Analysis of trace organic pollutants in wastewater to assess biodegradation using wrong-way-round ionization in liquid chromatography/tandem mass spectrometry.

RATIONALE: Monitoring the concentrations of pharmaceuticals and personal care products (PPCPs) in wastewater is an integral step toward understanding the fate of these contaminants in wastewater treatment plants (WWTPs). This paper aims to develop a method that allows for the simultaneous analysis of multiple classes of PPCPs that can be used as tracers to assess the performance of WWTPs. METHODS: Five PPCP tracers - carbamazepine (CBZ), 17α-ethinylestradiol (EE2), nonylphenol (NP), salicylic acid (SA), and trimethoprim (TMP) - were analyzed by liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) using a highly basic mobile phase (pH 10.3). Conventionally, TMP (pKa 7.12) and CBZ (pKa 13.94) are analyzed in positive ion mode using an acidic mobile phase. However, the high pH mobile phase allowed the quantification of all the tracers by polarity switching, with TMP undergoing wrong-way-round (WWR) ionization. RESULTS: The instrument limits of detection for the five tracers, without solid-phase extraction, were in the range of 1.3 to 5.9 ng/mL, except for NP, which was 238 ng/mL. The signal-to-noise (S/N) ratios for TMP and CBZ with the mobile phase at pH 10.3 were higher than the S/N ratios observed at pH 2.7 under positive electrospray ionization. The mechanism of WWR ionization for TMP was investigated, and we propose that a charge transfer from solvent clusters to TMP molecules due to electrolytic reactions at the surface of the droplet leads to WWR ionization in electrospray. CONCLUSIONS: A method to simultaneously analyze five representative PPCP tracers with a wide range of pKa values using WWR ionization in LC/MS/MS with polarity switching was developed. The method was successfully used to monitor the selected PPCPs in samples from full-scale WWTPs to assess their biodegradation under various treatment conditions.

Divergent microbial structure still results in convergent microbial function during arrested anaerobic digestion of food waste at different hydraulic retention times.

In this study, two arrested anaerobic digestion bioreactors, fed with food waste, operated under different hydraulic retention times (HRTs) exhibited similar total volatile fatty acid (VFA) yields (p = 0.09). 16S rRNA gene sequencing revealed distinct microbial structure (p = 0.02) at the two HRTs. However, between the two HRTs, there were no differences in potential (DNA) and extant (mRNA) functionality for the production of acetic (AA)-, propionic (PA)-, butyric (BA)- and valeric-acid (VA), as indicated by the metagenome and metatranscriptome data, respectively. The highest potential and extant functionality for PA production in the reactor microbiomes mirrored the highest abundance of PA in the reactor effluents. Meta-omics analysis of BA production indicated possible metabolite exchange across different community members. Notably, the basis for similar VFA production performance observed under the HRTs tested lies in the community-level redundancy in convergent acidification functions and pathways, rather than trends in community-level structure alone.

Low diversity and microdiversity of comammox bacteria in wastewater systems suggest specific adaptations within the Ca. Nitrospira nitrosa cluster.

Studies have found Ca. Nitrospira nitrosa-like bacteria to be the principal or sole comammox bacteria in nitrogen removal systems for wastewater treatment. In contrast, multiple populations of strict ammonia and nitrite oxidizers co-exist in similar systems. This apparent lack of diversity is surprising and could impact the feasibility of leveraging comammox bacteria for nitrogen removal. We used full-length 16S rRNA gene sequencing and genome-resolved metagenomics to compare the species-level diversity of comammox bacteria with that of strict nitrifiers in full-scale wastewater treatment systems and assess whether this comparison is consistent or diverged at the strain-level. Full-length 16S rRNA gene sequencing indicated that Nitrosomonas-like bacteria exhibited higher species-level diversity in comparison with other nitrifying bacteria, while the strain-level diversity (also called microdiversity) of most Nitrospira-like bacteria were higher than Nitrosomonas-like bacteria with few exceptions (one Nitrospira lineage II population). Comammox bacterial metagenome assembled genomes (MAGs) were associated with Ca. Nitrospira nitrosa. The average amino acid identity between principal comammox bacterial MAGs (93% ± 3) across systems was significantly higher than that of the Nitrosomonas-like ammonia oxidizers (73% ± 8), the Nitrospira_A-like nitrite oxidizer (85% ± 4), and the Nitrospira_D-like nitrite oxidizer (83% ± 1). This demonstrated the low species-level diversity of comammox bacteria compared with strict nitrifiers and further suggests that the same comammox population was detected in all systems. Comammox bacteria (Nitrospira lineage II), Nitrosomonas and, Nitrospira_D (Nitrospira lineage II) MAGs were significantly less microdiverse than the Nitrospira_A (lineage I) MAGs. Interestingly, strain-resolved analysis also indicates that different nitrogen removal systems harbor different comammox bacterial strains within the Ca. Nitrospira nitrosa cluster. These results suggest that comammox bacteria associated with Ca. Nitrospira nitrosa have low species- and strain-level diversity in nitrogen removal systems and may thus harbor specific adaptations to the wastewater ecosystem.

