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Urine bacterial community convergence through fertilizer production: Storage, pasteurization, and struvite precipitation Rebecca H Lahr, Heather E Goetsch, Sarah-Jane Haig, Abraham Noe-Hays, Nancy G. Love, Diana S Aga, Charles B. Bott, Betsy Foxman, Jose Jimenez, Ting Luo, Kim Nace, Kirtana Ramadugu, and Krista R. Wigginton Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02094 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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Environmental Science & Technology

Aged Urine

Fresh Urine

Pasteurized Urine

Struvite Fertilizer ACS Paragon Plus Environment


1

Urine bacterial community convergence through fertilizer

2

production: Storage, pasteurization, and struvite

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precipitation

4 5 6 7 8 9 10 11

Rebecca H. Lahr1,2, Heather E. Goetsch1, Sarah J. Haig1, Abraham Noe-Hays3, Nancy G. Love1, Diana S. Aga4, Charles B. Bott5, Betsy Foxman6, Jose Jimenez7, Ting Luo6, Kim Nace3, Kirtana Ramadugu6, Krista R. Wigginton1,* 1

Department of Civil and Environmental Engineering, University of Michigan, 1351 Beal Ave, EWRE, Ann Arbor, MI 48109 2

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Department of Civil and Environmental Engineering, Michigan State University, 1449 Engineering Research Court, East Lansing, MI 48824 3

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4

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Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260 5

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Rich Earth Institute, 44 Fuller Drive, Brattleboro VT

Hampton Roads Sanitation District, 1434 Air Rail Ave, Virginia Beach, VA 23455

Epidemiology Department, University of Michigan 1415 Washington Heights, Ann Arbor, MI 48109 7

Brown and Caldwell, 850 Trafalgar Court, Suite 300, Maitland, FL 32751

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*Corresponding author.

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Tel.: (734) 763-9661

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Fax: (734) 764-4292

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E-mail addresses: [email protected]

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Keywords: urine diversion, source-separated urine, bacterial community analysis, urine

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pasteurization, 16S rRNA gene sequencing

ACS Paragon Plus Environment

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TOC ART

Fresh Urine

Aged Urine

Pasteurized Urine

Struvite Fertilizer

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ABSTRACT

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Source-separated human urine was collected from six pulic events to study the impact of urine

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processing and storage on bacterial community composition and viability. Illumina 16S rRNA

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gene sequencing revealed a complex community of bacteria in fresh urine that differed across

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collection events. Despite the harsh chemical conditions of stored urine (pH >9 and total

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ammonia nitrogen >4000 mg N/L), bacteria consistently grew to 5±2×108 cells/mL. Storing

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hydrolyzed urine for any amount of time significantly reduced the number of operational

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taxonomic units (OTUs) to 130 ± 70, increased Pielou evenness to 0.60 ± 0.06, and produced

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communities dominated by Clostridiales and Lactobacillales. After 80 days of storage, all six

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urine samples from different starting materials converged to these characteristics. Urine

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pasteurization or struvite precipitation did not change the microbial community, even when

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pasteurized urine was stored for an additional 70 days. Pasteurization decreased metabolic

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activity by 50±10% and additional storage after pasteurization did not lead to recovery of

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metabolic activity. Urine-derived fertilizers consistently contained 16S rRNA genes belonging to

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Tissierella, Erysipelothrix, Atopostipes, Bacteroides, and many Clostridiales OTUs; additional

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experiments must determine whether pathogenic species are present, responsible for observed

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metabolic activity, or regrow when applied.

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INTRODUCTION

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The diversion of urine from municipal waste streams can provide a number of environmental and

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economic benefits, namely a concentrated stream of nitrogen (N), phosphorus (P), and potassium

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(K) for nutrient management and recovery. Although urine only contributes 1% of the volume in

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domestic wastewater, it contains ~85% of the N and ~55% of the P from the total municipal

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wastewater stream.1 Compared to conventional wastewater collection and treatment, the



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widespread adoption of urine-diversion practices could lead to significant reductions in the

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consumption of energy and water.1,2

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Nutrient-rich urine is a sustainable source of fertilizer. The average person’s yearly urine

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contains 2.5 – 4.0 kg of total nitrogen (TN) and 0.22 – 0.37 g of total phosphorus (TP), enough to

