Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes.

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na+). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as Nε-acetyl-ß-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments. IMPORTANCE Using genome-centric metagenomics, we discovered a new Methanothrix harundinacea subspecies that appears to be a halotolerant acetoclastic methanogen with the flexibility for adaptation in the anaerobic digestion process both at low (5 g/L Na+) and high salinity conditions (20 g/L Na+). Annotation of the recovered M. harundinacea genome revealed salinity stress-related functions, including the modification of EPS glycoconjugates and the production of compatible solutes. This is the first study reporting these genomic features within a Methanothrix sp., a milestone further supporting previous studies that identified M. harundinacea as a key-driver in anaerobic granulation under high salinity stress.

Recovery Techniques Enabling Circular Chemistry from Wastewater.

In an era where it becomes less and less accepted to just send waste to landfills and release wastewater into the environment without treatment, numerous initiatives are pursued to facilitate chemical production from waste. This includes microbial conversions of waste in digesters, and with this type of approach, a variety of chemicals can be produced. Typical for digestion systems is that the products are present only in (very) dilute amounts. For such productions to be technically and economically interesting to pursue, it is of key importance that effective product recovery strategies are being developed. In this review, we focus on the recovery of biologically produced carboxylic acids, including volatile fatty acids (VFAs), medium-chain carboxylic acids (MCCAs), long-chain dicarboxylic acids (LCDAs) being directly produced by microorganisms, and indirectly produced unsaturated short-chain acids (USCA), as well as polymers. Key recovery techniques for carboxylic acids in solution include liquid-liquid extraction, adsorption, and membrane separations. The route toward USCA is discussed, including their production by thermal treatment of intracellular polyhydroxyalkanoates (PHA) polymers and the downstream separations. Polymers included in this review are extracellular polymeric substances (EPS). Strategies for fractionation of the different fractions of EPS are discussed, aiming at the valorization of both polysaccharides and proteins. It is concluded that several separation strategies have the potential to further develop the wastewater valorization chains.

Effect of carbohydrates on protein hydrolysis in anaerobic digestion.

This study aimed to assess the effect of carbohydrates on protein hydrolysis and potential implications for the design of anaerobic reactors for treatment of protein-rich wastewaters. Batch experiments were carried out with dissolved starch (Sta) and gelatine (Gel) at different chemical oxygen demand (COD) ratios ranging from 0 to 5.5 under methanogenic conditions for methane production and up to 3.8 under non-methanogenic conditions for volatile fatty acids (VFA), both at 35 °C. The Sta/Gel did not have a direct effect on the gelatine hydrolysis rate constants under methanogenic (0.51 ± 0.05 L g VSS-1 day-1) and non-methanogenic conditions (0.48 ± 0.05 L g VSS-1 day-1). However, under non-methanogenic conditions, gelatine hydrolysis was inhibited by 64% when a spectrum of VFA was added at a VFA/Gel (COD) ratio of 5.9. This was not caused by the ionic strength exerted by VFA but by the VFA itself. These results imply that methanogenesis dictates the reactor design for methane production but hydrolysis does for VFA production from wastewater proteins.

The Effect of Bioinduced Increased pH on the Enrichment of Calcium Phosphate in Granules during Anaerobic Treatment of Black Water.

Simultaneous recovery of calcium phosphate granules (CaP granules) and methane in anaerobic treatment of source separated black water (BW) has been previously demonstrated. The exact mechanism behind the accumulation of calcium phosphate (Ca x(PO4) y) in CaP granules during black water treatment was investigated in this study by examination of the interface between the outer anaerobic biofilm and the core of CaP granules. A key factor in this process is the pH profile in CaP granules, which increases from the edge (7.4) to the center (7.9). The pH increase enhances supersaturation for Ca x(PO4) y phases, creating internal conditions preferable for Ca x(PO4) y precipitation. The pH profile can be explained by measured bioconversion of acetate and H2, HCO3- and H+ into CH4 in the outer biofilm and eventual stripping of CO2 and CH4 (biogas) from the granule. Phosphorus content and Ca x(PO4) y crystal mass quantity in the granules positively correlated with the granule size, in the reactor without Ca2+ addition, indicating that the phosphorus rich core matures with the granule growth. Adding Ca2+ increased the overall phosphorus content in granules >0.4 mm diameter, but not in fine particles (<0.4 mm). Additionally, H+ released from aqueous phosphate species during Ca x(PO4) y crystallization were buffered by internal hydrogenotrophic methanogenesis and stripping of biogas from the granule. These insights into the formation and growth of CaP granules are important for process optimization, enabling simultaneous Ca x(PO4) y and CH4 recovery in a single reactor. Moreover, the biological induction of Ca x(PO4) y crystallization resulting from biological increase of pH is relevant for stimulation and control of (bio)crystallization and (bio)mineralization in real environmental conditions.

