Evaluating Microbial and Chemical Hazards in Commercial Struvite

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Ecotoxicology and Human Environmental Health

Evaluating Microbial and Chemical Hazards in Commercial Struvite Recovered from Wastewater Rachel A. Yee, Mats Leifels, Candis Scott, Nicholas J. Ashbolt, and Yang Liu Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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Environmental Science & Technology

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Evaluating Microbial and Chemical Hazards in Commercial Struvite

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Recovered from Wastewater

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Rachel A. Yee1, Mats Leifels2, Candis Scott3, Nicholas J. Ashbolt4, Yang Liu5,*

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Contact info:

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1 Department

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Alberta, Canada, T6G 2R3, [email protected]

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2 Centre

of Civil and Environmental Engineering, University of Alberta, Edmonton,

for Water and Environmental Research (ZWU), University Duisburg-Essen, Essen,

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Germany and School of Public Health, University of Alberta, Edmonton, Alberta, Canada, T6G

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2R3, [email protected]

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3

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[email protected]

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4 School

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[email protected]

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5 Department

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Alberta, Canada, T6G 2R3, [email protected]

School of Public Health, University of Alberta, Edmonton, Alberta, Canada, T6G 2R3,

of Public Health, University of Alberta, Edmonton, Alberta, Canada, T6G 2R3,

of Civil and Environmental Engineering, University of Alberta, Edmonton,

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*Corresponding author: Yang Liu

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Address: 116 St & 85 Ave, Edmonton, AB T6G 2R3

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Email: [email protected]

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Telephone: 780-492-5115

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1 ACS Paragon Plus Environment


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Submitted to Environmental Science & Technology for Consideration for Publication as a

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Research Article

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Rationale

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Wastewater recovered struvite is not a new process for recovery of residual phosphorus and

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nitrogen evidenced by the development of several commercial operations worldwide. However,

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these processes were developed and optimized with the main goal being nutrient recovery. Very

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little is published on potential co-contaminating hazards with recovered struvite, such as heavy

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metals and antibiotic-resistant genes/bacteria, for which there is increasing public health concerns

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generally associated with wastewaters. The purpose of this study was to show that there is a public

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health aspect that needs further evaluation in addition to optimization processes to generate a safe

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product.

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The authors confirm that the all graphics, including the TOC was created by the authors and has

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not been previously published.


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Abstract

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Controlled struvite (NH4MgPO4·6H2O) precipitation has become a well-known process for

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nutrient recovery from wastewater treatment systems to alleviate the pressures of diminishing,

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finite rock phosphate reservoirs. Nonetheless, co-precipitation of potential microbial and chemical

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hazards is poorly understood. On the other hand, antimicrobial resistance (AMR) is a major global

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public health concern and wastewater is thought to disseminate resistance genes within bacteria.

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Fecal indicator bacteria (FIB) are typically used as measures of treatment quality and with multi-

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resistant E. coli and Enterococcus spp. rising in concern, the quantification of FIB can be used as

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a preliminary method to assess the risk of AMR. Focusing on struvite produced from full-scale

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operations, culture and qPCR methods were utilized to identify FIB, antibiotic resistance genes

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and human enteric viruses in the final product. Detection of these hazards occurred in both wet

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and dry struvite samples indicating that there is a potential risk that needs further consideration.

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Chemical and biological analyses support the idea that the presence of other wastewater

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components can impact struvite formation through ion and microbial interference. While heavy

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metal concentrations met current fertilizer standards, the presence of K, Na, Ca and Fe ions can

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impact struvite purity yet provide benefit for agricultural uses. Additionally, the quantified hazards

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detected varied among struvite samples produced from different methods and sources, thus

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indicating that production methods could be a large factor in the risk associated with wastewater-

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recovered struvite. In all, co-precipitation of metals, fecal indicator bacteria, antimicrobial

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resistance genes and human enteric viruses with struvite was shown to be likely and future

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engineered wastewater systems producing struvite may require additional step(s) to manage these

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newly identified public health risks.



