Wendell O. Khunjar

Elucidating the Relative Roles of Ammonia Oxidizing and Heterotrophic Bacteria during the Biotransformation of 17α-Ethinylestradiol and Trimethoprim

The biological fate of 17α-ethinylestradiol (EE2; 500 ng/L to 1 mg/L) and trimethoprim (TMP; 1 μg/L to 1 mg/L) was evaluated with flow through reactors containing an ammonia oxidizing bacterial (AOB) culture, two enriched heterotrophic cultures devoid of nitrifier activity, and nitrifying activated sludge (NAS) cultures. AOBs biotransformed EE2 but not TMP, whereas heterotrophs mineralized EE2, biotransformed TMP, and mineralized EE2-derived metabolites generated by AOBs. Kinetic bioassays showed that AOBs biotransformed EE2 five times faster than heterotrophs. The basal expression of heterotrophic dioxygenase enzymes was sufficient to achieve the high degree of transformation observed at EE2 and TMP concentrations ≤ 1 mg/L, and enhanced enzyme expression was not necessary. The importance of AOBs in removing EE2 and TMP was evaluated further by performing NAS experiments at lower feed concentrations (500-1000 ng/L). EE2 removal slowed markedly after AOBs were inhibited, while TMP removal was not affected by AOB inhibition. Two key EE2 metabolites formed by AOB and heterotrophic laboratory-scale chemostats were also found in independent laboratory-scale mixed culture bioreactors; one of these, sulfo-EE2, was largely resistant to further biodegradation. AOBs and heterotrophs may cooperatively enhance the reliability of treatment systems where efficient removal of EE2 is desired.

Technologies to Recover Nutrients from Waste Streams: A Critical Review

Technologies to recover nitrogen, phosphorus, and potassium from waste streams have undergone accelerated development in the past decade, predominantly due to a surge in fertilizer prices and stringent discharge limits on these nutrients. This review provides a critical state of art review of appropriate technologies which identifies research gaps, evaluates current and future potential for application of the respective technologies, and outlines paths and barriers for adoption of the nutrient recovery technologies. The different technologies can be broadly divided into the sequential categories of nutrient accumulation, followed by nutrient release, followed by nutrient extraction. Nutrient accumulation can be achieved via plants, microorganisms (algae and prokaryotic), and physicochemical mechanisms including chemical precipitation, membrane separation, sorption, and binding with magnetic particles. Nutrient release can occur by biochemical (anaerobic digestion and bioleaching) and thermochemical treatment. Nutrient extraction can occur via crystallization, gas-permeable membranes, liquid–gas stripping, and electrodialysis. These technologies were analyzed with respect to waste stream type, the product being recovered, and relative maturity. Recovery of nutrients in a concentrated form (e.g., the inorganic precipitate struvite) is seen as desirable because it would allow a wider range of options for eventual reuse with reduced pathogen risk and improved ease of transportation. Overall, there is a need to further develop technologies for nitrogen and potassium recovery and to integrate accumulation–release–extraction technologies to improve nutrient recovery efficiency. There is a need to apply, demonstrate, and prove the more recent and innovative technologies to move these beyond their current infancy. Lastly, there is a need to investigate and develop agriculture application of the recovered nutrient products. These advancements will reduce waterway and air pollution by redirecting nutrients from waste into recovered nutrient products that provides a long-term sustainable supply of nutrients and helps buffer nutrient price rises in the future. Graphical Abstract:

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.

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.

Characterizing the metabolic trade-off in Nitrosomonas europaea in response to changes in inorganic carbon supply

