In methanogenic communities, two main pathways drive methanogenesis: acetoclastic methanogenesis, which converts acetate into CH4 and CO2, and hydrogenotrophic methanogenesis, which reduces CO2 with H2 to CH4. Under high-pH conditions, a shift in dominance from acetoclastic to hydrogenotrophic methanogenesis is often observed. The goal of this work was to identify the pH tipping point for this metabolic shift and to elucidate the influence of alkalinity on this transition in a haloalkaliphilic methanogenic community enriched from anaerobic soda lake sediments. To this end, a haloalkaliphilic microbial community was cultivated across a pH range (8.20–10.00) at three different alkalinities (0.1, 0.6, 1.2 eq/L). Specific qPCR probes were developed to quantify the two dominant methanogens for each catabolism: “Ca. Methanocrinis natronophilus” (acetoclastic) and Methanocalculus alkaliphilus (hydrogenotrophic). Results showed that the relative abundance of Methanocalculus increased with the rise of pH for all alkalinities, with alkalinity exerting a stronger influence than pH. At low alkalinity (0.1 eq/L), Methanocalculus abundance doubled from 5.14 ± 1.95% to 9.15 ± 0.77% (pH 8.40–10.35). At moderate alkalinity (0.6 eq/L), it increased from 8.33 ± 1.34% to 47.92 ± 3.76% (pH 8.41–10.00), and at the highest alkalinity (1.2 eq/L), it increased from 6.78 ± 1.06% to 60.25 ± 2.00% (pH 8.26–9.68). 16S rRNA gene amplicon sequencing further identified “Candidatus Contubernalis” as a putative syntrophic acetate-oxidizing bacterium likely partnering with Methanocalculus in indirect hydrogenotrophic methanogenesis. This work highlights that haloalkaliphilic hydrogenotrophic methanogens offer a promising strategy to integrate CO2 capture in alkaline solutions with biomethanation.

The role of high-pH conditions in anaerobic digestion (AD) has traditionally been confined to it's use in pre-treatment processes. However, operating AD at elevated pH and alkalinity offers significant advantages, including in-situ upgrading of biogas to biomethane. This study examines the potential and scalability of AD under these conditions (pH ∼ 9.3; alkalinity ∼ 0.5 eq/L). The substrate used was the alkaline waste generated from the extraction of extracellular polymeric substances (EPS) from aerobic granular sludge (AGS), and the inoculum used was a haloalkaliphile microbial community from soda lake sediments. To evaluate the system’s performance, the organic loading rate (OLR) was incrementally increased. The highest methane production obtained was 8.4 ± 0.1 mL/day/gVSadded at a hydraulic retention time (HRT) of 15 days and an OLR of 1 kgVS/day/m3. At this loading rate, methanogenesis became the rate limiting conversion. The maximum volatile solids conversion was 48.1 ± 1.1 %. Throughout the reactor operation, methane purity in the biogas consistently exceeded 90 % peaking at 96.0 ± 0.2 %, showcasing the potential for in-situ biogas purification under these conditions. In addition, no ammonia inhibition was observed, even with free-ammonia (NH3) concentrations reaching up to 14 mM. This study underscores the potential of high-pH anaerobic digestion as a sustainable method for both waste treatment and energy recovery.

Kaumera are extracellular polymeric substances (EPS) extracted from excess aerobic granular sludge from Nereda® wastewater treatment plants. Kaumera exhibits significant market potential across diverse applications, fostering rapid research and business development. Furthermore, it will begin to be extracted from numerous installations worldwide. This calls for standard methods as analogue to (waste)water and sludge characterization. Due to lack of standardization, stakeholders are currently using different extraction and characterization protocols, impeding the development of a more uniform product and comparison of results across research studies. To address this, this report compiles the standard protocol for Kaumera extraction in the laboratory and for on-site and lab characterization to be used by researchers, the public Dutch water authorities, and the private industry. The procedures detailed in this document are in accordance with EPS research conducted at TU Delft and methodologies employed in Kaumera production facilities. This report aids in monitoring Kaumera characteristics worldwide and for optimizing the extraction process (including up and downstream processing). This will help maximize repeatability, interoperability, and quality and therefore accelerate business and research development, paving the way to develop a product that meets the needs of the endusers. Through the widespread adoption of this manual, our aim is to foster greater coordination and collaboration among stakeholders, thereby expediting the realization of Kaumera's full potential.