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Introduction

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Phosphate rock is the primary but finite source of phosphorus used in agricultural production1 in

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which alternative sources need to be identified to sustain humanity.2 In the future, industries such

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as agriculture are expected to become limited by phosphorus availability and wastewater treatment

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plants (WWTPs) are currently point sources contributing to eutrophication through the release of

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excess nutrients (phosphorus and nitrogen) within effluent streams.3 While initial wastewater

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studies addressed struvite (NH4MgPO4·6H2O) scale blockages of pipes and fixture,4 the focus has

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since shifted to examining methods for maximum struvite precipitation for economic benefits in

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regards to nutrient recovery.5 Hence, integrating nutrient recovery methods into existing WWTPs

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has the potential to recover otherwise wasted resources, thus providing a solution to alleviating

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receiving water nutrient pollution as well as gaining recoverable nitrogen and phosphorus for use

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in agriculture.6

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Extensive research has been conducted on struvite precipitation from various waste streams to

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optimize recovery conditions and to date there are several commercial processes operating to

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recover nutrients from wastewater.7–9 Worldwide, the Airprex® Process, the Pearl® Process, the

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PHOSPAQ™ process and the NuReSys® Process are currently operating, each with a different


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process mechanism. Other notable commercial processes in Asia include the Phosnix® Process

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in Japan (Unitika Ldt.) and the Crystalactor® in China (DHV).

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Though the process has been commercialized, a limited number of studies have been conducted

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that examine the potential co-precipitation occurrence of the recovered products. Wastewater is a

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reservoir for many potential hazards, such as enteric pathogens, heavy metals and organics of

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emerging concern that could co-precipitate and pose a threat to public health following the

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application of struvite fertilizer.10–12 Chemical hazards of potential concern include heavy metals,

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various endocrine disrupting organics and pharmaceutical residuals. Various macro- and micro-

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nutrients (including Ca2+, Na+, K+, Fe2+, Zn2+ and Mg2+) may also be problematic, as they could

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interfere with struvite crystallization. In particular, Ca2+ has repeatedly been shown to be a

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competitor with Mg2+ in the formation of struvite, resulting in calcium phosphate and carbonate

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products.13

93 94

Typical microbial parameters for WWTP effluent discharges include fecal indicator bacteria (FIB),

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such as Escherichia coli and enterococci.14 While Enterococcus spp. can be more resilient to

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environmental stressors than E. coli,15 most regulations for wastewater discharge are currently

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based on coliforms (e.g. not to exceed 200/100 mL).16 Spore-forming FIB, such as Clostridium

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perfringens may also be important due their spore’s greater resistance to disinfection/treatment

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than vegetative FIB cells.15,17 WWTPs have also been identified as potential reservoirs for

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antimicrobial resistant bacteria (ARB) and antimicrobial resistance genes (ARGs)18 including their

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FIB,19,20 which are considered a significant hazard to manage.21 Though naturally found in the

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environmental microbiota, increasing levels of ARB and ARGs are being detected worldwide in



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association with engineered water systems22 and thus has topped the WHO concerns list of the

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decade.23 Mobile genetic elements (such as class 1 integrons) are important for horizontal gene

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transfer (HGT) of ARGs between different bacterial species within the aquatic environment and

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biofilms.18,22,24 HGT may be more evident within WWTPs as the system provides a means of

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selective pressures that favour the transfer of ARGs to indigenous bacteria.18,25 However, the lack

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of antibiotic, ARB and ARG regulations in wastewater effluents results in an unclear view of the

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target levels required to reduce potential downstream impacts.26 It is only recently that ARB and

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ARGs within recovered struvite have been examined,1,27 where evidence of wastewater-derived

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struvite can increase the ARG levels in soil, the rhizosphere and phyllosphere following

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application as fertilizer.1 The occurrence of residual antibiotics in recovered struvite is not well

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characterized, however, as current WWTPs do not efficiently remove all residual antibiotics,28

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chemical interactions of the matrix also increase the likelihood for detection in recovered struvite.

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Hence, with residual antibiotics, AMR bacteria and ARGs could potentially end up in struvite and

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contribute to the growing issues of AMR via the environment.29–31

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Human enteric viruses related to waterborne outbreaks could also be a concern in nutrient recovery

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from wastewater.32,33

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adenoviruses (HAdVs), both associated with acute gastrointestinal infections in children and adults

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via environmental routes,34,35 along with Enterovirus (EV) members,36 which can lead to various

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chronic diseases.37 While WWTPs may reduce viral loads to receiving environments, persistent

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virions,38–40 such as noroviruses have been associated with spinach crop outbreaks from reclaimed

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wastewater uses.41 Furthermore, analysis of a human virus surrogate (somatic bacteriophage

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ɸX174) in struvite recovered from urine shows that inactivation and infectivity can be affected by