Effect of low concentrations of dissolved oxygen on the activity of denitrifying methanotrophic bacteria.

Chemical energy can be recovered from municipal wastewater as biogas through anaerobic treatment. Effluent from direct anaerobic wastewater treatment at low temperatures, however, still contains ammonium and considerable amounts of dissolved methane. After nitritation, methane can be used as electron donor for denitrification by the anaerobic bacterium 'Candidatus Methylomirabilis oxyfera'. It was shown that in the presence of 0.7% O2, denitrifying methanotrophic activity slightly increased and returned to its original level after oxygen had been removed. At 1.1% O2, methane consumption rate increased 118%, nitrite consumption rate increased 58%. After removal of oxygen, methane consumption rate fully recovered, and nitrite consumption rate returned to 88%. Therefore, traces of oxygen that bacteria are likely to be exposed to in wastewater treatment are not expected to negatively affect the denitrifying methanotrophic process. 2.0% O2 inhibited denitrifying activity. Nitrite consumption rate decreased 60% and did not recover after removal of oxygen. No clear effect on methane consumption was observed. Further studies should evaluate if intermittent addition of oxygen results in increased growth rates of the slow-growing 'Candidatus Methylomirabilis oxyfera'.

Effect of temperature on denitrifying methanotrophic activity of 'Candidatus Methylomirabilis oxyfera'.

The activity of denitrifying methanotrophic bacteria at 11-30 °C was assessed in short-term experiments. The aim was to determine the feasibility of applying denitrifying methanotrophic bacteria in low-temperature anaerobic wastewater treatment. This study showed that biomass enriched at 21 °C had an optimum temperature of 20-25 °C and that activity dropped as temperature was increased to 30 °C. Biomass enriched at 30 °C had an optimum temperature of 25-30 °C. These results indicated that biomass from low-temperature inocula adjusted to the enrichment temperature and that low-temperature enrichment is suitable for applications in low-temperature wastewater treatment. Biomass growth at ≤20 °C still needs to be studied.

Fractionation of Extracellular Polymeric Substances by Aqueous Three-Phase Partitioning Systems.

Extracellular polymeric substances (EPS) are natural polymers secreted by microorganisms and represent a key chemical for the development of a range of circular economy applications. The production of EPS comes with notable challenges such as downstream processing. In this work, a three-phase partitioning (TPP) system was investigated as a fractionation technique for the separation of polysaccharides and proteins, both present in the EPS culture broth. The effect of the type of phase-forming compounds (alcohol, polymer, or ionic liquid, in combination with salt) and its concentration were evaluated and compared to the results previously obtained with model systems. The recyclability of phase-forming compounds used to form the fractionation platform was assessed by ultrafiltration. The best fractionation of EPS was achieved using a TPP system composed of 23 wt % ethanol and 25% K3C6H5O7 as 82% EPS-PS partitioned to the salt-rich/bottom phase, and 76% EPS-PN was recovered as an interfacial precipitate, which could be readily resolubilized in water. This represented an increase of 1.24 and 2.83-fold in the purity of EPS-PS and EPS-PN, respectively, in relation to the initial feed concentration. Finally, high recovery yields of phase-forming compounds (>99%) and fractionated EPS (>80%) were obtained using ultrafiltration/diafiltration (UF/DF) as the regeneration technique. The substantial fractionation yields, selectivity, and recyclability of the phase-forming compounds confirm the potential of TPP systems in combination with UF/DF as the separation method for real biopolymer mixtures. Key contributions of this study include the demonstration of the applicability of a readily scalable and cost-effective separation technique for the fractionation of EPS from real EPS-containing broths, while also the limitations of prescreening with model systems became clear through the observed deviating trends between model system studies and real broth studies.

Variation of viscoelastic properties of extracellular polymeric substances and their relation to anaerobic granule's mechanical strength in full-scale treatment plants.

