Looking for lipases and lipolytic organisms in low-temperature anaerobic reactors treating domestic wastewater

Poor lipid degradation limits low-temperature anaerobic treatment of domestic wastewater even when psychrophiles are used. We combined metagenomics and metaproteomics to find lipolytic bacteria and their potential, and actual, cold-adapted extracellular lipases in anaerobic membrane bioreactors treating domestic wastewater at 4℃ and 15℃. Of the 40 recovered putative lipolytic metagenome-assembled genomes (MAGs), only three (Chlorobium, Desulfobacter, and Mycolicibacterium) were common and abundant (relative abundance ≥ 1%) in all reactors. Notably, some MAGs that represented aerobic autotrophs contained lipases. Therefore, we hypothesised that the lipases we found are not always associated with exogenous lipid degradation and can have other roles such as polyhydroxyalkanoates (PHA) accumulation/degradation and interference with the outer membranes of other bacteria. Metaproteomics did not provide sufficient proteome coverage for relatively lower abundant proteins such as lipases though the expression of fadL genes, long-chain fatty acid transporters, was confirmed for four genera (Dechloromonas, Azoarcus, Aeromonas and Sulfurimonas), none of which were recovered as putative lipolytic MAGs. Metaproteomics also confirmed the presence of 15 relatively abundant (≥1%) genera in all reactors, of which at least 6 can potentially accumulate lipid/polyhydroxyalkanoates. For most putative lipolytic MAGs, there was no statistically significant correlation between the read abundance and reactor conditions such as temperature, phase (biofilm and bulk liquid), and feed type (treated by ultraviolet light or not). Results obtained by metagenomics and metaproteomics did not confirm each other and further work is required to identify the true lipid degraders in these systems. Keywords: Anaerobic treatment, domestic wastewater, psychrophilic extracellular lipases, metagenomics, metaproteomics

Examination of Extracellular Polymer (EPS) Extraction Methods for Anaerobic Membrane Bioreactor (AnMBR) Biomass

Membrane bioreactor fouling is a complex process, which is typically driven by extracellular polymeric substances (EPS), a complex mixture of polysaccharides, proteins, lipids, humic substances, and other intercellular polymers. While much is known about fouling in aerobic membrane reactors, far less is known about fouling in anaerobic membrane bioreactors (AnMBR). Much of this knowledge, including EPS extraction methods, has been extrapolated from aerobic processes and is commonly assumed to be comparable. Therefore, several extraction methods commonly used for aerobic EPS quantification, including ultrasonication, ethylenediaminetetraacetic acid (EDTA), and formaldehyde plus sodium hydroxide (CH2O+NaOH), were evaluated to determine the most suitable extraction method for EPS of anaerobic microorganisms in an AnMBR. To maximize EPS yields, each extraction was performed four times. Experimental results showed that the EDTA method was best for EPS quantification, based on chemical oxygen demand (COD), dissolved organic carbon (DOC), and protein yields: 1.43 mg COD/mg volatile suspended solids (VSS), 0.14 mg DOC/mg VSS, and 0.11 mg proteins/mg VSS. In comparison, the CH2O+NaOH method maximized the extraction of carbohydrates (0.12 mg carbohydrates/mg VSS). However, multiple extraction cycles with EDTA and ultrasonication exhibited lower extracellular adenosine triphosphate (ATP) concentrations compared to CH2O+NaOH extractions, indicating lower levels of released intracellular substances. Successive EPS extractions over four cycles are better able to quantify EPS from anaerobic microorganisms, since a single extraction may not accurately reflect the true levels of EPS contents in AnMBRs, and possibly in other anaerobic processes.

Operation of Submerged Anaerobic Membrane Bioreactors at 20 °C: Effect of Solids Retention Time on Flux, Mixed Liquor Characteristics and Performance

Four flat-sheet submerged anaerobic membrane bioreactors ran for 242 days on a simulated domestic wastewater with low Chemical Oxygen Demand (COD) and high suspended solids. Organic loading was maintained around 1.0 g COD L−1 day−1, while solids retention time (SRT) was varied from 20–90 days. This was achieved at a constant membrane flux, maintained by adjusting transmembrane pressure (TMP) in the range 1.8-9.8 kPa. Membrane fouling was assessed based on the required TMP, with mixed liquors characterised using capillary suction time, frozen image centrifugation and quantification of extracellular polymeric substances (EPS). SRT had a significant effect on these parameters: fouling was least at an SRT of 30 days and highest at 60 days, with some reduction as this extended to 90 days. Operation at SRT <30 days showed no further benefits. Although operation at a short SRT was optimal for membrane performance it led to lower specific methane productivity, higher biomass yields and higher effluent COD. Short SRT may also have accelerated the loss of essential trace elements, leading to reduced performance under these conditions. A COD-based mass balance was conducted, including both biomass and methane dissolved in the effluent.