These viruses include noroviruses GI and GII (NoVs) and human


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moisture content.12

The study12 suggested post-processing methods involving high drying

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temperatures and storage to help further reduce infectious virions in struvite, although it was based

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on testing of virus surrogates whose behavior and characteristics in the environment may differ

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from human enteric viruses.42 While likely abundant in the source material, the presence of EVs,

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HAdVs and NoVs have yet to be reported in struvite. Even though viruses require a host to

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reproduce, their high persistence towards disinfection methods and low infectious dose make them

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a highly relevant hazard group for quantitative microbial risk assessment (QMRA) of residual

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pathogen risk associated with wastewater-recovered struvite. Hence, to provide initial scoping

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data to assess potential risks with agricultural use of recovered struvite, the current study focused

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on yields of plant nutrients and potential metal toxins43 as well as human pathogens and selected

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AMR genes in precipitated struvite.

137 138

Materials and Methods

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Struvite Samples

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Commercial struvite samples that achieved phosphorus recovery greater than 85% were obtained

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from a decentralized treatment system utilizing blackwater (toilet flush water collected from a

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vacuum collection system that was treated by anaerobic digestion and then an anammox reactor)

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(Sample 1), and from a full-scale system utilizing a side-stream (wastewater generated from

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anaerobic digestion of sewage sludge) from a municipal wastewater source (Sample 2).

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Commercial struvite Sample 1 was received as a wet, brown slurry sent in a plastic bottle and

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stored at 4-8 °C. Subsamples were stored in 10 % glycerol at -80 °C until analysed as the sample

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did not appear to dissolve in glycerol and the microbial community could be preserved.

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Commercial struvite Sample 2 was received as a dry, white commercial product and was stored in



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a sterile plastic bag at room temperature in the dark until analysed. Due to the sensitivity of the

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process, details of struvite production mechanisms cannot be disclosed, however, measured

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parameters of the wastewater sources are provided in the supporting information (Table S1).

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For comparison purposes, lab-produced struvite (Sample 3) was synthetically prepared in a 1 L

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sterile glass bottle using 700 mL of sterile distilled H2O containing 600 mg/L MgSO4 and 550

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mg/L (NH4)2HPO4 (from 1 M sterile stock solutions) and was adjusted to pH 9 with sterile 1 M

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NH4OH. The preparation was mixed with a stir bar at 400 RPM for 15 min at room temperature,

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then decreased to 200 RPM for 1 h to allow for precipitation. Wet precipitates were recovered

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using a 0.45 µm glass fiber filter (Sigma-Aldrich, USA) and dried at 150 °C overnight. Higher

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drying temperatures were required as lower temperatures resulted in microbial replication that

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interfered with examining microbial presence in struvite, a main goal of the study. The white

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synthetic struvite recovered was stored in a sterile, air-tight glass vial at room temperature in the

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dark until analysed and was used as a control.

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Chemical Composition of Struvite Samples

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Samples were analyzed via X-ray diffraction (XRD; Rigaku Ultimate IV, Japan), X-ray

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photoelectron spectroscopy – for Samples 1 and 2 only (XPS, Kratos AXIS Ultra) and a scanning

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electron microscope (SEM; Zeiss Sigma 300 VP-FESEM, USA) with energy dispersive X-ray

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spectroscopy (EDS; Bruker EDS system, USA). The peaks of the XRD figures were compared to

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the Inorganic Crystal Structure Database (ICSD) for confirmation of struvite using the following

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cards - PDF #97-006-0626 and PDF #98-000-0419. CasaXPS and the Handbook of X-ray

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Photoelectron Spectroscopy44 were used as confirmation of the chemical bonds present on the


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surface of the samples following calibration of peaks in reference to carbon binding energy of

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282.4eV. EDS analysis was based on point analysis of the SEM images and a series of six images

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were taken for each sample. For the above analysis, Sample 1 was oven-dried at 150 °C overnight.

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The struvite samples were analyzed for metals via ICP-OES (Thermo iCAP 6000 series, Thermo

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Scientific, USA). Dry samples (0.04-0.07 g) were digested in 5 mL of trace metal grade nitric acid

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(Thermo Fisher Scientific, USA) following Method 3051A as set by the U.S. EPA.45 Digested

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samples were diluted to a volume of 25 mL with distilled water prior to ICP-OES analysis in

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triplicate. The results of the ICP-OES analysis were converted to microgram of metal per gram of

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struvite for ease of comparison between samples.