Extracellular polymeric substances (EPS) are considered to play a pivotal role in shaping granules' physical properties. In this contribution, we characterized the viscoelastic properties of EPS from granules of 9 full-scale industrial anaerobic reactors; and quantitatively investigated whether these properties correlate with granules' resistance to compression (Egranule) and shear strength (Sgranule). Most granules with a higher shear strength, also exhibited a stronger resistance to compression (r = 0.96, p = 0.002), except those granules that contained relatively more proteins in their EPS. Interestingly, these granules were also the most resistant to shear stress (Sgranule ≥ 110 ± 40 h). Furthermore, the EPS hydrogels of these granules had slower softening rates (κ < 0.9) compared to the others (κ ranged between 0.95 and 1.20), indicating stronger gels were formed. These findings suggest that the EPS hydrogel softening rate could be a key parameter to explain granule's shear strength.

De novo anaerobic granulation with varying organic substrates: granule growth and microbial community responses.

Anaerobic granulation from dispersed inoculum is recognized as a slow process. However, studies under saline conditions have shown that adding complex proteinaceous substrates can accelerate this process. To explore whether this holds true also under non-saline conditions, we conducted a 262-days experiment with four lab-scale upflow anaerobic sludge blanket reactors inoculated with digested sewage sludge. Each reactor received a synthetic feed containing varying amount of carbohydrate/protein substrate: glucose (RGlu), acetate/tryptone (RAc+Try), glucose/tryptone (RGlu+Try), and glucose/starch (RGlu+Sta). Development of granules with different influent composition was monitored with macroscopy, analysis of the extracellular polymeric substances, and microbial diversity. Granulation was faster in reactors RGlu+Try and RGlu+Sta. Increasing granule diameters positively correlated with the occurrence of bacteria from Muribaculaceae and Lachnospiraceae families, suggesting their involvement in de novo granulation. Granules of RGlu+Try also had high relative abundances of both fermenting bacteria (e.g. Lactococcus, Streptococcus, Trichococcus) and bacteria involved in the oxidation of volatile fatty acids (Smithella, Acetobacteroides). The results of this study provide a basis for strategies to enhance the sludge granulation rate in practice when granular inoculum is not available. Specifically, supplementing small amounts of waste protein during reactor start-up can be effective.

Effect of low dosages of powdered activated carbon on membrane bioreactor performance.

Previous research has demonstrated that powdered activated carbon (PAC), when applied at very low dosages and long SRTs, reduces membrane fouling in membrane bioreactors (MBRs). This effect was related to the formation of stronger sludge flocs, which are less sensitive to shear. In this contribution the long-term effect of PAC addition was studied by running two parallel MBRs on sewage. To one of these, PAC was dosed and a lower fouling tendency of the sludge was verified, with a 70% longer sustainable filtration time. Low PAC dosages showed additional advantages with regard to oxygen transfer and dewaterability, which may provide savings on operational costs.

The effect of sludge recirculation rate on a UASB-digester treating domestic sewage at 15 °C.

The anaerobic treatment of low strength domestic sewage at low temperature is an attractive and important topic at present. The upflow anaerobic sludge bed (UASB)-digester system is one of the anaerobic systems to challenge low temperature and concentrations. The effect of sludge recirculation rate on a UASB-digester system treating domestic sewage at 15 °C was studied in this research. A sludge recirculation rate of 0.9, 2.6 and 12.5% of the influent flow rate was investigated. The results showed that the total chemical oxygen demand (COD) removal efficiency rose with increasing sludge recirculation rate. A sludge recirculation rate of 0.9% of the influent flow rate led to organic solids accumulation in the UASB reactor. After the sludge recirculation rate increased from 0.9 to 2.6%, the stability of the UASB sludge was substantially improved from 0.37 to 0.15 g CH4-COD/g COD, and the bio-gas production in the digester went up from 2.9 to 7.4 L/d. The stability of the UASB sludge and bio-gas production in the digester were not significantly further improved by increasing sludge recirculation rate to 12.5% of the influent flow rate, but the biogas production in the UASB increased from 0.37 to 1.2 L/d. It is recommended to apply a maximum sludge recirculation rate of 2-2.5% of the influent flow rate in a UASB-digester system, as this still allows energy self-sufficiency of the system.

The effect of intermittent anode potential regimes on the morphology and extracellular matrix composition of electro-active bacteria.