The link between the nitrogen and one-carbon cycles forms the metabolic basis for energy and biomass synthesis in autotrophic nitrifying organisms, which in turn are crucial players in engineered nitrogen removal processes. To understand how autotrophic nitrifying organisms respond to inorganic carbon (IC) conditions that could be encountered in engineered partially nitrifying systems, we investigated the response of one of the most extensively studied model ammonia oxidizing bacteria, Nitrosomonas europaea (ATCC19718), to three IC availability conditions: excess gaseous and excess ionic IC supply (40× stoichiometric requirement), excess gaseous IC supply (4× stoichiometric requirement in gaseous form only), and limiting IC supply (0.25× stoichiometric requirement). We found that, when switching from excess gaseous and excess ionic IC supply to excess gaseous IC supply, N. europaea chemostat cultures demonstrated an acclimation period that was characterized by transient decreases in the ammonia removal efficiency and transient peaks in the specific oxygen uptake rate. Limiting IC supply led to permanent reactor failures (characterized by biomass washout and failure of ammonia removal) that were preceded by similar decreases in the ammonia removal efficiency and peaks in the specific oxygen uptake rate. Notably, both excess gaseous IC supply and limiting IC supply elicited a previously undocumented increase in nitric and nitrous oxide emissions. Further, gene expression patterns suggested that excess gaseous IC supply and limiting IC supply led to consistent up-regulation of ammonia respiration genes and carbon assimilation genes. Under these conditions, interrogation of the N. europaea proteome revealed increased levels of carbon fixation and transport proteins and decreased levels of ammonia oxidation proteins (active in energy synthesis pathways). Together, the results indicated that N. europaea mobilized enhanced IC scavenging pathways for biosynthesis and turned down respiratory pathways for energy synthesis, when challenged with excess gaseous IC supply and limiting IC supply.

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.

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.

Developing a Standardized Protocol for Assessing the Biodegradability of Trace Organic Compounds

This work was performed to better understand the factors that impact trace organic compound (TOrC) biodegradation kinetics in mixed microbial cultures. To accomplish this goal, optimized short-term batch tests using TOrC and identified TOrC surrogates were conducted using lab-scale activated sludge cultures. In these optimization experiments, the initial TOrC/surrogate concentration, readily biodegradable carbon concentration (rbCOD) and dissolved oxygen (DO) concentrations were varied. Once developed, these batch tests were deployed at three full-scale wastewater treatment facilities to estimate in-situ biotransformation kinetics. This work confirms that TOrC biotransformation is best described using pseudo-first order kinetics. In contrast, results from surrogate experiments indicate that the current approach to aerobic respirometry is not sufficiently sensitive for resolving TOrC biotransformation versus endogenous respiration. Results from short-term batch test optimization also indicate that parameters estimated from depletion tests performed at artificially high TOrC concentrations are statistically similar to parameters estimated at low TOrC concentrations. This suggests that interrogation of the biomass can be performed at high TOrC concentrations, which would reduce the cost of sample processes associated with working at lower concentrations. It was also found that the presence of excess readily biodegradable substrates suppress biotransformation rates for specific TOrC under aerobic and anoxic conditions. These results indicate that accurate estimation of biotransformation parameters requires careful design of the batch test to match the in-situ carbon conditions. An initial attempt to incorporate existing biotransformation and sorption parameters into current activated sludge process models was also performed. Results from this simulation indicate that modeling results are highly sensitive to the processes of sorption and desorption. This title belongs to WERF Research Report Series . ISBN: 9781780405162 (eBook)

Towards a renewable future: assessing resource recovery as a viable treatment alternative state of the science and market assessment

This report presents a review of extractive nutrient recovery technologies with an emphasis on bridging the knowledge gap faced by utilities when considering nutrient recovery for nutrient management. The report provides a framework for selecting a nutrient recovery option and, depending on the conditions at a water resource recovery facility, establishes whether keeping phosphorus in biosolids is more or less beneficial than concentrating it in an inorganic phase such as struvite.

Biotransformation of Pharmaceuticals and Personal Care Products (PPCPs) during Nitrification: The Role of Ammonia Oxidizing Bacteria versus Heterotrophic Bacteria

Preliminary experiments to investigate the fate of 17α-ethinylestradiol (EE2) during nitrification were conducted. Sorption experiments were performed by incubating inactivated ammonia oxidizing bacteria and various heterotrophic cultures in the presence of 14 C-EE2 at 10 μg/L. Biotransformation experiments utilized LC-MS and LC-ITMS analysis of supernatant collected during batch cultivation of monoculture of Nitrosomonas europaea with EE2. Results confirmed that sorption of EE2 onto heterotrophic biomass is an important mechanism that must be considered when describing the fate of EE2. During batch incubation with N. europaea , 98% of EE2 was transformed, producing a unique metabolite corresponding to a mass/charge ratio of — 386 m/z. Short term inhibition study results also suggest that inhibition of respiration in N. europaea as measured through nitrite generation rates is not significant at concentrations at or below 1 mg EE2/L.