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Fecal Indicator Bacteria, Enteric Viruses and ARG Detection by qPCR

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For bacterial and ARG gene detections, DNA was extracted from 0.25 g of Samples 1 (wet) and 2

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(dry) using the PowerSoil DNA extraction kitTM (Qiagen, Germany) following the manufacturer’s

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protocol. Genes chosen for analysis were based on the interest for targeting ARB as outlined in

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the WHO’s list of public health concerns.23 As measures for total E. coli, Enterococcus spp. and

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C. perfringens, qPCR methods to detect uidA, entero1 and cpn60 gene targets were developed and

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optimized, respectively (Table 1). By reviewing the most prevalent ARGs in wastewater sources,46

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tetA and vanB assays were developed, based on a wild E. coli isolate and E. faecalis ATCC 51299.

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The class 1 integron gene, intI1, commonly associated with gene transfer was also developed using

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a wild E. coli isolate (primers and probes given in Table 1). In total, DNA was extracted from

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three different samples of each struvite source and samples assayed by qPCR in duplicate. Wild

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type E. coli and E. faecalis ATCC 51299 isolates were added to determine DNA extraction



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recovery. Results of Sample 1 are shown as per wet weight due to the DNA extraction from a wet

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sample without prior drying.

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To detect enteric viruses, total nucleic acids from six different 0.25 g of Sample 1 (wet) were

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extracted using the DNeasy Blood and Tissue Kit (Qiagen, Germany) according to manufacturer’s

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protocol. The kit was chosen due its ability to remove co-concentrated inhibitors as previously

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described by Hamza et al.47,48

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complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit (Thermo

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Fischer, USA) according to the manufacturer’s protocol.

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cell cultures were added prior to whole genome extraction.39 Additionally, an internal

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amplification control was added to quantify the influence of inorganic inhibitory substances on

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amplification efficiency. The extraction efficiency from bacterial and virus extraction was

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determined to be between 50-75%. All primer/probe sets used are described in Table 1. The

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development of standards and qPCR assay conditions can be found in the supporting information.

For EV and NoV GII, extracted RNA was converted to

Known concentrations of HAdv 40/41

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Table 1. List of primer and probe sequences used for qPCR assays for bacteria, ARG and virus

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detection. Target

E. coli

Primer/Probe

Sequence

Amplicon Reference

(5’  3’)

size (bp)

uidA - F

CGC AAG GTG CAC GGG AAT A

143

49

uidA - R

CAG GCA CAG CAC ATC AAA GAG A

uidA - P

[FAM] ACC CGA CGC GTC CGA TCA CCT

90

50

[MGBNFQ]

Enterococcus

entero1- F

GAG AAA TTC CAA ACG AAC TTG


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spp.

entero1 - R

CAG TGC TCT ACC TCC ATC ATT

entero1 - P

[FAM] TGG TTC TCT CCG AAA TAG CTT TAG GGC TA [TAMRA]

C. perfringens

cpn60 - F

AAA TGT AAC AGC AGG GGC A

(validated

cpn60 - R

TGA AAT TGC AGC AAC TCT AGC

with wild

cpn60 - P

[FAM] ATG TCT TCT TTT CCA TTT ACA GGC

139

This study

71

This study

69

This study

69

This study

TTA GAA [MGBNFQ]

C. perfringens isolate) Tetracycline

tetA – F

CCT GCC TGG ACA ACA TTG CT

(validated

tetA – R

CCC GAT CAT GGT CCT GCT T

with wild

tetA - P

[FAM] CAT TCC GAT GCC ACC C [MGBNFQ]

Vancomycin

vanB - F

ACG GTC AGG TTC GTC CTT TG

(validated

vanB - R

GCT TCT ATC GCA GCG TTT AGT TC

with ATCC

vanB - P

[FAM] CGT AAC CAA AGT AAA CAG TAC G

E. coli isolate)

[MGBNFQ]

E. faecalis 51299) Class 1

intI1 – F

GCC GAG GTC TTC CGA TCT C

Integron

intI1 - R

TGC TGT TCT TCT ACG GCA AGG T

(validated

intI1 - P

[FAM] CAG GGC AGA TCC GTG CA

with wild

[MGBNFQ]

E. coli isolate)



Internal

IAC - F

CTA ACC TTC GTG ATG AGC AAT CG

Amplification

IAC - R

GAT CAG CTA CGT GAG GTC CTA C

Control

IAC - P

[VIC] AGC TAG TCG ATG CAC TCC AFT CCT

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200

51

89

52,53

139

54

178

48

CCT [MGBNFQ]