Recalcitrant dissolved organic nitrogen formation in thermal hydrolysis pretreatment of municipal sludge.

Thermal hydrolysis pretreatment (THP) has been considered as an advanced approach to enhance the performance of anaerobic digestion treating municipal sludge. However, several drawbacks were also identified with THP including the formation of brown and ultraviolet-quenching compounds that contain recalcitrant dissolved organic nitrogen (rDON). Melanoidins produced from the Maillard reaction between reducing sugar and amino group have been regarded as a representative of such compounds. This review presented the state-of-the-art understanding of the mechanism of melanoidin formation derived from the research of sludge THP, food processing, and model Maillard reaction systems. Special attentions were paid to factors affecting melanoidin formation and their implications to the control of rDON in the sludge THP process. These factors include reactant availability, heating temperature and time, pH, and the presence of metallic ions. It was concluded that efforts need to be focused on elucidating the extent of the Maillard reaction in sludge THP. This paper aims to provide a mechanistic recommendation on the research and control of the THP-resulted rDON in municipal wastewater treatment plants.

Nitrogen removal from water resource recovery facilities using partial nitrification, denitratation-anaerobic ammonia oxidation (PANDA).

Nitrogen removal from wastewater is an energy and chemical intensive process that is becoming increasingly more common around the world. To address the cost and complexity issues associated with biological nitrogen removal from wastewater, an alternative approach for achieving next generation nitrogen removal via partial nitrification, denitratation and anaerobic ammonia oxidation (PANDA) has been developed. The PANDA process relies on converting 50% of influent ammonia load to nitrate via aerobic ammonia (AerAOB) and nitrite oxidizing bacteria (NOB). The nitrate is reduced to nitrite (denitratation), followed by the removal of ammonia and nitrite by heterotrophic denitrifiers and anaerobic ammonia oxidizing biomass (AnAOB). Results from a pilot-scale sidestream PANDA demonstration at nitrogen loadings of 0.2-0.25 kg N/m3-day illustrated that up to 80% ammonia removal could be achieved. Testing in the mainstream process at initial ammonia concentrations of ~25 mg N/L indicated that 90% removal of total inorganic nitrogen could be achieved and that nitrogen removal was ultimately dependent on operating factors including aeration time, supplemental carbon dosing, hydraulic retention time and nitrate concentrations. Results cumulatively indicated that there was inherent resiliency within the PANDA systems when responding to variable environmental and operational conditions. This is hypothesized to be due to the fact that nitrogen removal is due to the combined synergistic activity of AerAOB, NOB, heterotrophic denitrifiers and AnAOB. Accordingly, utilization of PANDA based treatment processes may allow Water Resource Recovery Facilities (WRRFs) to achieve more sustainable and cost effective nitrogen removal in sidestream and mainstream processes without the need for NOB suppression and complex operational controls.

Long solids retention times and attached growth phase favor prevalence of comammox bacteria in nitrogen removal systems.

The discovery of the complete ammonia oxidizing (comammox) bacteria overturns the traditional two-organism nitrification paradigm which largely underpins the design and operation of nitrogen removal during wastewater treatment. Quantifying the abundance, diversity, and activity of comammox bacteria in wastewater treatment systems is important for ensuring a clear understanding of the nitrogen biotransformations responsible for ammonia removal. To this end, we conducted a yearlong survey of 14 full-scale nitrogen removal systems including mainstream conventional and simultaneous nitrification-denitrification and side-stream partial nitrification-anammox systems with varying process configurations. Metagenomics and genome-resolved metagenomics identified comammox bacteria in mainstream conventional and simultaneous nitrification-denitrification systems, with no evidence for their presence in side-stream partial nitrification-anammox systems. Further, comammox bacterial diversity was restricted to clade A and these clade A comammox bacteria were detected in systems with long solids retention times (>10 days) and/or in the attached growth phase. Using a newly designed qPCR assay targeting the amoB gene of clade A comammox bacteria in combination with quantitation of other canonical nitrifiers, we show that long solids retention time is the key process parameter associated with the prevalence and abundance of comammox bacteria. The increase in comammox bacterial abundance was not associated with concomitant decrease in the abundance of canonical nitrifiers; however, systems with comammox bacteria showed significantly better and temporally stable ammonia removal compared to systems where they were not detected. Finally, in contrast to recent studies, we do not find any significant association of comammox bacterial prevalence and abundance with dissolved oxygen concentrations in this study.