Electro-active bacteria (EAB) can form biofilms on an anode (so-called bioanodes), and use the electrode as electron acceptor for oxidation of organics in wastewater. So far, bioanodes have mainly been investigated under a continuous anode potential, but intermittent anode potential has resulted in higher currents and different biofilm morphologies. However, little is known about how intermittent potential influences the electron balance in the anode compartment. In this study, we investigated electron balances of bioanodes at intermittent anode potential regimes. We used a transparent non-capacitive electrode that also allowed for in-situ quantification of the EAB using optical coherence tomography (OCT). We observed comparable current densities between continuous and intermittent bioanodes, and stored charge was similar for all the applied intermittent times (5 mC). Electron balances were further investigated by quantifying Extracellular Polymeric Substances (EPS), by analyzing the elemental composition of biomass, and by quantifying biofilm and planktonic cells. For all tested conditions, a charge balance of the anode compartment showed that more electrons were diverted to planktonic cells than biofilm. Besides, 27-43% of the total charge was detected as soluble EPS in intermittent bioanodes, whereas only 15% was found as soluble EPS in continuous bioanodes. The amount of proteins in the EPS of biofilms was higher for intermittent operated bioanodes (0.21 mg COD proteins mg COD biofilm-1) than for continuous operated bioanodes (0.05 mg COD proteins mg COD biofilm-1). OCT revealed patchy morphologies for biofilms under intermittent anode potential. Overall, this study helped understanding that the use of a non-capacitive electrode and intermittent anode potential deviated electrons to other processes other than electric current at the electrode by identifying electron sinks in the anolyte and quantifying the accumulation of electrons in the form of EPS.

Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge.

Recently discovered microorganisms affiliated to the bacterial phylum NC10, named "Candidatus Methylomirabilis oxyfera", perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were screened for the endogenous presence of M. oxyfera using molecular diagnostic methods. We could identify NC10 bacteria with 98% similarity to M. oxyfera in nine out of ten WWTPs tested. Sludge from one selected WWTP was used to start a new enrichment culture of NC10 bacteria. This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes. After 112 days, the enrichment consumed up to 0.4 mM NO(2)(-) per day. The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature.

Bioflocculants from wastewater: Insights into adsorption affinity, flocculation mechanisms and mixed particle flocculation based on biopolymer size-fractionation.

HYPOTHESIS: Microbial extracellular polymeric substances (EPS) produced from wastewater are generally heterodispersed, which is expected to influence their flocculation performances and mechanism, particularly in mixed particle systems. The different molecular weight (MW) fractions should contribute to the overall adsorption affinity and flocculation mechanism of EPS in single and dual clay systems. EXPERIMENTS: EPS harvested from bioreactors were size-fractionated into high, medium and low MW fractions (HMW, MMW, LMW, respectively). The harvested mixed EPS and its fractions were characterised by diverse analytical techniques coupled with optical reflectometry to investigate the role of each EPS fraction in the overall flocculation mechanism of EPS in kaolinite and montmorillonite clay systems. FINDINGS: In single clay systems, both the harvested mixed EPS and the HMW-EPS fraction showed comparable flocculation performances. However, mixed EPS proved to be more efficient than the HMW-EPS fraction for dual clay flocculation. Site blocking effects were observed in mixed EPS: the LMW and MMW EPS first adsorbed to the surface due to higher diffusivities and faster mass transfer to the interface, while the HMW-EPS were slowly transported but were attached to the surface irreversibly and stronger than the LMW/MMW-EPS. We propose from this, a mixed EPS adsorption mechanism: extended anionic polymer tails in solution, thereby enhancing particle flocculation.

Occurrence of xenobiotics in gray water and removal in three biological treatment systems.

Eighteen selected xenobiotics related to personal care and household chemicals (UV-filters, fragrances, preservatives, biocides, surfactants) were measured in gray water from 32 houses and in effluents of three different biological treatment systems (aerobic, anaerobic, and combined anaerobic+aerobic). All selected xenobiotics were detected in gray water samples in the low microg L(-1) range. Generally, lower concentrations were measured after biological treatment and removal efficiencies were higher under aerobic conditions than under anaerobic conditions. However, most of the xenobiotics were still detected in biologically treated gray water. The most persistent compounds were the fragrance tonalide and the UV-filters 2-phenyl-5-benzimidazolesulfonic acid and ethylhexyl methoxycinnamate. Estimated estrogenic potential of the effluent ranged between 0.07 and 0.72 ng L(-1) of 17beta-estradiol equivalents. Depending on the application of the effluent and its environmental risk, physical-chemical processes might be required to increase the removal efficiency of these compounds from gray water.

Feasibility of bioflocculation in a high-loaded membrane bioreactor for improved energy recovery from sewage.

The feasibility of a high-loaded membrane bioreactor to improve methane recovery from sewage was investigated. Although the process needs further optimization, it already is feasible to recover at least 35% of the sewage COD. Important aspects for further research are the occurrence of membrane fouling, and the optimum process conditions for bioflocculation, i.e. the proper SRT/HRT ratio, dissolved oxygen concentration and shear and overall energy production and consumption under optimised conditions.

