Bioremediation of rapid sand filters for removal of organic micropollutants during drinking water production.

Rapid sand filtration (RSF) is used during drinking water production for removal of particles, possible harmful microorganisms, organic material and inorganic compounds such as iron, manganese, ammonium and methane. However, RSF can also be used for removal of certain organic micropollutants (OMPs). In this study, it was investigated if OMP removal in columns packed with sand from full scale RSFs could be stimulated by bioaugmentation (i.e. inoculating RSFs with sand from another RSF) and/or biostimulation (i.e. addition of nutrients, vitamins and trace-elements that stimulate microbial growth). The results showed that removal of PFOA, carbamazepine, 1-H benzotriazole, amidotrizoate and iopamidol in the columns was low (< 20 %). Propranolol and diclofenac removal was higher (50-60 %) and propranolol removal likely occurred via sorption processes, whereas for diclofenac it was unclear if removal was a combination of physical-chemical and biological processes. Moreover, bioaugmentation and biostimulation resulted in 99 % removal of gabapentin and metoprolol after 38 days and 99 % removal of acesulfame after 52 days of incubation. The bioaugmented column without biostimulation showed 99 % removal for gabapentin and metoprolol after 52 days, and for acesulfame after 80 days. In contrast, the non-bioaugmented column did not remove gabapentin, removed < 40 % metoprolol and showed 99 % removal of acesulfame only after 80 days of incubation. Removal of these OMPs was negatively correlated with ammonium oxidation and the absolute abundance of ammonia-oxidizing bacteria. 16S rRNA gene sequencing showed that OMP removal of acesulfame, gabapentin and metoprolol was positively correlated to the relative abundance of specific bacterial genera that harbor species with a heterotrophic and aerobic or denitrifying metabolism. These results show that bioaugmentation of RSF can be successful for OMP removal, where biostimulation can accelerate this removal.

Identifying Eukaryotes and Factors Influencing Their Biogeography in Drinking Water Metagenomes.

The biogeography of eukaryotes in drinking water systems is poorly understood relative to that of prokaryotes or viruses, limiting the understanding of their role and management. A challenge with studying complex eukaryotic communities is that metagenomic analysis workflows are currently not as mature as those that focus on prokaryotes or viruses. In this study, we benchmarked different strategies to recover eukaryotic sequences and genomes from metagenomic data and applied the best-performing workflow to explore the factors affecting the relative abundance and diversity of eukaryotic communities in drinking water distribution systems (DWDSs). We developed an ensemble approach exploiting k-mer- and reference-based strategies to improve eukaryotic sequence identification and identified MetaBAT2 as the best-performing binning approach for their clustering. Applying this workflow to the DWDS metagenomes showed that eukaryotic sequences typically constituted small proportions (i.e., <1%) of the overall metagenomic data with higher relative abundances in surface water-fed or chlorinated systems with high residuals. The α and ß diversities of eukaryotes were correlated with those of prokaryotic and viral communities, highlighting the common role of environmental/management factors. Finally, a co-occurrence analysis highlighted clusters of eukaryotes whose members' presence and abundance in DWDSs were affected by disinfection strategies, climate conditions, and source water types.

Monitoring the exposure and emissions of antibiotic resistance: Co-occurrence of antibiotics and resistance genes in wastewater treatment plants.

The COVID-19 pandemic has brought new momentum to 'wastewater-based epidemiology' (WBE). This approach can be applied to monitor the levels of antibiotic-resistant genes (ARGs), which in terms are used to make inferences about the burden of antimicrobial resistance (AMR) in human settlements. However, there is still little information about temporal variability in ARG levels measured in wastewater streams and how these influence the inferences made about the occurrence of AMR in communities. The goal of this study was hence to gain insights into the variability in ARG levels measured in the influent and effluent of two wastewater treatment plants in The Netherlands and link these to levels of antibiotic residues measured in the same samples. Eleven antibiotics were detected, together with all selected ARGs, except for VanB. Among the measured antibiotics, significant positive correlations (p > 0.70) with the corresponding resistance genes and some non-corresponding ARGs were found. Mass loads varied up to a factor of 35 between days and in concomitance with rainfall. Adequate sampling schemes need to be designed to ensure that conclusions are drawn from valid and representative data. Additionally, we advocate for the use of mass loads to interpret levels of AMR measured in wastewater.

Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate, formate and/or hydrogen as electron donor.

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO2 fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO2 , and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.

Organic micropollutant removal in full-scale rapid sand filters used for drinking water treatment in The Netherlands and Belgium.

Biological treatment processes have the potential to remove organic micropollutants (OMPs) during water treatment. The OMP removal capacity of conventional drinking water treatment processes such as rapid sand filters (RSFs), however, has not been studied in detail. We investigated OMP removal and transformation product (TP) formation in seven full-scale RSFs all treating surface water, using high-resolution mass spectrometry based quantitative suspect and non-target screening (NTS). Additionally, we studied the microbial communities with 16S rRNA gene amplicon sequencing (NGS) in both influent and effluent waters as well as the filter medium, and integrated these data to comprehensively assess the processes that affect OMP removal. In the RSF influent, 9 to 30 of the 127 target OMPs were detected. The removal efficiencies ranged from 0 to 93%. A data-driven workflow was established to monitor TPs, based on the combination of NTS feature intensity profiles between influent and effluent samples and the prediction of biotic TPs. The workflow identified 10 TPs, including molecular structure. Microbial community composition analysis showed similar community composition in the influent and effluent of most RSFs, but different from the filter medium, implying that specific microorganisms proliferate in the RSFs. Some of these are able to perform typical processes in water treatment such as nitrification and iron oxidation. However, there was no clear relationship between OMP removal efficiency and microbial community composition. The innovative combination of quantitative analyses, NTS and NGS allowed to characterize real scale biological water treatments, emphasizing the potential of bio-stimulation applications in drinking water treatment.

Effect of methanethiol on process performance, selectivity and diversity of sulfur-oxidizing bacteria in a dual bioreactor gas biodesulfurization system.

This study provides important new insights on how to achieve high sulfur selectivities and stable gas biodesulfurization process operation in the presence of both methanethiol and H2S in the feed gas. On the basis of previous research, we hypothesized that a dual bioreactor lineup (with an added anaerobic bioreactor) would favor sulfur-oxidizing bacteria (SOB) that yield a higher sulfur selectivity. Therefore, the focus of the present study was to enrich thiol-resistant SOB that can withstand methanethiol, the most prevalent and toxic thiol in sulfur-containing industrial off gases. In addition, the effect of process conditions on the SOB population dynamics was investigated. The results confirmed that thiol-resistant SOB became dominant with a concomitant increase of the sulfur selectivity from 75 mol% to 90 mol% at a loading rate of 2 mM S methanethiol day-1. The abundant SOB in the inoculum - Thioalkalivibrio sulfidiphilus - was first outcompeted by Alkalilimnicola ehrlichii after which Thioalkalibacter halophilus eventually became the most abundant species. Furthermore, we found that the actual electron donor in our lab-scale biodesulfurization system was polysulfide, and not the primarily supplied sulfide.

Development of quantitative PCR for the detection of Alkalilimnicola ehrlichii, Thioalkalivibrio sulfidiphilus and Thioalkalibacter halophilus in gas biodesulfurization processes.

Chemolithoautotrophic sulfur-oxidizing bacteria (SOB) are crucial key players in biotechnological processes to remove hydrogen sulfide from sour gas streams. Several different haloalkaliphilic SOB have been detected and isolated from lab- and full-scale facilities, which all performed differently considering end product yields (sulfur and sulfate) and conversion rates. Understanding and regulating bacterial community dynamics in biodesulfurization processes will enable optimization of the process operation. We developed quantitative PCR (qPCR) assays to quantify haloalkaliphilic sulfur-oxidizing gammaproteobacterial species Alkalilimnicola ehrlichii, Thioalkalivibrio sulfidiphilus, and Thioalkalibacter halophilus that dominate bacterial communities of biodesulfurization lab- and full-scale installations at haloalkaline conditions. The specificity and PCR efficiency of novel primer sets were evaluated using pure cultures of these target species. We further validated the qPCR assays by quantification of target organisms in five globally distributed full-scale biodesulfurization installations. The qPCR assays perform a sensitive and accurate quantification of Alkalilimnicola ehrlichii, Thioalkalivibrio sulfidiphilus and Thioalkalibacter halophilus, thus providing rapid and valuable insights into process performance and SOB growth dynamics in gas biodesulfurization systems.