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grow cereal to feed one person (250 kg).3,4 Converting urine to fertilizer products can be carried

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out in a number of ways, the simplest approach being storage in a sealed container for several

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months.5 Nitrogen in fresh urine is primarily in the form of urea (~85%).6 As it is stored, urea is

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hydrolyzed into ammonia with the aid of bacterial urease enzymes:

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CH 4 N 2O + 2H 2O !Urease !! → NH 4+ + NH 3 + HCO3−

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This transformation causes the pH to increase from approximately 6 when it exits the body to > 9

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after one day to a few weeks at room temperature.7 At this point, ammonia concentrations in

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stored urine reach thousands of parts per million (as mg N/L). The unprotonated form of

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ammonia (NH3) is biocidal, and makes up approximately 50% of the total ammonia nitrogen in

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stored urine.8 This harsh chemical environment is harnessed to sanitize the source-separated

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urine.7-9 Additional approaches to creating fertilizer from urine include precipitating struvite

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minerals (NH4MgPO4·6H2O), nutrient concentration via reverse osmosis or distillation,

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pasteurization, and other techniques.5

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Source-separated urine is commonly contaminated with feces.10 As a result, research on

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pathogens in collected urine has focused primarily on the presence and survival of fecal indicator

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microorganisms, such as coliforms or Streptococci.7,11-14 Fecal indicator organisms are poor

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indicators for microbial risk,15 and only represent a fraction of the bacteria present in urine

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diversion systems. In addition to inoculation by fecal bacteria when it exits the urethra, urine can

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be inoculated with vulvovaginal bacteria in women, and environmental bacteria where the urine

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is collected and stored. Furthermore, urine contains bacteria before it even exits the body, as

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made evident by the recent advancement of rapid microbial sequencing techniques.16,17 The

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limited urine samples that have been sequenced to date suggest there is a core urinary

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microbiome that is dependent on the age and sex of the individual.17,18

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In the cases where human waste products are applied as fertilizers, risk assessments help

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contextualize the associated hazards. Before this can be conducted for urine fertilizers, additional

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information is needed on the presence and fate of pathogenic microorganisms through fertilizer

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production and application. This study served to identify the quantities and types of bacteria that

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survive or flourish in source-separated urine and urine-derived fertilizers. By collecting urine

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from different events and two regions of the U.S., we were also able to assess if the bacterial

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community in one source-separated urine sample was representative of other samples.

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MATERIALS AND METHODS

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Urine collection, processing, and characterization.

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We apply definitions as described by Udert and colleagues;6 in particular, the term “source-

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separated urine” describes urine that was collected with urine-diverting toilets or urinals and

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“stored urine” describes source-separated urine that has undergone complete urea hydrolysis.

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Urine was collected at six public events (Events A – F) from different communities in Michigan

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and Vermont (n = 6 urine collection events). Portable toilets were modified with screens below

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the seat opening to exclude paper or other solids (Figure SI-1). The event duration, daily

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temperatures, number of toilet users, and volume of urine were recorded for each event.

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Although collection and storage was not conducted under sterile conditions, the portable toilet

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collection tanks, fifty-five gallon storage tanks, urine pumps, and pump tubing were cleaned with

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either water or detergent and nitric acid solution, depending on resources available at the

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collection event. Immediately after each collection event, urine was transported or shipped on ice

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to the laboratories at the University of Michigan where fertilizer production, chemical and

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biological analyses took place. Urine was stored at room temperature in sealed carboys with

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spigots. Samples were mixed thoroughly prior to collecting aliquots, as previous research

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suggests that microorganism concentrations become stratified in stored urine.19

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Pasteurization was carried out by raising the urine temperature to 80 °C, holding at this

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temperature for 30 minutes without mixing, and then allowing the sample to cool to room

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temperature. Struvite was produced as described previously20 and detailed in the SI. In brief,

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urine was pumped into a 35-gallon reactor and stirred while a 0.5 M magnesium chloride



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solution was added to achieve a phosphate to magnesium molar ratio of 1:1.5. After reacting for

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30 minutes, struvite crystals were captured in a 50 µm nylon mesh filter bag.

