The potential of solar disinfection in combination with bacteriophage biocontrol to reduce the health risks associated with contaminated harvested rainwater

Thesis (PhD)--Stellenbosch University, 2020.ENGLISH ABSTRACT: Domestic rainwater harvesting may aid developing countries in achieving Sustainable Development Goal 6, which aims to ensure universal access to safe and affordable water. However, numerous microbial pathogens are associated with rainwater and treatment is required to reduce the potential health risk associated with using this water source. Solar disinfection (SODIS) has subsequently been identified as an easy-to-use and cost-effective treatment, where natural sunlight inactivates microorganisms through ultraviolet (UV) radiation and solar mild-heat. The small volumes of water that can effectively be treated (2 L) and decreased efficiency under poor weather conditions, have however been identified as key limitations of SODIS. The aim of Chapter 2 (published in Science of the Total Environment) was thus to monitor the efficiency of two large-volume batch solar reactor prototypes designed by the research consortium of the European Union (EU) Horizon 2020 project, Water Sustainable Point-of-Use Treatment Technologies (WATERSPOUTT). The systems were installed in a local informal settlement (Site 1; 88 L Prototype I) and rural farming community (Site 2; 140 L Prototype II) and were connected to rainwater harvesting systems. Due to space availability, a first-flush diverter system was installed at site 2. Conventional water quality monitoring techniques [chemical (anions, cations, physico-chemical parameters) and microbial (indicator organism and opportunistic pathogen culture-based analysis)] then indicated that the chemical quality of the untreated and treated rainwater adhered to national and international drinking water quality guidelines. While the microbial quality of the untreated rainwater exceeded the guideline limits at both sites, an 8 h solar exposure (6 and 8 h assessed) effectively reduced the indicator organism and monitored opportunistic pathogen counts to below the detection limit, with the exception of 43% of the samples collected from the Prototype I reactor (site 1), where heterotrophic bacteria still exceeded the guideline limit. An increased treatment efficiency was subsequently obtained at site 2 and was attributed to the first-flush diverter system (installed at this site) reducing the level of microbial contaminants entering the rainwater harvesting tank. As microorganisms may enter a viable but non culturable state under unfavourable conditions (such as those experienced during SODIS treatment), nucleic acid binding dyes were combined with quantitative polymerase chain reaction (qPCR) assays to monitor the removal of various opportunistic pathogens, including Escherichia coli (E. coli), Legionella spp. and Pseudomonas spp., amongst others. A mean overall reduction of 75% in the target organisms was then obtained for both solar reactors. It is however important to note that based on drinking water quality guidelines, the solar reactor prototypes were able to provide small households with the minimum essential daily water requirement for human activities of 25 L per person per day. Additionally, a generic Water Safety Plan was compiled for use in combination with the solar reactor prototypes to aid in ensuring the quality of the treated water. The detection of intact and potentially viable cells following solar reactor treatment however, highlighted the need for quantitative microbial risk assessment (QMRA) of the rainwater. However, as numerous microbial pathogens may be present within harvested rainwater, Illumina next-generation sequencing was coupled with ethidium monoazide (EMA) viability pre-treatment to elucidate the total viable microbial community in the untreated and treated rainwater (Chapter 3). Legionella, Pseudomonas, Mycobacterium, Clostridium sensu stricto and Escherichia were subsequently identified as the most frequently detected pathogenic or opportunistic pathogenic genera within the untreated and treated rainwater samples collected from both sites. Legionella pneumophila (L. pneumophila), Pseudomonas aeruginosa (P. aeruginosa) and E. coli were then selected as the target organisms for the health-risk assessment of the untreated and treated rainwater. Treatment with the solar reactor prototypes effectively reduced the health risk posed by E. coli and P. aeruginosa, when using rainwater for bathing/washing, garden work, cleaning and washing laundry (by hand), to below the one extra infection per 10 000 persons per year (1 × 10-4) benchmark