Isolation and characterization of Sphingomonadaceae from fouled membranes.

Membrane filtration systems are widely applied for the production of clean drinking water. However, the accumulation of particles on synthetic membranes leads to fouling. Biological fouling (i.e., biofouling) of reverse osmosis and nanofiltration membranes is difficult to control by existing cleaning procedures. Improved strategies are therefore needed. The bacterial diversity on fouled membranes has been studied, especially to identify bacteria with specialized functions and to develop targeted approaches against these microbes. Previous studies have shown that Sphingomonadaceae are initial membrane colonizers that remain dominant while the biofilm develops. Here, we characterized 21 Sphingomonadaceae isolates, obtained from six different fouled membranes, to determine which physiological traits could contribute to colonization of membrane surfaces. Their growth conditions ranged from temperatures between 8 and 42 oC, salinity between 0.0 and 5.0% w/v NaCl, pH from 4 and 10, and all isolates were able to metabolize a wide range of substrates. The results presented here show that Sphingomonadaceae membrane isolates share many features that are uncommon for other members of the Sphingomonadaceae family: all membrane isolates are motile and their tolerance for different temperatures, salt concentrations, and pH is high. Although relative abundance is an indicator of fitness for a whole group, for the Sphingomonadaceae it does not reveal the specific physiological traits that are required for membrane colonization. This study, therefore, adds to more fundamental insights in membrane biofouling.

Controlling Ethanol Use in Chain Elongation by CO2 Loading Rate.

Chain elongation is an open-culture biotechnological process which converts volatile fatty acids (VFAs) into medium chain fatty acids (MCFAs) using ethanol and other reduced substrates. The objective of this study was to investigate the quantitative effect of CO2 loading rate on ethanol usages in a chain elongation process. We supplied different rates of CO2 to a continuously stirred anaerobic reactor, fed with ethanol and propionate. Ethanol was used to upgrade ethanol itself into caproate and to upgrade the supplied VFA (propionate) into heptanoate. A high CO2 loading rate (2.5 LCO2·L-1·d-1) stimulated excessive ethanol oxidation (EEO; up to 29%) which resulted in a high caproate production (10.8 g·L-1·d-1). A low CO2 loading rate (0.5 LCO2·L-1·d-1) reduced EEO (16%) and caproate production (2.9 g·L-1·d-1). Heptanoate production by VFA upgrading remained constant (∼1.8 g·L-1·d-1) at CO2 loading rates higher than or equal to 1 LCO2·L-1·d-1. CO2 was likely essential for growth of chain elongating microorganisms while it also stimulated syntrophic ethanol oxidation. A high CO2 loading rate must be selected to upgrade ethanol (e.g., from lignocellulosic bioethanol) into MCFAs whereas lower CO2 loading rates must be selected to upgrade VFAs (e.g., from acidified organic residues) into MCFAs while minimizing use of costly ethanol.

Metabolism and Occurrence of Methanogenic and Sulfate-Reducing Syntrophic Acetate Oxidizing Communities in Haloalkaline Environments.

Anaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners, typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions were investigated, using highly enriched methanogenic (M-SAO) and sulfate-reducing (S-SAO) cultures from south-western Siberian hypersaline soda lakes. Activity tests with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that H2 and formate are important IECs in both SAO cultures. Metagenomic analysis of the M-SAO cultures showed that the dominant SAOB was 'Candidatus Syntrophonatronum acetioxidans,' and a near-complete draft genome of this SAOB was reconstructed. 'Ca. S. acetioxidans' has all genes necessary for operating the Wood-Ljungdahl pathway, which is likely employed for acetate oxidation. It also encodes several genes essential to thrive at haloalkaline conditions; including a Na+-dependent ATP synthase and marker genes for 'salt-out' strategies for osmotic homeostasis at high soda conditions. Membrane lipid analysis of the M-SAO culture showed the presence of unusual bacterial diether membrane lipids which are presumably beneficial at extreme haloalkaline conditions. To determine the importance of SAO in haloalkaline environments, previously obtained 16S rRNA gene sequencing data and metagenomic data of five different hypersaline soda lake sediment samples were investigated, including the soda lakes where the enrichment cultures originated from. The draft genome of 'Ca. S. acetioxidans' showed highest identity with two metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the highly abundant and diverse Syntrophomonadaceae family present in the soda lake sediments. The 16S rRNA gene amplicon datasets of the soda lake sediments showed a high similarity of reads to 'Ca. S. acetioxidans' with abundance as high as 1.3% of all reads, whereas aceticlastic methanogens and acetate oxidizing sulfate-reducers were not abundant (≤0.1%) or could not be detected. These combined results indicate that SAO is the primary anaerobic acetate oxidizing pathway at extreme haloalkaline conditions performed by haloalkaliphilic syntrophic consortia.

Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation.

Since the discovery that anaerobic methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane coupled to sulfate reduction in marine sediments, different primers and probes specifically targeting the 16S rRNA gene of these archaea have been developed. Microbial investigation of the different ANME subtypes (ANME-1; ANME-2a, b, and c; and ANME-3) was mainly done in sediments where specific subtypes of ANME were highly enriched and methanogenic cell numbers were low. In different sediments with higher archaeal diversity and abundance, it is important that primers and probes targeting different ANME subtypes are very specific and do not detect other ANME subtypes or methanogens that are also present. In this study, primers and probes that were regularly used in AOM studies were tested in silico on coverage and specificity. Most of the previously developed primers and probes were not specific for the ANME subtypes, thereby not reflecting the actual ANME population in complex samples. Selected primers that showed good coverage and high specificity for the subclades ANME-1, ANME-2a/b, and ANME-2c were thoroughly validated using quantitative polymerase chain reaction (qPCR). From these qPCR tests, only certain combinations seemed suitable for selective amplification. After optimization of these primer sets, we obtained valid primer combinations for the selective detection and quantification of ANME-1, ANME-2a/b, and ANME-2c in samples where different ANME subtypes and possibly methanogens could be present. As a result of this work, we propose a standard workflow to facilitate selection of suitable primers for qPCR experiments on novel environmental samples.

Reverse Methanogenesis and Respiration in Methanotrophic Archaea.

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., "trace methane oxidation"). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.

Thiosulphate conversion in a methane and acetate fed membrane bioreactor.

The use of methane and acetate as electron donors for biological reduction of thiosulphate in a 5-L laboratory membrane bioreactor was studied and compared to disproportionation of thiosulphate as competing biological reaction. The reactor was operated for 454 days in semi-batch mode; 30 % of its liquid phase was removed and periodically replenished (days 77, 119, 166, 258, 312 and 385). Although the reactor was operated under conditions favourable to promote thiosulphate reduction coupled to methane oxidation, thiosulphate disproportionation was the dominant microbial process. Pyrosequencing analysis showed that the most abundant microorganisms in the bioreactor were phototrophic green sulphur bacteria (GSB) belonging to the family Chlorobiaceae and thiosulphate-disproportionating bacteria belonging to the genus Desulfocapsa. Even though the reactor system was surrounded with opaque plastic capable of filtering most of the light, the GSB used it to oxidize the hydrogen sulphide produced from thiosulphate disproportionation to elemental sulphur. Interrupting methane and acetate supply did not have any effect on the microbial processes taking place. The ultimate goal of our research was to develop a process that could be applied for thiosulphate and sulphate removal and biogenic sulphide formation for metal precipitation. Even though the system achieved in this study did not accomplish the targeted conversion using methane as electron donor, it does perform microbial conversions which allow to directly obtain elemental sulphur from thiosulphate.

Growth and activity of ANME clades with different sulfate and sulfide concentrations in the presence of methane.