Norovirus

QNIF2

ATG TTC AGR TGG ATG AGR TTC TCW GA

GII

COG2R

TCG ACG CCA TCT TCA TTC ACA

(NoV GII)

QNIFs

[FAM] AGC ACG TGG GAG GGC GAT CG [TAMRA]

Human

AQ1

GCC ACG GTG GGG TTT CTA AAC TT

Adenoviruses

AQ2

GCC CCA GTG GTC TTA CAT GCA CATC

(HAdV)

AdVP

[Hex]-TGC ACC AGA CCC GGG CTC AGG TAC TCC GA-[BHQ1]

Enteroviruses

EV 444

CCT CCG GCC CCT GAA TG

(EV)

EV 621

ACC GGA TGG CCA ATC CAA

EV P

[FAM] ACG GAC ACC CAA AGT CGG TTC CG [BHQ1]

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Viability/Infectivity Assays for E. coli, Enterococcus spp. and Human Enteric Viruses

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Fresh, wet samples of Sample 1 were inoculated into Colilert and Enterolert (IDEXX, USA) media

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and quantified using Quanti-Tray® 2000 according to manufacturer’s protocols and detection limits

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of one most probable number (MPN) per 100 mL to estimate culturable E. coli and Enterococcus

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spp. concentrations, respectively.

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Enterococcus spp. viability, the plate method using CM0587 Perfringens agar base (TSC & SFP)

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media (Oxoid, USA) with 85 mg/L 4-methylumbelliferyl phosphate (Invitrogen, USA) was used

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to quantify C. perfringens via colony forming unit (CFU) counts. For plate counts, 1:10 serial

While MPN counts were used to quantify E. coli and


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dilutions of fresh, wet samples of Sample 1 were conducted. Plates were incubated in an anaerobic

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box at 37 °C overnight.

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For enteric viruses, infectivity was determined based on an adapted capsid-integrity (ci-) PMA-

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qPCR as previously described.40,55 Furthermore, the culture-based integrated cell culture (ICC)

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qPCR using buffalo green monkey (BGM) cells was utilized to assess the infectivity of HAdV and

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EV according to Hamza et al.48 For ci-qPCR, a stock solution of PMA (Biotium Inc, USA) was

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reconstituted with 20 % dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) to a final concentration

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of 10 mM and stored at -20 °C. A working solution of 1 mM was prepared in aliquots (to avoid

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freezing and thawing). The PMA pre-treatment was undertaken using a modified protocol56 as

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follows; PMA in the working solution was added to 200 µL of samples up to a final concentration

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of 0.04 mM per reaction, mixed gently and incubated in the dark for 30 min at room temperature

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to allow the dye to penetration into virions with permeable capsids. Afterwards, samples were

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exposed to daylight for 15 min to enable photo-induced crosslinking of the reagent before 15 min

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exposure to a 4-Watt LED lamp to develop irreversible and polymerase inhibiting azo-dye genome

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complexes. HAdV whole genome was added as an internal control and showed qPCR signal

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reduction between 4-6 log10 for each RT-ci-qPCR run (data not shown).

238 239

Results and Discussion

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Struvite Sample Morphology

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The presence of struvite in Samples 1-3 was confirmed by XRD analysis (Figure 1). Utilizing the

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Inorganic Crystal Structure Database (ICSD), the observed peaks aligned with crystalline struvite

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as indicated by the red dots in Figure 1, however the intensity of the peaks differ (quantitative data



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shown in supporting information Figure S1) While the peak positions correspond with the

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diffraction angles calculated by Bragg’s law57, the peak intensities differed between the samples,

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likely due to the X-ray absorbance and different sample volumes used in the analysis.57 Though

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co-precipitates are indicated by the mineral analyses (described later), these were not detected by

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XRD, presumably due to relatively low levels and the crystalline structure of struvite. Regardless

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of the process utilized and the wastewater source, struvite was efficiently recovered and phase

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identified.