Calcium effect on microbial activity and biomass aggregation during anaerobic digestion at high salinity.

The potential effect of different Ca2+ additions (150, 300, 450, 600 and 1000 mg/L) on microbial activity and aggregation, during anaerobic digestion at moderate (8 g/L Na+) and high salinity (20 g/L Na+) has been investigated. Batch tests were carried out in duplicate serum bottles and operated for 30 days at 37 °C. At 8 g/L Na+, methanogenic activity and protein degradation were comparable from 150 to 450 mg/L Ca2+, and a significant inhibition was only observed at a Ca2+concentration of 1000 mg/L. In contrast, at 20 g/L Na+, 150 to 300 mg/L were the only Ca2+ concentrations to maintain chemical oxygen demand (COD) removal, protein hydrolysis and methane production. Overall, increasing Ca2+ concentrations had a larger impact on acetotrophic methanogenesis at 20 g/L than at 8 g/L Na+. Increasing Ca2+ had a negative effect on the aggregation behaviour of the dominant methanogen Methanosaeta when working at 8 g/L Na+. At 20 g/L Na+ the aggregation of Methanosaeta was less affected by addition of Ca2+ than at 8 g/L Na+. The negative effect appeared to be connected with Ca2+ precipitation and its impact on cell-to cell communication. The results highlight the importance of ionic balance for microbial aggregation at high salinity, bringing to the forefront the effect on Methanosaeta cells, known to be important to obtain anaerobic granules.

Regeneration and reuse of microbial extracellular polymers immobilised on a bed column for heavy metal recovery.

Microbial extracellular polymeric substances (EPS) have gained increasing attention for various water treatment applications. In this study, EPS produced from nitrogen-limited glycerol/ethanol-rich wastewater were used to recover Cu2+ and Pb2+ from aqueous solutions. Continuous flow-through tests were conducted on a column packed with silica gel coated with polyethyleneimine, to which EPS were irreversibly attached as shown by optical reflectometry. These immobilised EPS excellently adsorbed Cu2+ and Pb2+, with 99.9% of influent metal adsorbed before the breakthrough points. Metal desorption was achieved with 0.1M HCl, with an average recovery of 86% for Cu2+ and 90% recovery for Pb2+. For the first time, we successfully showed the possibility to regenerate and reuse the immobilised EPS for five adsorption-desorption cycles (using Cu2+ as an example) with no reduction in the adsorbed amount at the breakthrough point (qbp). Based on the mass balance of the associated metal ions participating in the adsorption process, ion exchange was identified as the major mechanism responsible for Cu2+ and Pb2+ adsorption by EPS. The results demonstrate the potential of wastewater-produced EPS as an attractive and perhaps, cost-effective biosorbent for heavy metal removal (to trace effluent concentrations) and recovery (86-99%).

Microbial Community Drivers in Anaerobic Granulation at High Salinity.

In the recent years anaerobic sludge granulation at elevated salinities in upflow anaerobic sludge blanket (UASB) reactors has been investigated in few engineering based studies, never addressing the microbial community structural role in driving aggregation and keeping granules stability. In this study, the combination of different techniques was applied in order to follow the microbial community members and their structural dynamics in granules formed at low (5 g/L Na+) and high (20 g/L Na+) salinity conditions. Experiments were carried out in four UASB reactors fed with synthetic wastewater, using two experimental set-ups. By applying 16S rRNA gene analysis, the comparison of granules grown at low and high salinity showed that acetotrophic Methanosaeta harundinacea was the dominant methanogen at both salinities, while the dominant bacteria changed. At 5 g/L Na+, cocci chains of Streptoccoccus were developing, while at 20 g/L Na+ members of the family Defluviitaleaceae formed long filaments. By means of Fluorescence in Situ Hybridization (FISH) and Scanning Electron Microscopy (SEM), it was shown that aggregation of Methanosaeta in compact clusters and the formation of filaments of Streptoccoccus and Defluviitaleaceae during the digestion time were the main drivers for the granulation at low and high salinity. Interestingly, when the complex protein substrate (tryptone) in the synthetic wastewater was substituted with single amino acids (proline, leucine and glutamic acid), granules at high salinity (20 g/L Na+) were not formed. This corresponded to a decrease of Methanosaeta relative abundance and a lack of compact clustering, together with disappearance of Defluviitaleaceae and consequent absence of bacterial filaments within the dispersed biomass. In these conditions, a biofilm was growing on the glass wall of the reactor instead, highlighting that a complex protein substrate such as tryptone can contribute to granules formation at elevated salinity.