Evaluating the fate of bacterial indicators, viral indicators, and viruses in water resource recovery facilities.

A year-long sampling campaign at nine water resource recovery facilities (WRRFs) was conducted to assess the treatability and fate of bacterial indicators, viral indicators, and viruses. Influent concentrations of viral indicators (male-specific and somatic coliphages) and bacterial indicators (Escherichia coli and enterococci) remained relatively constant, typically varying by one order of magnitude over the course of the year. Annual average bacterial indicator reduction ranged from 4.0 to 6.7 logs, and annual average viral indicator reduction ranged from 1.6 to 5.4 logs. Bacterial and viral indicator reduction depended on the WRRF's treatment processes, and bacterial indicator reduction was greater than viral indicator reduction for many processes. Viral reduction (adenovirus 41, norovirus GI, and norovirus GII) was more similar to viral indicator reduction than bacterial indicator reduction. Overall, this work suggests that viral indicator reduction in WRRFs is variable and depends on specific unit processes. Moreover, for the same unit treatment process, viral indicator reduction and bacterial indicator reduction can vary. PRACTITIONER POINTS: A year-long sampling campaign was conducted at nine water resource recovery facilities (WRRFs). The treatability and fate of bacterial indicators, viral indicators, and viruses were assessed. Viral indicator reduction in WRRFs is variable and depends on specific unit processes. For the same unit treatment process, viral indicator reduction and bacterial indicator reduction can vary.

Mathematical modeling of biologically active filtration (BAF) for potable water production applications.

In this study, a modeling framework was developed to simulate biologically active filtration (BAF) headloss buildup in response to organic removal and nitrification. This model considered not only the biofilm growth on the BAF media but also the particle deposition in the BAF bed. In addition, the model also took temperature effect into consideration. It was calibrated and validated with data collected from a pilot-scale study used for potable water reuse and a full-scale facility used for potable water treatment. The model prediction provided insights that biofilm growth rather than particle deposition primarily contributes to the headloss buildup. Therefore, biofilm control is essential for managing headloss buildup and reducing the backwash frequency. Model simulation indicated that the BAF performance in terms of pollutant removal per unit headloss is insensitive to the BAF bed depth but can be effectively improved by increasing the media size. The partial biofilm coverage of the media is confirmed in this study and was mathematically verified to be a prerequisite for the model fitness.

Effect of advanced oxidation on N-nitrosodimethylamine (NDMA) formation and microbial ecology during pilot-scale biological activated carbon filtration.

Water treatment combining advanced oxidative processes with subsequent exposure to biological activated carbon (BAC) holds promise for the attenuation of recalcitrant pollutants. Here we contrast oxidation and subsequent biofiltration of treated wastewater effluent employing either ozone or UV/H2O2 followed by BAC during pilot-scale implementation. Both treatment trains largely met target water quality goals by facilitating the removal of a suite of trace organics and bulk water parameters. N-nitrosodimethylamine (NDMA) formation was observed in ozone fed BAC columns during biofiltration and to a lesser extent in UV/H2O2 fed columns and was most pronounced at 20 min of empty bed contact time (EBCT) when compared to shorter EBCTs evaluated. While microbial populations were highly similar in the upper reaches, deeper samples revealed a divergence within and between BAC filtration systems where EBCT was identified to be a significant environmental predictor for shifts in microbial populations. The abundance of Nitrospira in the top samples of both columns provides an explanation for the oxidation of nitrite and corresponding increases in nitrate concentrations during BAC transit and support interplay between nitrogen cycling with nitrosamine formation. The results of this study demonstrate that pretreatments using ozone versus UV/H2O2 impart modest differences to the overall BAC microbial population structural and functional attributes, and further highlight the need to evaluate NDMA formation prior to full-scale implementation of BAC in potable reuse applications.

Factors impacting biotransformation kinetics of trace organic compounds in lab-scale activated sludge systems performing nitrification and denitrification.