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Fresh, stored, and pasteurized urines were analyzed for pH, total N, total ammonia N, and total P

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with standard chemical methods (Table SI-1). Conventionally, “stored” urine specifies any urine

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over pH 9. Further classification was required to document trends in the bacterial community

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over time; urine samples were classified as “young” when pH was less than 9 and “hydrolyzed”

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when pH was greater than 9. Subsets called “fresh” described urine that was less than 24 hours

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old and “converged” described urine that was over 80 days old (determined by analysis

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documented in the results). Stored urine was analyzed from all six public events (Events A-F).

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Urine from three events was analyzed when fresh, through storage, and following pasteurization

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(Events D, E, F). Events D and E were analyzed until >100 days and event F which was

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monitored until 62 days.

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Bacterial cell counts. Hemocytometer counts were conducted in disposable hemocytometer cells (Fisher Scientific C-

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chip DHC-N01-5). Counts with SYBR® Gold Nucleic Acid Stain (Invitrogen) and fluorescence

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microscopy were based on a published method and described in the SI.21 The slides were stored

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at -20 °C until they were analyzed with a fluorescence microscope. Objects with diameters of 0.4

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– 4 µm were identified as bacteria.

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Bacterial cultivability and viability. Three methods were used to collectively define bacterial viability in this study, namely live-dead

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staining, culture-based techniques, and cell viability assays. Bacteria with intact membranes were

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quantified in urine samples with a BacLight “Live/Dead” stain (Molecular Probes) according to

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the manufacturer’s protocol, and optimized for our purposes. Select urine samples were plated on

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various media (lysogeny broth (LB), brain-heart infusion (BHI), MacConkey, eosin methylene

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blue agar (EMB), reasoner’s 2A agar (R2A)) and incubated overnight at 37 oC to isolate

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heterotrophs, enteric bacteria, and E. coli colonies. The capacity for microorganisms to respire in

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the hydrolyzed and pasteurized urine samples were assessed in triplicate with the artificial

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electron acceptor sodium 3,3'-[5-(phenylcarbamoyl)-2H-tetrazol-3-ium-2,3-diyl]bis(4-methoxy-

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6-nitrobenzenesulfonate) (XTT) according to the kit manufacturer’s protocol. XTT measured


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bacterial cell viability by the activity of a dehydrogenase enzyme present in live cells, which

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reduced XTT into a colored product.22,23 In brief, 100 µL of BHI broth, 10 µL of urine, and 25

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µL activated XTT were mixed in microplate wells and incubated at 37 °C for three hours. The

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absorbance of the samples at 620 nm and 450 nm were measured with a microplate reader

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(Biotek uQuant). The activity levels of samples were calculated by first subtracting the

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absorbance at 620 nm from the absorbance at 450 nm. The background absorbance was

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measured from a control containing media and the XTT reagents that was incubated for 3 hours.

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Negative controls, consisting of autoclaved Escherichia coli K12, and positive controls,

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consisting of live E. coli K12, were included in the analyses.

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Bacterial DNA extraction and sequencing.

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Bacteria were separated from 10-15 mL urine samples with 0.2 µm polycarbonate membrane

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filters (Fisher Scientific GTTP04700) and stored at -80 °C. DNA was extracted from the filtered

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samples with Maxwell 16 blood DNA kits according to manufacturer instructions, after an initial

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bead-beating step described in the SI.24 DNA in struvite was extracted using FastDNA Spin Kit

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for Soil (MP Biomedical) following the manufacturer’s instructions. DNA extracts were stored at

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-20 °C for short periods and -80 °C for long periods. DNA concentrations were measured with a

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Qubit or Nanodrop 3300 fluorometer with a picogreen dsDNA quantification kit. DNA quality

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was assessed with a Nanodrop 1000.

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The 16S rRNA gene amplicon of 65 samples was sequenced using primers for the V4 region

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following the procedure outlined previously.25,26 Miseq (Illumina) sequenced samples were

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quality filtered and demultiplexed. Taxonomy was assigned against the Silva database (Release

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119),27 with operational taxonomic unit (OTU) identified after pre-clustered at ≥97% similarity

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using the Mothur MiSeq pipeline.26 Relative abundances were rarefied to the sample with the

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lowest number of sequences (21,956 reads).