limit. However, for both organisms the risk associated with using treated rainwater for drinking, still exceeded the benchmark limit. Additionally, while the solar reactor treatment reduced the risk associated with garden hosing and showering based on the presence of L. pneumophila, the risk estimate for using the treated rainwater for showering still exceeded the annual benchmark limit. The water produced by the large-volume batch solar reactor prototypes may thus safely be used for various domestic activities commonly practised within the target communities (e.g. washing/bathing, cleaning the home, etc.) in water scarce regions of sub-Saharan Africa. As Pseudomonas was identified as one of the dominant bacterial genera persisting following treatment using the solar reactor prototypes, bacteriophages targeting Pseudomonas spp. were isolated and characterised for the biocontrol pre-treatment of rainwater (Chapter 4; published in Environmental Science: Water Research and Technology). Bacteriophage PAW33 was isolated and characterised as a Podoviridae bacteriophage, with broad-spectrum activity against P. aeruginosa strains, and was applied in small-scale rainwater pre-treatment trials for 8 h and 24 h, to restrict the proliferation of an environmental P. aeruginosa S1 68 strain. The bacteriophage pre-treated samples were then subjected to treatment in small-scale SODIS compound parabolic collector (SODIS-CPC) systems for 4 h under natural sunlight. Although culture- and molecular-based analyses for the 8 h trial indicated similar total log reductions of P. aeruginosa S1 68 for the bacteriophage pre-treated and non-pre-treated samples, results for the 24 h trial (followed by SODIS) indicated that a higher log reduction was recorded for the pre-treated sample (4.61 log) in comparison to the non-pre-treated sample (3.91 log), using culture-based analysis, with comparable results obtained using EMA-qPCR. Gene expression analysis indicated that PAW33 pre-treatment for 24 h influenced the ability of P. aeruginosa S1 68 to initiate stress response mechanisms (decreased expression of the recA and lexA genes) during the SODIS-CPC treatment and resulted in the decreased expression of the phzM gene (virulence factor responsible for pyocyanin production). Bacteriophage PAW33 thus displays promise as a pre-treatment strategy of rainwater as it restricts the proliferation of P. aeruginosa and may increase the efficiency of primary disinfection methods.AFRIKAANSE OPSOMMING: Huishoudelike reënwater-oesting kan ontwikkelende lande help om Volhoubare Ontwikkelings Doel 6 te bereik, wat daarna streef om universele toegang tot veilige en bekostigbare water te verseker. Verskeie patogene mag egter met reënwater geassosieerd wees en daarom is waterbehandeling noodsaaklik om die moontlike gesondheidsrisiko’s te elimineer. Sonkrag ontsmetting (SODIS) is as ‘n maklike en bekostigbare strategie geïdentifiseer en behels die gebruik van natuurlike sonlig om mikroörganismes te inaktiveer deur ultra-violet bestraling en matige-temperature. Die klein volume water wat behandel kan word deur hierdie tegniek (2 L) en verlaagde effektiwiteit tydens slegte weersomstandighede is egter geïdentifiseer as beperkings van SODIS. Die doel van Hoofstuk 2 (gepubliseer in “Science of the Total Environment” vir publikasie) was dus om die effektiwiteit van twee groot-volume sonkragreaktors te monitor wat deur die navorsingskonsortium van die Europese Unie (EU) Horison 2020 projek, “Water Sustainable Point-of-Use Treatment Technologies” (WATERSPOUTT), ontwerp is. Die sisteme is onderskeidelik in ‘n plaaslike informele nedersetting (Terrein 1; 88 L Prototipe I) en plaasgemeenskap (Terrein 2; 140 L Prototipe II) geïnstalleer en gekoppel aan reënwater-oesting sisteme. ‘n Eerste-spoel-wegleiding sisteem kon ook geïnstalleer word by terrein 2, omdat daar genoeg spasie beskikbaar was. Die konvensionele waterkwaliteit analises [chemies (anione, en katione) en mikrobies (groei van indikator-organismes en opportunistiese patogene)] het aangedui dat die chemiese kwaliteit van die onbehandelde en behandelde reënwater aan nasionale en internasionale drinkwater riglyne voldoen. Alhoewel die mikrobiese kwaliteit van die onbehandelde reënwater die riglyne oorskry het vir beide terreine, het resultate daarop gedui dat ‘n 8 uur sonkrag behandeling voldoende was om die indikator-organismes en opportunistiese patogene tot onder die opsporingslimiet te verminder, met die uitsondering van 43% van die monsters van die Prototipe I reaktor (Terrein 1), waar die vlak van heterotrofiese bakterieë steeds die riglyn oorskry het. Die verbeterde behandelings