Fate of Extracellular DNA in the Production of Fertilizers from Source-Separated Urine.

The practice of urine source-separation for fertilizer production necessitates an understanding of the presence and impact of extracellular DNA in the urine. This study examines the fate of plasmid DNA carrying ampicillin and tetracycline resistance genes in aged urine, including its ability to be taken up and expressed by competent bacteria. Plasmid DNA incubated in aged urine resulted in a >2 log loss of bacterial transformation efficiency in Acinetobacter baylyi within 24 h. The concentration of ampicillin and tetracycline resistance genes, as measured with quantitative polymerase chain reaction, did not correspond with the observed transformation loss. When the plasmid DNA was incubated in aged urine that had been filtered (0.22 µm) or heated (75 °C), the transformation efficiencies were more stable than when the plasmids were incubated in unfiltered and unheated aged urine. Gel electrophoresis results indicated that plasmid linearization by materials larger than 100 kDa in the aged urine caused the observed transformation efficiency decreases. The results of this study suggest that extracellular DNA released into aged urine poses a low potential for the spread of antibiotic resistance genes to bacteria once it is released to the environment.

Fate of the Urinary Tract Virus BK Human Polyomavirus in Source-Separated Urine.

Human polyomaviruses are emerging pathogens that infect a large percentage of the human population and are excreted in urine. Consequently, urine that is collected for fertilizer production often has high concentrations of polyomavirus genes. We studied the fate of infectious double-stranded DNA (dsDNA) BK human polyomavirus (BKPyV) in hydrolyzed source-separated urine with infectivity assays and quantitative PCR (qPCR). Although BKPyV genomes persisted in the hydrolyzed urine for long periods of time (T90 [time required for 90% reduction in infectivity or gene copies] of >3 weeks), the viruses were rapidly inactivated (T90 of 1.1 to 11 h) in most of the tested urine samples. Interestingly, the infectivity of dsDNA bacteriophage surrogate T3 (T90 of 24 to 46 days) was much more persistent than that of BKPyV, highlighting a major shortcoming of using bacteriophages as human virus surrogates. Pasteurization and filtration experiments suggest that BKPyV virus inactivation was due to microorganism activity in the source-separated urine, and SDS-PAGE Western blots showed that BKPyV protein capsid disassembly is concurrent with inactivation. Our results imply that stored urine does not pose a substantial risk of BKPyV transmission, that qPCR and infectivity of the dsDNA surrogate do not accurately depict BKPyV fate, and that microbial inactivation is driven by structural elements of the BKPyV capsid.IMPORTANCE We demonstrate that a common urinary tract virus has a high susceptibility to the conditions in hydrolyzed urine and consequently would not be a substantial exposure route to humans using urine-derived fertilizers. The results have significant implications for understanding virus fate. First, by demonstrating that the dsDNA (double-stranded DNA) genome of the polyomavirus lasts for weeks despite infectivity lasting for hours to days, our work highlights the shortcomings of using qPCR to estimate risks from unculturable viruses. Second, commonly used dsDNA surrogate viruses survived for weeks under the same conditions that BK polyomavirus survived for only hours, highlighting issues with using virus surrogates to predict how human viruses will behave in the environment. Finally, our mechanistic inactivation analysis provides strong evidence that microbial activity drives rapid virus inactivation, likely through capsid disassembly. Overall, our work underlines how subtle structural differences between viruses can greatly impact their environmental fate.

Urine Bacterial Community Convergence through Fertilizer Production: Storage, Pasteurization, and Struvite Precipitation.

Source-separated human urine was collected from six public events to study the impact of urine processing and storage on bacterial community composition and viability. Illumina 16S rRNA gene sequencing revealed a complex community of bacteria in fresh urine that differed across collection events. Despite the harsh chemical conditions of stored urine (pH > 9 and total ammonia nitrogen > 4000 mg N/L), bacteria consistently grew to 5 ± 2 × 108 cells/mL. Storing hydrolyzed urine for any amount of time significantly reduced the number of operational taxonomic units (OTUs) to 130 ± 70, increased Pielou evenness to 0.60 ± 0.06, and produced communities dominated by Clostridiales and Lactobacillales. After 80 days of storage, all six urine samples from different starting materials converged to these characteristics. Urine pasteurization or struvite precipitation did not change the microbial community, even when pasteurized urine was stored for an additional 70 days. Pasteurization decreased metabolic activity by 50 ± 10% and additional storage after pasteurization did not lead to recovery of metabolic activity. Urine-derived fertilizers consistently contained 16S rRNA genes belonging to Tissierella, Erysipelothrix, Atopostipes, Bacteroides, and many Clostridiales OTUs; additional experiments must determine whether pathogenic species are present, responsible for observed metabolic activity, or regrow when applied.

Virus Emissions from Toilet Flushing: Comparing Urine-Diverting to Mix Flush Toilets.

High levels of viruses can be found in human excrement from infected individuals, a fraction of which can be emitted from toilet flushing. Unlike the common mix flush toilet (MFT), the urine-diverting toilet (UDT) separates urine from the toilet water. Specific focus on urine-associated viruses is needed because the UDT can emit different levels of urine-associated and fecal-borne viruses and urine has different properties compared to feces that can affect emission levels (e.g., protein content). In this work, we quantified emission levels of surrogate bacteriophages for urine-associated and fecal-borne viruses, MS2 and T3, from flushing a UDT and an MFT, with and without protein in the water. Emission levels of viruses in the water of the UDT were lower than that of the MFT by up to 1.2-log10 and 1.3-log10 for T3 and MS2, respectively. If urine is completely diverted in the UDT, virus emissions can be reduced by up to 4-log10. Based on these results and typical levels in urine and feces, we estimate that up to 107 and 108 gene copies of human viruses per flush can be released from the UDT and MFT, respectively. Lower emissions observed with the UDT suggest reduced exposure to viruses from flushing the UDT.

Oxytetracycline interactions at the soil-water interface: effects of environmental surfaces on natural transformation and growth inhibition of Azotobacter vinelandii.

The mechanism of oxytetracycline (OTC) adsorption to a silty clay loam soil was investigated using sorption isotherm experiments, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction spectroscopy (XRD). Sorption data fit well to a cation-exchange capacity sorption model. Spectroscopic data indicate that the interactions between oxytetracycline and silty clay loam soil were primarily through electrostatic interactions between the protonated dimethylamino group of OTC and the negatively charged moieties on the surface of the soil. Based on XRD results, OTC adsorption appeared to inhibit the ethylene glycol solvation of the expandable clay minerals, suggesting that OTC had diffused into the clay interlayer space. The presence of adsorbed OTC did not significantly affect the transformation frequency of the soil bacterium Azotobacter vinelandii with plasmid DNA (soil alone 3 × 10(6) ± 4 × 10(6) and soil with adsorbed OTC 4 × 10(6) ± 0.5 × 10(6) ). Growth was inhibited by adsorbed OTC, although a greater mass of adsorbed OTC was required to achieve the same degree of inhibition as the system of dissolved OTC alone. These results suggest that the interactions of tetracyclines at the soil-water interface will affect the growth of sensitive microorganisms in soil microbial communities.