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Statistics. All statistical analyses were conducted in R with statistical significance identified with p-values

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2; LDA values of 4-5 signified a higher abundance of that OTU in the listed group.31

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Shannon diversity indices, Chao's diversity index, and Pielou's evenness were calculated on

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rarefied samples at the 3% nucleotide distance. The relationships between environmental

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variables and patterns in bacterial community structure were examined by canonical

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correspondence analysis (CCA) with significance tested by analysis of variance tests (ANOVA).

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The phyloseq package was used to plot relative abundance.33

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RESULTS AND DISCUSSION

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Chemical composition of fresh and hydrolyzed urine. Source-separated urine was collected from six events (Table 1). Samples experienced a pH shift

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from less than 7 to over 9 after 1 to 18 days of storage, which is within the time range reported in

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the literature7 (Figure SI-2). The time it takes for urea hydrolysis is dependent on the amount of

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urease in the urine and on the temperature.34 Hydrolyzed urine samples contained typical N and

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P concentrations (5500-9000 mg/L as N, and 440-1130 mg/L total phosphorus, n = 6 urine

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collection events). Total organic carbon concentrations were equal to 1600-2000 mg C/L (n = 2

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collection events). Pasteurized urine contained similar nutrient concentrations (4600-4800 mg/L

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as N, 380-510 mg/L as P, 4500-5100 mg/L TAN, and 1700-1900 mg C/L, n = 2 collection

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events). Chemical parameters and nutrient levels measured for fresh and hydrolyzed urine are in

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agreement with previous reports outside the U.S.6,19

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Table 1. Characteristics of urine collected in portable toilets from public events. Event A 1 City High: 49 Low: 32 1 yr) 5050±230 (>1 yr) nitrogen 4130 ± 150 (>1 yr)

NA 800 ± 70 (>1 yr) 5470±70 (>1 yr) 5230 ± 280 (> 1 yr)

NA 1240± 440 (>1 yr) 8150±70 (>1 yr) 7560 ± 370 (> 1 yr)

Duration (days) Event type Daily temperatures (°F) Approximate # toilet uses Volume collected (gallons) Timescale of analyses

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Time to pH > 9 Total phosphorus (mg/L as P) Total nitrogen (mg/L as N) Total ammonia (mg/L as N)

Event D 1 Parade High: 80 Low: 46 1400 84 0 days to >3 months 13-18 days 440 ± 30 (>1 yr) 5820±80 (>1 yr) 5200 ± 190 (>3 month)

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Event E 3 Rural festival High: 75, 75, 81 Low: 54, 55, 57 2900 210 0 days to >3 months ≤ 1 day 510 ± 80 (>1 yr) 5900±90 (>1 yr) 5150 ± 540 (>3 month)

Event F 1 University High: 61 Low: 32 200 13 0 days to 2 months ≈ 2 weeks 850 ± 210 (>1 month) 6090 ± 370 (>1 month) 5400 ± 280 (>1 month)


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Bacteria concentrations and viability. Both SYBR gold staining and hemocytometer counts revealed that the concentrations of total

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bacteria in fresh urine samples varied, but consistently increased to 5 ± 2 × 108 cells/mL within

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one to two weeks of storage in all samples (Figures 1a & SI-3). Three metrics were used to

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examine whether living bacteria remained after storage and/or pasteurization, including culturing

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on a variety of growth media, live/dead staining, and XTT cleavage assays. In terms of

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culturability, fresh urine samples displayed bacterial growth on LB, BHI, and E. coli on EMB

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media. After storage, no E. coli growth was observed on EMB media; however, even after

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several months of storage, organisms from urine grew on LB and BHI media. Since culturing

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neglects viable but nonculturable organisms, more effort was placed on live/dead assays and

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XTT cleavage assays than on culturing. Live/dead staining of all urine samples, including

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pasteurized samples, demonstrated that bacteria with intact membranes remained after three

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months of storage and, hence, may be viable (Figure SI-4). The XTT cleavage assay results

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showed that pasteurization reduced metabolic activity in urine by 50 ± 10 % (n = 4; Figure 1b).

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Storage after pasteurization for up to 167 days did not lead to an increase in metabolic activity. Collectively these results demonstrate that some bacteria can survive urine storage, that the

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number of viable bacteria is significantly reduced by pasteurization at 80 ºC for 30 minutes, and

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that storage after pasteurization does not lead to significant regrowth of bacteria.