aktiwiteit by terrein 2 is toegeskryf aan die gebruik van die eerste-spoel-wegleiding sisteem wat die konsentrasie van mikroörganismes wat die reënwatertenk binnedring, verminder het. Omdat mikroörganismes ‘n lewendig-maar-nie-kultiveerbaar toestand kan aanneem tydens ongunstige toestande (soos ervaar tydens SODIS), is nukleïensuur bindende kleurstowwe gekombineer met kwantitatiewe polimerase ketting reaksies (kPKR) om die inaktiveering van verskeie opportunistiese patogene, insluitend Escherichia coli (E. coli), Legionella spp. en Pseudomonas spp., te monitor. Die resultate het daaropvolgens aangedui dat die teiken organismes met ʼn gemiddeld van 75% verminder/geinaktiveer is, in beide sonkragreaktors. Volgens drinkwater riglyne, is die sisteme egter voldoende om klein huishoudings van hul minimum noodsaaklike daaglikse waterbehoefte vir menslike aktiwiteite (25 L/persoon/dag) te voorsien. In hierdie studie is ‘n generiese Water Veiligheids Plan ook saamgestel vir gebruik in samewerking met die sonkragreaktore om die kwaliteit van die behandelde water te verseker. Die feit dat van die teiken mikroorganismes moontlik sonkrag behandeling kan oorleef (kPKR analise), het die noodsaaklikheid vir kwantitatiewe mikrobiese risiko analise (KMRA) beklemtoon. Verskeie patogene mag egter teenwoordig wees in geoeste-reënwater, en dus is Illumina volgende generasie volgorde ontleding met ethidium monoasied (EMA) gekombineer om die totale lewendige mikrobiese gemeenskap in die onbehandelde en behandelde reënwater te ontleed (Hoofstuk 3). Gevolglik is Legionella, Pseudomonas, Mycobacterium, Clostridium sensu stricto en Escherichia geïdentifiseer as die patogeniese of opportunistiese patogeniese genera wat die meeste voorgekom het in die onbehandelde en behandelde reënwater by beide terreine. Legionella pneumophila (L. pneumophila), Pseudomonas aeruginosa (P. aeruginosa) en E. coli is dus geselekteer as die teiken organismes vir die gesondheidsrisiko ontleding van die reënwater. Behandeling met die sonkragreaktors het effektief die gesondheidsrisiko geassosieerd met E. coli en P. aeruginosa verminder tot onder die een ekstra infeksie per 10 000 persone per jaar (1 × 10-4) maatstaf limiet, wanneer die reënwater vir bad, tuinwerk, skoonmaak of wasgoed was (met die hand) gebruik word. Die risiko limiet vir die gebruik van die behandelde reënwater vir drink doeleindes is egter vir albei organismes steeds oorskry. Daarbenewens, alhoewel behandeling met die sonkragreaktors die risiko vir tuinbesproeing en stort doeleindes (as gevolg van die teenwoordigheid van L. pneumophila) verminder het, is die maatstaf limiet vir stort doeleindes steeds oorskry. Die water wat geproduseer word deur die sonkragreaktors kan dus veilig gebruik word vir verskillende huishoudelike aktiwiteite wat gereeld binne die teikengemeenskappe beoefen word (soos bad, skoonmaak van die huis, ens.), veral in waterskaars streke van Suider Afrika. Omdat Pseudomonas geïdentifiseer is as een van die dominante bakteriële genera wat oorleef na behandeling met die sonkragreaktors, is bakteriofage wat Pseudomonas spp. teiken geïsoleer en geïdentifiseer vir die biologiese behandeling van reënwater (Hoofstuk 4; gepubliseer in “Environmental Science: Water Research and Technology”). Bakteriofaag PAW33 is geïsoleer en geïdentifiseer as ‘n Podoviridae bakteriofaag, met breë-spektrum aktiwiteit teen P. aeruginosa stamme en is dus gebruik in kleinskaalse reënwater voorbehandelings proewe (vir 8 en 24 uur) om die groei van ‘n P. aeruginosa S1 68 te inhibeer. Die bakteriofaag-behandelde reënwater is daarna in ‘n paraboliese versamelaar SODIS sisteem vir 4 ure met natuurlike sonkrag behandel. Alhoewel kultuur- en molekulêre analises vir die 8 uur proef soortgelyke verminderings aangetoon het vir die bakteriofaag-behandelde en onbehandelde reënwater, het die resultate van die kultuur analise vir die 24 uur proef (gevolg deur SODIS) aangedui dat ‘n hoër logaritmiese vermindering verkry is in die baketeriofaag-behandelde reënwater (4.61 log) teenoor die onbehandelde reënwater (3.91 log), terwyl soortgelyke resultate met EMA-kPKR verkry is. Geenuitdrukking analise het toe aangedui dat PAW33 voorbehandeling vir 24 uur die vermoë van P. aeruginosa S1 68 om ‘n stresrespons te inisieer beïnvloed (verminderde uitdrukking van recA en lexA) en ook verminderde uitdrukking van die phzM geen (virulensie faktor verantwoordelik vir pyocyanin produksie) tot gevolg het. Bakteriofaag PAW33 kan moontlik as ‘n voorbehandeling strategie vir reënwater dien, omdat dit die groei van P. aeruginosa beperk en die effektiwiteit van primêre behandelings metodes kan verbeter.Doctora