degradation. The DIET between Geobacter and Methanosaeta might provide more energies or rapidly consume the oxidation products to promote the propionate degradation.

Microbial interactions regulated by the dosage of ferroferric oxide in the co-metabolism of organic carbon and sulfate.

Effects of ferroferric oxide (Fe3O4) and organic carbon on co-metabolism of sulfate and organic carbon were investigated. With Fe3O4, the degradation of acetate and sulfate was inhibited when fed with acetate, while the degradation of acetate and propionate produced from ethanol was promoted when fed with ethanol. The dominant sulfate reducing bacteria (SRB) of acetate-fed reactors were Desulfobacteraceae (complete oxidizing SRB, CO-SRB) and Desulfurmonas (incomplete oxidizing SRB, IO-SRB). IO-SRBs of Desulfobulbus and Desulfomicrobium were dominant in ethanol-fed reactors. CO-SRB had higher competitiveness than methanogens to utilize acetate, while IO-SRBs might cooperate with methanogens to produce methane when dosed with ethanol and Fe3O4. The dosage of Fe3O4 changed the dominant methanogen from Methanosarcina to Methanosaeta with acetate as the organic carbon, while increased the relative abundance of Methanosaeta with ethanol as the organic carbon.

Thermodynamic analysis of direct interspecies electron transfer in syntrophic methanogenesis based on the optimized energy distribution.

The aim of this study was to investigate the syntrophic methanogenesis from the perspective of energy transfer and competition. Effects of redox materials and redox potential on direct interspecies electron transfer (DIET) were examined through thermodynamic analysis based on the energy distribution principle. Types of redox materials could affect the efficiency of DIET via changing the total energy supply of the syntrophic methanogenesis. Decreasing system redox potential could facilitate DIET through increasing the total available energy. The competition between hydrogenotrophic methanogens and DIET methanogens might be the reason for the low proportion of the DIET pathway in the syntrophic methanogenesis. A facilitation mechanism of DIET was proposed based on the energy distribution. Providing sufficient electrons, inhibiting hydrogenotrophic methanogens and adding more competitive redox couples to avoid hydrogen generation might be beneficial for the facilitation of DIET.

Advances in direct interspecies electron transfer and conductive materials: Electron flux, organic degradation and microbial interaction.

Direct interspecies electron transfer (DIET) via electrically conductive pili (e-pili) and c-type cytochrome between acetogens and methanogens has been proposed as an essential pathway for methane production. Supplements of conductive materials have been extensively found to promote methane production in microbial anaerobic treatment systems. This review comprehensively presents recent findings of DIET and the addition of conductive materials for methanogenesis and summarizes important results through aspects of electron flux, organic degradation, and microbial interaction. Conductive materials improve DIET and methanogenesis by acting as either substitute of e-pili or electron conduit between e-pili and electron acceptors. Other effects of conductive materials such as the change of redox potential may also be important factors for the stimulation. The type and organic loading rate of substrates affect the occurrence of DIET and stimulating effects of conductive materials. Geobacter, which can participate in DIET, were less enriched in anaerobic systems cultivated with non-ethanol substrates, suggesting the existence of other syntrophs with the capability of DIET. The coupling of communication systems such as quorum sensing may be a good strategy to achieve the formation of biofilm or granule enriched with syntrophic partners capable of DIET.

Inhibition mitigation and ecological mechanism of mesophilic methanogenesis triggered by supplement of ferroferric oxide in sulfate-containing systems.

Methanogenesis can be inhibited by volatile fatty acids (VFAs) accumulation and sulfate during anaerobic wastewater treatment. In this study, effects of ferroferric oxide (Fe3O4) on VFAs degradation and methanogenesis in sulfate-containing environment were investigated. Methanogenesis in reactors with or without sulfate were both favored through the addition of Fe3O4. In reactors without sulfate, the dosage of Fe3O4 increased the maximum methane production rate by 21.7% accompanied with faster acetate and propionate degradation. Metagenomic analysis showed that Fe3O4 mainly promoted electron exchange between Mesotoga, Syntrophobacter, Smithella and Methanosaeta without altering the syntrophic patterns. However, in the sulfate-containing reactor with low methanogenic efficiency, syntrophic ethanol users and Methanosaeta were replaced by sulfate-reducing bacteria and Methanosarcina, respectively. The supplement of Fe3O4 re-enriched the syntrophic partners inhibited by sulfate and rebuilt a new syntrophic interaction with high efficiency similar to that in sulfate-free environment, leading to better methanogenic performance in sulfate-containing environment.

Impacts of environmental factors on microbial diversity, distribution patterns and syntrophic correlation in anaerobic processes.

Anaerobic processes are widely used for treating high-strength organic wastewater. Understanding the ecological patterns of the microorganisms involved and the effect of environmental factors on microbial community are important to manage the performance of anaerobic processes. Microbial communities of 12 anaerobic sludge samples acclimated under different environmental conditions were investigated. Genera detected from these anaerobic sludge samples generally presented three distribution patterns: frequently detected with high abundance, frequently detected with low abundance and occasionally detected with permanently low abundance. The type of feed stock was one of the most important process parameters affecting the shape of microbial community (e.g., Syntrophus, Methylomonas and Methylobacillus). Dye wastewater (Bacteroides) and the supplement of conductive materials (genus T78) were also found to shape the microbial community. Some syntrophic bacteria and methanogens were rare in many anaerobic samples. However, correlation analysis suggested that rare genera are potential syntrophic partners and are responsible for syntrophic methanogenesis.

System performance and microbial community in ethanol-fed anaerobic reactors acclimated with different organic carbon to sulfate ratios.

Sulfate influences the organics removal and methanogenic performance during anaerobic wastewater treatment. System performance, microbial community and metabolic pathways in ethanol-fed anaerobic reactors were investigated under different COD/SO42- ratios (2, 1 and 0.67) and control without sulfate addition. The sulfate removal percentages declined (99%, 60% and 49%) with decreasing COD/SO42- ratios, and methanogenesis was completely inhibited. Acetate accumulated to 903-734 mg/L, though propionate was constantly lower than 30 mg/L. Without sulfate, acetate and propionate did not accumulate, despite the extended time for propionate degradation. Incomplete oxidizing sulfate reducing bacteria (Desulfobulbus and Desulfomicrobium) and hydrolysis-acidification genera (Treponema and Bacteroidales) predominated but could not degrade acetate. Desulfobulbus was the key genus for propionate degradation through the pyruvate & propanoate metabolism pathway. Pseudomonas and Desulfobulbus, possessing genes encoding Type IV pili and cytochrome c6 OmcF, respectively, potentially participated in the direct interspecies electron transfer in sulfate-rich conditions.