To predict TOrC fate in biological activated sludge systems, there is a need to accurately determine TOrC biodegradation kinetics in mixed microbial cultures. Short-term batch tests with salicylic acid, 17α-ethinylestradiol, nonylphenol, trimethoprim and carbamazepine were conducted with lab-scale activated sludge cultures in which the initial TOrC concentration (1mg/L and 0.0005mg/L) and readily biodegradable substrate concentrations were varied. The results indicate that pseudo-first order kinetic estimates of TOrC are not sensitive (p>0.05) to the initial TOrC concentration as long as the initial TOrC concentration (S0) to biomass (X0) ratio (on COD basis) is below 2×10(-3). The presence of readily biodegradable organic matter suppresses TOrC biotransformation rates under nitrifying and denitrifying conditions, and this impact can be adequately described using a reversible non-competitive inhibition equation. These results demonstrate the importance of closely mimicking parent reactor conditions in batch testing because biotransformation parameters are impacted by in-situ carbon loading and redox conditions.

Biomass production from electricity using ammonia as an electron carrier in a reverse microbial fuel cell.

The storage of renewable electrical energy within chemical bonds of biofuels and other chemicals is a route to decreasing petroleum usage. A critical challenge is the efficient transfer of electrons into a biological host that can covert this energy into high energy organic compounds. In this paper, we describe an approach whereby biomass is grown using energy obtained from a soluble mediator that is regenerated electrochemically. The net result is a separate-stage reverse microbial fuel cell (rMFC) that fixes CO2 into biomass using electrical energy. We selected ammonia as a low cost, abundant, safe, and soluble redox mediator that facilitated energy transfer to biomass. Nitrosomonas europaea, a chemolithoautotroph, was used as the biocatalyst due to its inherent capability to utilize ammonia as its sole energy source for growth. An electrochemical reactor was designed for the regeneration of ammonia from nitrite, and current efficiencies of 100% were achieved. Calculations indicated that overall bioproduction efficiency could approach 2.7±0.2% under optimal electrolysis conditions. The application of chemolithoautotrophy for industrial bioproduction has been largely unexplored, and results suggest that this and related rMFC platforms may enable biofuel and related biochemical production.

Evaluating the solid retention time of bacteria in flocculent and granular sludge.

The specific solid retention time for different bacteria within flocculent and granular sludge was determined. Samples were collected from reactor and effluent sludge and the number of a specific bacterial group was evaluated in respect to the total bacterial community with quantitative polymerase chain reaction (qPCR). The ratio of the relative presence of a specific bacterial group in the reactor sludge and wasted sludge was established to observe if preferential wash-out occurred. From the data also the solid retention time for different microbial groups can be estimated. Using this tool, we were able to show that the SRT of populations found on the exterior of granules is slightly lower than the SRT for population in the interior. Archaea were not found in the flocculent system but were present in small amounts within the granular system. It was further observed that protozoa were grazing on the bacterial community within the system indicating that they have the potential to shorten the specific SRT of bacteria.

Sorption of carbamazepine, 17α-ethinylestradiol, iopromide and trimethoprim to biomass involves interactions with exocellular polymeric substances.

The sorption of carbamazepine (CBZ), iopromide (IOP), trimethoprim (TMP) and 17α-ethinylestradiol (EE2) was evaluated using four biomass types (pure ammonia oxidizing bacterial culture, two heterotrophic enrichment cultures with varying levels of oxygenase activity, and a full-scale nitrifying activated sludge (NAS) culture). CBZ and IOP did not sorb to the four biomass types. EE2 did not sorb to the pure culture but sorbed significantly to the heterotrophic cultures and NAS. TMP sorbed to the heterotrophic cultures and NAS, and was not evaluated for the pure culture. Three floc characteristics (hydrophobicity, median particle size, organic matter content) correlated moderately well with the EE2 organic matter sorption coefficient (KOM,EE2). Zeta potential did not correlate well with KOM,EE2 but did with KOM,TMP, indicating that TMP sorption is more influenced by electrostatic factors than EE2. Once divalent cation-linked exocellular polymeric substances (EPS) were removed from flocs, EE2 and TMP sorption to the non-EPS (cellular) fraction decreased by approximately 50%. The correlation between KOM,EE2 for the non-EPS cellular fraction deteriorated while the correlation between KOM,TMP improved. EE2 seemed to sorb more strongly to EPS protein whereas TMP sorbed equally to polysaccharide and protein EPS. Attempts to develop predictive models were not successful. Pharmaceuticals that sorbed to biomass samples underwent biodegradation whereas those that did not sorb were not biodegraded, suggesting a relationship between sorption and pharmaceutical biotransformation.