Humic substances as electron acceptor for anaerobic oxidation of methane (AOM) and electron shuttle in Mn (IV)-dependent AOM.

Anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae are crucial for the global carbon cycle and different biogeochemical processes, owing to their metabolic versatility to couple anaerobic oxidation of methane (AOM) with different electron acceptors. A universal feature of Methanoperedenaceae is the abundant genes encoded in their genomes associated with extracellular electron transfer (EET) pathways. Candidatus. 'Methanoperedens manganicus', an archaeon belonging to the family Methanoperedenaceae, was recently enriched in a bioreactor performing AOM coupled with Mn (IV) reduction. Using this EET-capable ANME, we tested the hypothesis in this study that ANME can catalyse the humic-dependent AOM for growth. A two-year incubation showed that AOM activity can be sustained by Ca. 'M. manganicus' consortium in a bioreactor fed only with humic acids and methane. An isotopic mass balance batch test confirmed that the observed AOM was coupled to the reduction of humic acids. The increase of relative abundance of Ca. 'M. manganicus', and the total archaea population in the microbial community suggested that Ca. 'M. manganicus' can grow on methane and humic acids. The observation of humic-dependent AOM led to a subsequent hypothesis that humic acids could be used as the electron shuttle to mediate the EET in dissimilatory Mn (IV) reduction by Ca. 'M. manganicus'. We tested this hypothesis by adding humic acids to a Ca. 'M. manganicus' dominated-culture, which showed that the AOM rate was doubled by the addition of humic acids. X-ray photoelectron spectroscopy (XPS) showed that quinone moieties were consumed when humic acids worked as electron acceptors while remaining stable when functioning as a shuttle for electron transfer. The results of our study suggest that humic acids may serve as electron shuttles to allow ANME to access more electron acceptors through long-range EET.

The origin of waste activated sludge affects the enhancement of anaerobic digestion by free nitrous acid pre-treatment.

Anaerobic digestion is a common stabilization method for treating primary sludge (PS) and waste activated sludge (WAS). However, its application is often limited by the degradation of WAS. Recent studies have demonstrated FNA to be an effective pre-treatment for enhancing WAS degradability, while having limited effect on PS degradability. WAS characteristics are impacted by wastewater treatment plant (WWTP) configuration and this study is the first to compare the effectiveness of FNA pre-treatment on WAS from WWTP with and without primary treatment. In this study, WAS samples were collected from four full-scale WWTPs with or without primary treatment. Sludge characterization, biomethane potential tests and mathematical modeling were conducted to assess the impacts of FNA pre-treatment on anaerobic digestion. The results showed that FNA pre-treatment was consistently effective for WAS from different WWTPs, while the extent of enhancement varied between WWTPs. For WAS from WWTPs without primary treatment, FNA pretreatment increased the rate of hydrolysis by 54-66% compared to 22-33% increase for WAS without primary treatment. In contrast, WAS from WWTPs with primary treatment experienced greater increases in methane potential (22-24%) compared to WAS from WWTPs without primary treatment (14-16%). These variances could be associated with primary treatment impacting the wastewater COD/N ratio and thus portion of extracellular polymetric substances (EPS) and cells in WAS. FNA pre-treatment targets the destruction of polymetric substances and cells, therefore WAS with a higher proportion of cells (i.e., WAS with primary treatment) experienced greater improvements in methane yield. Similarly, greater improvements in hydrolysis rate were observed for WAS from WWTP without primary sedimentation which contain higher proportions of large EPS molecules. Despite its consistent effectiveness on WAS samples, FNA pre-treatment was ineffective for improving the digestibility of high-rate activated sludge (HRAS).

Stoichiometric and kinetic characterization of an acid-tolerant ammonia oxidizer 'Candidatus Nitrosoglobus'.

Recently, acidic (i.e. pH<5) nitrification in activated-sludge is attracting attention because it enables stable nitritation (NH4+ â†’ NO2-), and enhances sludge reduction and stabilization. However, the key acid-tolerant ammonia oxidizers involved are poorly understood. In this study, we performed stoichiometric and kinetic characterization of a new acid-tolerant ammonia-oxidizing bacterium (AOB) belonging to gamma-proteobacterium, Candidatus Nitrosoglobus. Ca. Nitrosoglobus was cultivated in activated-sludge in a laboratory membrane bioreactor over 200 days, with a relative abundance of 55.1 ± 0.5% (indicated by 16S rRNA gene amplicon sequencing) at the time of the characterization experiments. Among all known nitrifiers, Ca. Nitrosoglobus bears the highest resistance to nitrite, low pH, and free nitrous acid (FNA). These traits define Ca. Nitrosoglobus as an adversity-strategist that tends to prosper in acidic activated-sludge, where the low pH (< 5.0) and high levels of FNA (at parts per million levels) sustained and inhibited all other nitrifiers. In contrast, in the conventional pH-neutral activated-sludge process, Ca. Nitrosoglobus is less competitive with canonical AOB (e.g. Nitrosomonas) due to the relatively slow specific growth rate and low affinities to both oxygen and total ammonia. These results advance our understanding of acid-tolerant ammonia oxidizers, and support further development of the acidic activated-sludge process in which Ca. Nitrosoglobus can play a critical role.

Novel Multiplexed Amplicon-Based Sequencing to Quantify SARS-CoV-2 RNA from Wastewater.

The application of wastewater-based epidemiology (WBE) to support the global response to the COVID-19 pandemic has shown encouraging outcomes. The accurate, sensitive, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in municipal wastewater is critical for WBE. Here, we present a novel approach based on multiplexed amplicon-based sequencing, namely the ATOPlex platform, for detecting SARS-CoV-2. The ATOPlex platform is capable of quantifying SARS-CoV-2 RNA at concentrations that are at least 1 order of magnitude lower than the detection limit of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Robust and accurate phylogenetic placement can be done at viral concentrations 4 times lower than the detection limit of RT-qPCR. We further found that the solid fraction in wastewater harbors a considerable amount of viral RNA, highlighting the need to extract viral RNA from the solid and liquid fractions of wastewater. This study delivers a highly sensitive, phylogenetically informative, and high-throughput analytical workflow that facilitates the application of WBE.