251 252 253 254 255 256 257 258 259 260 261 262

Figure 1. XRD analysis of Sample 1 (commercial struvite), Sample 2 (commercial struvite) and

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Sample 3 (lab produced struvite)

264 265

The morphology described by SEM imaging shows the orthorhombic shaped crystal of Sample 1

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with an approximate size of 120 µm (Figure 2). However, as seen in the left-hand corner of the

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Sample 1 image, there also appeared to be an amorphous, non-struvite structure, which was 12 ACS Paragon Plus Environment

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obscured by the large struvite crystalline structure during XRD. Sample 2 consisted of larger (few

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mm), round crystals, which were fractured with a mortar and pestle into smaller pieces prior to

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undertaking SEM analysis, thus presenting a rougher surface appearance. Sample 3 differed in

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structure to the commercial struvite products as crystal twinning was observed. This is likely due

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to equal crystal growth in two directions under the laboratory controlled conditions.58

273 Sample 1

274

Sample 2

Sample 3

Figure 2. SEM images of Sample 1-3 at 1000x magnification.

275 276

Previous descriptions of crystalline struvite describe dendritic and orthorhombic crystal

277

structures,59–61 with several factors accounting for different crystal morphologies. In addition to

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the chemical factors that impact induction, physical factors, such as mixing speed, time and

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presence of a seed have been suggested factors to impact crystal growth. Mixing speed and time

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have been shown to alter the ion interactions within the matrix, thus playing a role in controlling

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crystal formation and growth.4 Additionally, seed materials with a lower specific gravity have been

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shown to play a role in the surface area and particle number available for nucleation; however,

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there will be an optimal seed amount associated with maximum phosphorus recovery.59 Thus,

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varying processes used for recovery will ultimately impact nucleation and crystal formation.

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Hence, the different shaped crystals are not surprising given the different conditions and feeds



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used to form the three struvite products examined.

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differentially embed during crystallization and/or form separate amorphous co-precipitates.

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As a result, chemical hazards could

288 289

Metal co-precipitation

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XPS analysis was performed for surface analysis of Mg, P, C, O and N as the likely major elements

291

(supporting information Figure S2), thus co-precipitates could not be directly detected using XPS.

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The resulting binding energies in comparison to the database confirmed the main N peaks at

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399.6eV (Sample 1) and 400.4 eV (Sample 2) as ammonia, while the P peaks at 132.4 eV (Sample

294

1) and 133.4 eV (Sample 2) were representative of phosphate.44 The oxygen peaks of both samples

295

overlap with binding energies that match with phosphates, required for struvite formation, as well

296

as carbonates that could account for potential co-precipitation.

297 298

Further surface chemical analysis by EDS showed that O, Mg and P were the predominant

299

elements detected, which is in line with the struvite chemical composition. Additionally, the

300

detection of C was omitted from the calculated relative mass percentage as it is often detected as

301

an artefact from the carbon paper used in the analysis. The observed oxygen detection is likely due

302

to the hydrate required for struvite formation. Specifically, the O:N:Mg:P:K ratio detected in

303

Sample 1 was 30:1.25:8.5:8.5:1, 30:2.5:7.5:9.5:0.025 in Sample 2 and 31.5:1:8:9:0.025 in Sample

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3. The potassium detected in Sample 1 could be due to very low levels of potassium struvite, where

305

K+ replaces NH4+ in the crystal structure.62 Trace amounts of Na, Ca and Cl were also detected in

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all samples further supporting the likelihood of co-precipitation. Additionally, EDS should not

307

solely be relied on as a quantification method as the measurements reported above are based on

308

relative mass percentage and only highlights the surface chemical composition. Despite this, the


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relative ratio of elements can be compared and detection of the abovementioned metals are

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expected due to the complex matrix of wastewater. Overall, the results supports the argument that

311

co-precipitation does occur during struvite precipitation and should be more carefully considered

312

in future struvite production intended as fertilizer.

313 314

Metals were further investigated using ICP-OES to confirm that elemental ions other than Mg and

315

P were present in the struvite samples (Figure 4).

316

similar, which is expected if struvite is the main product (given its 1:1 molar ratio of Mg:P). The

317

dominant metals observed in the commercial samples included Na, Ca, K and Fe. Other metals

318

that were detected include Zn, Cu, Mn and Cr, with only trace amounts of As, Cd, Pb and Ni.

319

Higher traces of micro- and macronutrients compared to Samples 2-3 were detected in Sample 1,

320

presumably due to the concentrated blackwater source. Precipitation from a municipal wastewater

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source, as in Sample 2, versus a blackwater source would be expected to result in a large dilution

322

factor so reducing metal concentrations. Sample 3 was produced from sterile stock chemical

323

solutions and therefore the detection for the above mentioned metals were well below the limit of

324

detection (0.001 – 0.019 µg metal/g of struvite), thus not illustrated in Figure 4.