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Figure 1. A) Bacterial counts in urine with SYBR gold staining and hemocytometer counts. The storage time (day 0) was initiated at the end of the public collection event when toilets were pumped into a storage tank. B) Bacterial viability of unpasteurized and pasteurized urine samples from Events D and E was measured using an XTT cleavage assay. The length of time between urine pasteurization and the time of the XTT assay is represented on the x-axis. The control consists of autoclaved E. coli K12. Error bars in both panels represent the standard deviation of triplicate samples taken from a single urine storage tank.

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Storage changes the diversity of bacterial communities in urine.

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Historically, and based on culturing methods, clinicians have assumed that clean-catch urine was

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sterile unless the individual had a urinary tract infection (UTI). However, numerous studies have

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now shown that this is not the case.17,18,35 Urine collected by clean-catch in healthy adults is

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typically composed of approximately 240 OTUs and is dominated by a few orders

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(Actinomycetales,

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Pseudomonadales), with gender influencing taxa abundance.17,18,35 Comparing these clinical

Bacteroidales,

Clostridiales,

Enterobacteriales,


Lactobacillales

and


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results to the aggregate urine samples collected in this study, similar taxonomic orders occur in

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the source-separated fresh urine as in clinical samples (Figure 2). Between 181 and 611 OTU’s

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(species-level (97% identity)) were observed in the fresh urine samples, with all samples having

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marginally even community structures (Pielou evenness value of 0.49 ± 0.09).

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Figure 2. The top 30 most abundant OTUs observed in fresh (left) and hydrolyzed (right) source-separated urine are color coded by genus. Relative abundance is plotted vs OTU order for each urine collection event. A simple bar chart of the top 10 OTUs for fresh urine and hydrolyzed urine is shown in Figure SI-6.

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Compared to fresh urine, hydrolyzed urine that had been stored for long periods of time (over 80

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days) was dominated by Clostridiales and Lactobacillales (Figure 2), contained fewer OTUs

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(130 ± 70), had a more even community structure (Pielou evenness value of 0.60 ± 0.06), and

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exhibited less variability between events (Figure 3 & SI-5). These diversity metric correlations

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are consistent with those found in other extreme environments where typically few organisms

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survive.36 Therefore, it is not surprising that there is low species richness in the high pH (>9) and

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ammonia (> 5 g/L) environment of hydrolyzed urine. Overall, fresh and hydrolyzed urine are

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dominated by different bacterial orders. Among the genera detected, only Bacteroides,


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Enterococcus, and Erysipelothrix occur in the top 30 OTUs for both fresh and hydrolyzed urine

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that was stored over 80 days (Figure 2).

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Figure 3. Diversity metrics of urine bacterial communities according to urine age and urine source for the rarefied 16S rRNA gene sequencing dataset. Additional diversity metrics are plotted in Figure SI-5.

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All six aged urines from different starting materials had consistent characteristics after > 80 days

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of storage, including 5 ± 2 × 108 cells/mL, reduced number of observed OTUs to 130 ± 70,

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increased Pielou evenness to 0.60 ± 0.06, communities dominated by Clostridiales and

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Lactobacillales, and viable organisms remaining that could cleave XTT. This suggests that the

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urines are changing in a similar way. Additional work must be conducted to determine if this

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trend holds true with different collection systems (e.g., waterless urinals, urine-diverting toilets,

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etc.) and at locations outside the U.S.

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Shifts in urine chemistry during storage correlate with bacterial community composition.

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A transition in community composition occurred as pH increased and eventually exceeded 9

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(Figure 4a). Canonical correspondence analysis (CCA) was applied to statistically resolve the

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factors shaping the bacterial community in the urine samples. The environmental and sampling

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parameters used in the CCA were urine age (number of days old), the urine collection event, pH,

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time over pH 9, and urine classification (young, hydrolyzed < 80 days, and hydrolyzed > 80

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days). Each of these factors significantly impacted the community structure, with urine age and

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pH being the major drivers of the bacterial community structure (Figure 4b; Table SI-2). This

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finding is consistent with previous research that found a positive correlation between pH and

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ammonia concentrations with the removal of certain bacteria.7,11,37

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292 293 294 295 296 297 298 299 300 301

Figure 4. Urine bacterial communities analyzed over storage time. A) Order of the 20 most abundant OTUs in source-separated urine at various storage times in three urine collection events. Day zero represents the transition of sample pH to > 9. Black arrows indicate the day of urine collection for each event. B) Canonical-correlation analysis (CCA) plot with biplots (solid black arrows) showing significant variables that influence community structure (stress value = 0.11). Colored, dotted arrows indicate urine aging, with arrows pointing from the freshest sample to an older sample. The classification of “young” implies pH less than 9 and “hydrolyzed” signifies pH over 9.

