Anaerobic digestion (AD) has been commercially operated worldwide in full scale as a resource recovery technology underpinning a circular economy. However, problems such as a long start-up time, or system instability, have been reported in response to operational shocks. These issues are usually linked to the dynamics of the functional microbiota in AD. Exploring the microbiota-functionality nexus (MFN) could be pivotal to understand the reasons behind these difficulties, and hence improving AD performance. Here we present a systematic MFN study based on 138 samples taken from 20 well-profiled lab-scale AD reactors operated for up to two years. All the reactors were operated in the same lab within the same period of time using the same methodology to harvest physio-chemical and molecular data, including key monitoring parameters, qPCR, and 16S sequencing results. The results showed a core bacterial microbiota prevailing in all reactor types, including Bacillus, Clostridium, Bacteroides, Eubacterium, Cytophaga, Anaerophaga, and Syntrophomonas, while various methanogens dominated different communities due to different inocula origins, reactor temperatures, or salinity levels. This core bacterial microbiota well correlated with biogas production (Pearson correlation coefficient of 0.481, p < 0.0001). Such strong correlation was even comparable to that between the biogas production and the methanogenic 16S rRNA gene content (Pearson correlation coefficient of 0.481, p < 0.0001). The results indicated that AD performance only modestly correlated with microbial diversity, a key governing factor. AD microbiota was neither functionally redundant nor plastic, and a high variety in communities can exhibit a strong difference in reactor performance. Our study demonstrates the importance of a core bacterial microbiota in AD and supports inspiring considerations for design, bioaugmentation, and operational strategies of AD reactors in the future.

Anaerobic ammonium-oxidizing (anammox)bacteria convert ammonium and nitrite into N ₂ in a chemolithoautotrophic way, meaning that they utilize CO ₂ /HCO ₃ solely as their carbon sources. Such autotrophic behavior limits their competitiveness with heterotrophic microorganisms in both natural environments and engineered systems. Recently, environmental metagenomic results have indicated the capability of anammox bacteria to metabolize short-chain fatty acids, further confirmed by limited experimental evidence based on highly enriched cultures. However, clear evidence is difficult to get because of the limits of traditional methodologies which rely on the availability of a pure anammox culture. In this study, we identified and quantified the uptake of acetate and propionate, on a single-cell level, by an anammox consortium that was dominated by Candidatus Jettenia asiatica (relative abundance of 96%). The consortium, growing in granular form with an average relative abundance of anammox bacteria of 96.0%, was firstly incubated in a ¹³ C-labelled acetate or propionate medium; then microtome sections were scanned by a nanometer-scale secondary ion mass spectrometer (NanoSIMS). The NanoSIMS scannings revealed that the consortium enriched acetate and propionate at a >10 times higher efficiency than bicarbonate incorporation. Our results also suggest that acetate or propionate was likely not assimilated by J. asiatica directly, but firstly oxidized to CO ₂ , which then served as carbon sources for the follow-up autotrophy in J. asiatica cells. Furthermore, more [ ¹⁵ N]ammonium was enriched by the propionate-fed consortium than the acetate-fed consortium despite that exactly the same amount of ¹³ C atoms were supplied. Our study strongly indicates an alternative lifestyle, namely organotrophy, in addition to chemolithoautotrophy of anammox bacteria, making it more versatile than often expected. It suggests that the niche of anammox bacteria in both natural and engineered ecosystems can be much broader than usual assumed. Recognising this is important for their role in wastewater treatment and the global nitrogen turn-over rates.

This study provides a comparison of the sludge characteristics along the height of an upflow anaerobic sludge blanket (UASB) reactor in terms of sludge morphology, activity and stability. The main aim of this study was to identify the best location (i.e. where sludge is of lowest stability and/or highest concentration) in the sludge bed for conveying the sludge from the low temperature UASB reactor to a digester. The sludge profile was investigated by collecting sludge samples along the different heights of the UASB-anaerobic membrane bioreactor treating municipal wastewater. Results showed that total solids and volatile solids concentrations decreased with height, and the highest chemical oxygen demand concentration was observed at the bottom of the reactor. Active biomass remained near inlet of the reactor; whereas, non-active biomass consisted of loose, suspended particles and flocculents moved towards the top. This was confirmed by the high specific COD consumption rate near the inlet and poor specific COD biodegradation in the remaining portions of the bioreactor. Apparently, the assumption of a completely mixed sludge bed behavior for the UASB reactor, being part of an AnMBR system, does not hold for this type of reactor systems even at low temperatures, which makes the location in sludge bed from where the sludge is to be conveyed to the digester of operational importance. Considering the observed sludge bed stratification, the sludge to be recirculated from the UASB reactor to the digester is recommended to be taken from 40 to 50% of the sludge bed height.

Submerged and external anaerobic dynamic membrane bioreactors (AnDMBRs) have been compared in terms of removal efficiency, filtration characteristics and microbial community structure. High COD removal efficiencies were obtained with both submerged and external AnDMBRs. To obtain an effective dynamic membrane (DM) layer enabling high quality permeate, longer time was required in the external AnDMBR configuration compared to the submerged one. A difference in microbial community structure was identified using pyrosequencing analyses between the submerged and external AnDMBRs. The number of archaeal types decreased in the bulk sludge of the external AnDMBR. External sludge recirculation might have had a negative effect on the archaeal community in the bulk sludge of the external AnDMBR. However, the sludge recirculation in the external AnDMBR configuration led to a filtration at lower total filtration resistance and TMP in comparison to the submerged one at the same gas sparging rate. Results showed that the submerged AnDMBR system can provide a shorter start-up period, slightly better permeate quality in terms of COD concentration, and higher biogas production in comparison to the external one in gas-lift mode.

A laboratory scale external anaerobic dynamic membrane bioreactor (AnDMBR) treating high strength wastewater (influent COD ≈ 20 g/L) was operated to assess the effect of biogas sparging velocity (GSV) and hydraulic retention time (HRT) on removal efficiency and dynamic membrane (DM) filtration characteristics. An increase in GSV resulted in a decrease in DM filtration resistance. DM or cake layer was identified as the main contributor to the total filtration resistance. The external AnDMBR achieved over 99% COD removal efficiency irrespective of the GSV. The results showed that the DM formation process proceeded until a stable cake layer was reached. Reducing of HRT resulted in an increase in protein/carbohydrate ratio in soluble microbial products (SMP) and an increase in biomass concentration in the bioreactor. Therefore, HRT affected TMP and total filtration resistance in the AnDMBR. A high permeate quality was obtained by an effective DM layer at organic loading rates (OLRs) between 2 and 3.6 kg COD/m³ d. Based on the fluxes observed in this research, the filter cloth costs would be in the range of 0.17 €/m³ of treated wastewater. The investment and operational costs of the AnDMBRs are expected to be substantially lower than that of conventional membrane filtration.