Extensive geochemical data showed that significant methane oxidation activity exists in marine sediments. The organisms responsible for this activity are anaerobic methane-oxidizing archaea (ANME) that occur in consortia with sulfate-reducing bacteria. A distinct zonation of different clades of ANME (ANME-1, ANME-2a/b, and ANME-2c) exists in marine sediments, which could be related to the localized concentrations of methane, sulfate, and sulfide. In order to test this hypothesis we performed long-term incubation of marine sediments under defined conditions with methane as a headspace gas: low or high sulfate (±4 and ±21 mM, respectively) in combination with low or high sulfide (±0.1 and ±4 mM, respectively) concentrations. Control incubations were also performed, with only methane, high sulfate, or high sulfide. Methane oxidation was monitored and growth of subtypes ANME-1, ANME-2a/b, and ANME-2c assessed using qPCR analysis. A preliminary archaeal community analysis was performed to gain insight into the ecological and taxonomic diversity. Almost all of the incubations with methane had methane oxidation activity, with the exception of the incubations with combined low sulfate and high sulfide concentrations. Sulfide inhibition occurred only with low sulfate concentrations, which could be due to the lower Gibbs free energy available as well as sulfide toxicity. ANME-2a/b appears to mainly grow in incubations which had high sulfate levels and methane oxidation activity, whereas ANME-1 did not show this distinction. ANME-2c only grew in incubations with only sulfate addition. These findings are consistent with previously published in situ profiling analysis of ANME subclusters in different marine sediments. Interestingly, since all ANME subtypes also grew in incubations with only methane or sulfate addition, ANME may also be able to perform anaerobic methane oxidation under substrate limited conditions or alternatively perform additional metabolic processes.

High rates of anaerobic oxidation of methane, ethane and propane coupled to thiosulphate reduction.

Anaerobic methane oxidation coupled to sulphate reduction and the use of ethane and propane as electron donors by sulphate-reducing bacteria represent new opportunities for the treatment of streams contaminated with sulphur oxyanions. However, growth of microbial sulphate-reducing populations with methane, propane or butane is extremely slow, which hampers research and development of bioprocesses based on these conversions. Thermodynamic calculations indicate that the growth rate with possible alternative terminal electron acceptors such as thiosulphate and elemental sulphur may be higher, which would facilitate future research. Here, we investigate the use of these electron acceptors for oxidation of methane, ethane and propane, with marine sediment as inoculum. Mixed marine sediments originating from Aarhus Bay (Denmark) and Eckernförde Bay (Germany) were cultivated anaerobically at a pH between 7.2 and 7.8 and a temperature of 15 °C in the presence of methane, ethane and propane and various sulphur electron acceptors. The sulphide production rates in the conditions with methane, ethane and propane with sulphate were respectively 2.3, 2.2 and 1.8 µmol S L(-1) day(-1). For sulphur, no reduction was demonstrated. For thiosulphate, the sulphide production rates were up to 50 times higher compared to those of sulphate, with 86.2, 90.7 and 108.1 µmol S L(-1) day(-1) for methane, ethane and propane respectively. This sulphide production was partly due to disproportionation, 50 % for ethane but only 7 and 14 % for methane and propane respectively. The oxidation of the alkanes in the presence of thiosulphate was confirmed by carbon dioxide production. This is, to our knowledge, the first report of thiosulphate use as electron acceptor with ethane and propane as electron donors. Additionally, these results indicate that thiosulphate is a promising electron acceptor to increase start-up rates for sulphate-reducing bioprocesses coupled to short-chain alkane oxidation.

Growth of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a high-pressure membrane capsule bioreactor.

Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-m-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the Desulfuromonadales and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.

Enrichment of ANME-1 from Eckernförde Bay sediment on thiosulfate, methane and short-chain fatty acids.

The microorganisms involved in sulfate-dependent anaerobic oxidation of methane (AOM) have not yet been isolated. In an attempt to stimulate the growth of anaerobic methanotrophs and associated sulfate reducing bacteria (SRB), Eckernförde Bay sediment was incubated with different combinations of electron donors and acceptors. The organisms involved in AOM coupled to sulfate reduction (ANME-1, ANME-2, and Desulfosarcina/Desulfococcus) were monitored using specific primers and probes. With thiosulfate as sole electron acceptor and acetate, pyruvate or butyrate as the sole electron donor, ANME-1 became the dominant archaeal species. This finding suggests that ANME-1 archaea are not obligate methanotrophs and that ANME-1 can grow on acetate, pyruvate or butyrate.