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Adonis analysis confirmed that significant differences existed in the bacterial communities

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between urine classified as young, hydrolyzed < 80 days, and hydrolyzed > 80 days (p-value =


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0.001). From the time when urine was over 80 days old, there was little or no statistical

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difference in community composition amongst samples (p-value > 0.05). In other words, urine

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bacterial communities converged from this time point onwards (Figure 4b). Lefse and indicator

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analyses identified 243 and 155 OTUs respectively that discriminated between fresh urine (< 24

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hours) and converged urine (hydrolyzed and over 80 days old; Table SI-3). In particular, Lefse

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analysis linked fresh urine with various organisms that are associated with UTIs and with enteric

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pathogenic organisms, such as Escherichia, Shigella, Proteus, Providencia, and Aeromonas

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(Table SI-3, LDA score above 4). The presence of these organisms in fresh urine is not

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unexpected; fecal contamination or donors with asymptomatic or symptomatic UTIs are likely

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within the large sample sizes. Importantly, several human health associated groups were

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successfully removed by storage over 80 days, including Staphylococcus, Lactobacillus and

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Prevotella (Table SI-3). Fewer OTUs (27) were associated with converged urine based on the

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Lefse and indicator analyses, and many of those OTUs were members of the Firmicutes phylum

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(Figure 4, Table SI-3). The dominance of Firmicutes is consistent with studies performed in

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other harsh alkaline environments such as soda lakes38 and with culture-based studies that have

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shown Clostridium can survive in the presence of chemical stressors such as those in hydrolyzed

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urine.12,39,40

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There are several veterinary and human pathogenic genera within the Firmicutes OTUs detected

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in converged urine, including Clostridium sensu stricto, Enterococcus, Erysipelothrix, and

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Tissierel (Figure 2, Table SI-4). Future research should assess if pathogenic strains are present

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in these populations, if the organisms are alive, and if they can regrow when applied as fertilizer.

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These results highlight issues with the use of enteric indicator organisms to assess the bacterial

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risks involved with urine derived-fertilizer products. For example, traditional indicator groups

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such as Streptococcus and Escherichia decreased very quickly during urine storage (Figure SI-

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7) and hence would indicate that stored source-separated urine posed a low risk, despite the fact

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that many potentially pathogenic OTUs (Morganella, Pseudomonas, Tissierella, Erysipelothrix,

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or Atopostipes) are still present. It should be noted that organisms in the genera that persisted in

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source-separated urine also exist in the gastrointestinal tract of healthy adults (genera

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Clostridium, Lactobacillus, and Bacteriodes), many of which are important for normal gut

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function.41



335 336

Pasteurization reduced metabolic activity, but did not alter community composition.

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Pasteurization was applied to hydrolyzed urine that had been stored for over 80 days, and thus to

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a bacterial community that had converged. Urine containers were sealed during pasteurization,

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and total ammonia nitrogen analysis verified that 93-95% of total ammonia nitrogen was retained

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in stored urine after pasteurization. No difference was detected in the relative abundance of taxa

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based on 16S rRNA genes immediately after pasteurization (Figure SI-8). A similar result was

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seen when milk samples were sequenced immediately after pasteurization and was likely due to

343

the DNA of inactivated bacteria in the samples.42 In contrast, XTT cleavage assays showed a

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reduction in metabolic activity within five days of pasteurization (Figure 1b). Therefore,

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pasteurized samples were stored for two to ten additional weeks at room temperature and

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reanalyzed. Metabolic activity did not increase back to pre-pasteurization levels with additional

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storage after pasteurization. Interestingly, no significant difference in community composition

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was detected at any levels of classification by Adonis analysis, even when samples were stored

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two to ten weeks after pasteurization (Figure 5). Additional research is needed to evaluate

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changes in organism function before and after pasteurization (i.e., analysis of RNA instead of

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DNA) and potential regrowth of organisms upon application to soil as fertilizer (Figure SI-9,

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Table SI-5).