The ratios of P and Mg for each sample were

325 326



328 329 330 331 332

Metal detection (µg metal/g struvite)

327

338 339 340

Metal detection (µg metal/g struvite

337

Sample 1 Sample 2

20000 15000 10000 5000

Na

334

336

25000

0

333

335

Page 18 of 33

Ca

Metal

K

Fe

250 200 150 100 50 0 Zn

Cu

As

Mn

Metal

Cr

Cd

Pb

Ni

341 342

Figure 4. ICP-OES results of Sample 1 and Sample 2. The dominant metals are shown in the

343

top graph of the figure (N=3, standard deviation bars not shown).

344 345

Based on previous reports and surface analysis, the presence of calcium carbonates and phosphates

346

as an amorphous compound is likely to be an observed co-precipitate.13 The remaining P and Mg

347

not accounted for in struvite can be used with Ca to estimate the residuals Ca3(PO4)2 and CaCO3.

348

With Mg as the limiting reagent, residual P could be assumed to form Ca3(PO4)2 and equate to

349

approximately 0.06 g/g of struvite and 0.03 g/g of struvite for Sample 1 and 2 respectively.


Page 19 of 33


350 351

Furthermore, according to the fertilizer regulations used by the Canadian Food Inspection Agency

352

(CFIA), the metals added to soils must meet fertilizer/supplement standards, which are similar to

353

those used in other countries such as in the US.63,64 Several macro- and micronutrients are essential

354

plant growth components at low levels,65 which are reflected in the ranges provided in the

355

standards. Based on the annual acceptable metal concentrations, the detected metals in the samples

356

were all within the allowable range despite potentially affecting the purity of the final product.

357

Limits regarding Na, Ca and K levels are not addressed in the CFIA safety guidelines, but higher

358

levels of these nutrients are generally acceptable. Surface metal presence and inclusion could be

359

important to understand the impact on struvite purity and may also indicate metal accumulation

360

and potential toxicity to crops upon downstream application.

361 362

Biological Analysis

363

Addressing gene presence, qPCR was performed to quantify FIB, ARGs and enteric viruses. Most

364

bacterial gene targets were detected in quantities above the limit of detection in all samples (Figure

365

5). Of unknown relative significance, Sample 1 (sourced from blackwater) had a higher overall

366

presence of ARG copies per gram of struvite than Sample 2 (sourced from municipal wastewater).

367

The highest target gene detected in each sample was the class 1 integron gene (intI1), which is of

368

potential concern given its role in HGT of AMR genes.66 Of the FIB, enterococci was the most

369

numerous in Sample 1, which fits with evolving practice to utilize qPCR targeting the more

370

persistent enterococci as a health-related marker for sewage-contaminated environments and

371

standard methods for its assay are also well described.67,68 Given the drying process step expected

372

for commercial struvite fertilizer production,69 the samples were also screened for highly resistant



Page 20 of 33

373

C. perfringens spores, though detection of the cpn60 gene was below the detection limit for

374

Sample 2. For all targets, only a portion of the entire gene is amplified via qPCR, thus the

375

functionality of the ARGs remains unknown, but could still be transferred to soil bacteria.70 A

376

previous study conducted by Chen and colleagues1, has shown that struvite with ARGs present

377

when applied to soils can increase the diversity and abundance of ARGS in soils, indicating that

378

struvite could play a large role in influencing the microbial soil community.1 As many ARB have

379

been detected in grown crops, increases in AMR are a risk that requires further consideration with

380

the use of wastewater recovered struvite as also identified for biosolid-amended soils.46

Average gene copies/g of struvite (log10)

381

382

uidA entero1 cpn60 tetA vanB intI1

7 6 5 4 3 2 1 Sample 1 (wet)

Samples

Sample 2 (dry)

383

Figure 5. qPCR analysis of Sample 1 (wet weight) and Sample 2 (dry weight) shown as average

384

gene copies/g of struvite (N=3, errors bar indicate standard deviation)

385 386

Bacterial viability assays detected enterococci within the wet Sample 1 material, with an estimated

387

quantification of 1.04x103 MPN.mL-1. In comparison, the MPN of enterococci in the dried

388

Sample 1 material was 215 MPN.g-1. Colilert results were negative indicating that viable E. coli 18 ACS Paragon Plus Environment

Page 21 of 33


389

cells, if present, were below detection. These results are consistent with previous knowledge that

390

enterococci are generally more resilient to changing environments than E. coli.71 Nonetheless,