353

354 355 356 357

Figure 5. Bacterial community after pasteurization. Three sets of Event E urine samples were pasteurized and left to age at room temperature for an additional 16, 57, or 70 days before 16S rRNA gene analysis.

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Struvite precipitation did not alter community composition.


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Like pasteurized samples, the bacterial community structure of precipitated struvite from Event F

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was similar to that of hydrolyzed urine. The most drastic difference between the struvite and

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hydrolyzed urine communities was the increase in the relative abundance of Pseudomonadales

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(Figure 6), the vast majority belonging to Pseudomonas. This is similar to what has been

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observed in the microbial community of kidney stones, which are composed of a struvite-like

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material.43 Although the survival of bacteria has not been previously studied through struvite

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precipitation, Ascaris suum eggs and bacteriophage ΦX174 were shown to survive struvite

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production.44 In that report, the parasite and virus survivals were influenced by the struvite

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drying process. Our results represent one batch of struvite production; therefore, further analysis

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is required to determine the statistical significance across different starting urines and the

369

viability of the remaining organisms.

370

371 372 373 374

Figure 6. The bacterial community was sequenced through each stage of urine processing for struvite production for urine from Event F after 1 month of storage.

375

FUTURE OUTLOOK

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The urine studied here was collected over one to three days and subsequently stored. In practice,

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urine-diverting systems often collect urine over weeks to months in a single tank, which may

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impact both the organisms inoculated in the fresh urine and the time it takes for a community to

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converge to the stored urine community reported here. Furthermore, this research used waterless

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collection systems, whereas low flow urine diversion systems add a small amount of water, thus

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decreasing ammonia concentrations. Previous work suggests that enteric bacteria survive for



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longer periods of time at lower ammonia and pH levels.7,11 Future work will need to explore how

383

differences in urine collection systems, as well as geography and local public health impact the

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bacterial communities in fresh and stored urine.

385 386

As researchers continue to develop urine-derived fertilizer products that rely upon storage as

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defense against potential pathogens, we recommend making a practice of using aged urine with

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converged bacterial communities. In this way, urine products have more predictable bacterial

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content. Increased sampling up to three months of storage may demonstrate that less than 80

390

days of storage is required for convergence of bacterial communities.

391 392

As demonstrated previously, E. coli and Streptococcus decreased consistently in hydrolyzed

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urine. These organisms, along with Shigella, Proteus, Providencia, Aeromonas, and

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Lactobacillus, rarely occur in hydrolyzed urine over 80 days old; therefore, urine treatment

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through storage was valuable. Tissierella, many genera of Clostridiales, Erysipelothrix,

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Atopostipes, Bacteroides, and the family Pseudomonadales consistently persisted in urine-

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derived fertilizers and should be further studied to determine whether pathogenic species are

398

present, which organisms are responsible for the observed metabolic activity, and which

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organisms regrow when applied to soil. If the converged urine is found to contain pathogens,

400

then organisms such as Pseudomonas, Morganella, or pathogenic Clostridiales will be more

401

conservative measures of risk than E. coli or Streptococcus. To expand on this work, additional

402

research on the viral and protozoan communities in urine will be necessary to understand all

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potential microbial risks associated with urine-derived fertilizers.

404 405 406

ACKNOWLEDGEMENTS

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We acknowledge our funding sources, including an EPA and Water Environment Research

408

Foundation grant (STAR N1R14), a University of Michigan Dow Sustainability Postdoctoral

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Fellowship to R.H.L., and a University of Michigan Researching Fresh Solutions to the

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Energy/Water/Food Challenge in Resource Constrained Environments grant (REFRESCH). We

411

also acknowledge Rick Burch for struvite reactor construction as well as Saloni Dagli, Mariah

412

Gnegy, and Lauren Eastes for lab work.


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SUPPORTING INFORMATION AVAILABLE

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Details regarding sample collection and analysis, pH versus urine age, cell counts, live/dead

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images, diversity metrics, relative abundance in fresh and hydrolyzed urine, CCA, indicator and

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LEfSe analysis, pathogenic potential, relative abundance of specific genera or families, and

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relative abundance of pasteurized urine are included in the SI. This information is available free

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of charge via the Internet at http://pubs.acs.org.

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