391

comparison of results to the CFIA maximum acceptable level of indicator organisms in fertilizers

392

(1000 MPN.g-1 solid) suggests that Sample 1 upon drying was within the acceptable limit.63 Such

393

FIB targets were developed before AMR became a global concern and without consideration of

394

more persistent enteric pathogens, such as human enteric viruses. While FIB and AMR can be

395

used to describe different biological aspects, antimicrobial resistant E. coli and enterococci have

396

been identified as top public health concerns by WHO.23 Hence, the presence of viable FIB in

397

conjunction with possible dissemination of ARGs (such as vancomycin resistance in enterococci)

398

could still pose a potential risk that requires further examination. Of increasing concern are the

399

culturable C. perfringens spores from the wet Sample 1 material that were estimated at 3.0x105

400

CFU/mL. Correlation between viability and qPCR results are not clear as not all spores may

401

germinate on culture of C. perfringens. Though the dry sample was not tested, post precipitation

402

processing steps will be integral for microbial reduction should spore-formers be considered an

403

important class of AMR hazard. It has been previously shown that an accumulation of bacteria can

404

occur during the filtration process of struvite production.72 The results of the study indicated that

405

heating under moist conditions followed by desiccation would increase the inactivation

406

efficiency.72 The drying temperature and moisture content were shown to play a role in the

407

bacterial inactivation where a low relative humidity allowed bacteria to persist, further indicating

408

the importance of post precipitation treatment steps.72

409 410

In addition to the hazard presented by enteric bacteria, more than 150 different genera of enteric

411

viruses have been described as waterborne,73 in which a relatively short list of viruses are



Page 22 of 33

412

considered representative in wastewater for QMRA studies,74 hence the focus on EV, HAdV and

413

NoV GII. While (RT)-qPCR was positive for all viruses in Sample 1, RT-ci-qPCR only confirmed

414

enterovirus virions with intact capsids (Figure 6), indicating infectivity potential. Should struvite

415

fertilizer be utilized in crops eaten raw, such as lettuce or carrots, the infectivity of these enteric

416

viruses is also of potential concern.75

417 418 419 420 421 422 423 424 425 426 427

Figure 6. (RT-) qPCR and RT-ci-qPCR analysis of Sample 1 for HAdV, EV and NoV GII

428

shown as average gene copies/100 mL of struvite (N=6, error bars indicate standard deviation).

429 430

In all, with the observation that chemical and microbial co-precipitates were present in the samples,

431

a connection to the morphological analysis can be predicted. Detection of unexpected metals also

432

illustrates that co-precipitation can occur in struvite production from wastewater. The metal co-

433

precipitation can interfere with the struvite structure, thus, resulting in chemical bonds that would

434

typically not be found in a pure sample. As a result of these additional bonds in the early stages of


Page 23 of 33


435

crystal growth, the morphology of the final crystal can be greatly impacted as observed (Figure 2).

436

Similarly, bacterial and virus detection indicates that microbial interference could also be a factor

437

that dictates struvite formation and overall morphology.

438 439

Based on the results obtained, there is reason to further determine the risks associated with

440

wastewater-recovered struvite. While all potential hazards were not assessed in the current study,

441

the co-precipitation of other chemical hazards such as pharmaceuticals and personal care products

442

would be useful in further understanding the hazards potentially present in struvite. Though there

443

were variations in wastewater collection, type and treatment between samples, it has been shown

444

that regardless of the process, co-precipitation could be a concern requiring further investigation.

445

In addition to determining the presence of hazards, the operational conditions and processes that

446

elevate or reduce these risks should also be documented to minimize downstream public health

447

concerns.

448 449

Supporting Information

450

XPS and XRD results along with further details regarding wastewater source parameters

451

development of standards and reaction conditions for qPCR assays are described in the supporting

452

information.

453 454

Acknowledgements

455

The work was supported by research grants provided by the Natural Sciences and Engineering

456

Research Council (NSERC), the City of Edmonton, the NSERC Industrial Research Chair (IRC)

457

Program in Sustainable Urban Water Development, the Canada Research Chair (CRC) in Future 21 ACS Paragon Plus Environment


Page 24 of 33

458

Community Water Services and Alberta Innovates via a grant supporting the Translational Health

459

Chair in Waterborne Diseases (#201300490). The authors wish to also thank Nancy Price (School

460

of Public Health, University of Alberta) for microbiological training and technical support.

461 462

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