TITLE: WATER PRODUCTION FOR AQUAPONICS
AUTHORS: Celia Manaia

[DESCRIPTION]
The protocol summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.

[GUIDELINES]
RECOMMENDED/ACCEPTED VALUE

#Step2.1: Mesophilic Bacteria in PCA (Plate Count Agar):
According to drinking water EU legislation 

#Step2.2: Total Coliforms, Fecal Enterocci and Escherichia Coli:
Total coliforms:0 MPN/ mL
E.coli: 0 MPN/ mL 
Fecal enterococci: 0 MPN mL 

#Step2.3 Total Bacteria and Antibiotic Resistance Genes:
No legal recommendation available.  
16S rRNA gene<3 log units/ mL; other faecal and antibiotic resistance genes < limit of detection (LOD) in2000 mL

#Step2.4: Virus:
Viruses not included in any normative. 
Contemplated in the EU proposal of a new directive on the Treatment of urban wastewater 

#Step2.5: Parasites:
Regulation 2020/741 Minimum requirements for water reuse. 
Guidelines to support the application of Regulation 2020/741 on minimum requirements for water reuse 
Council Directive 93/88/EEC of 12 October 1993 on the protection of workers from risks related to exposure to biological agents at work. 
Relevant food-borne parasites are not included in any normative. 

#Step2.6: Metals:
Directive 2008/105/EC of the European Parliament and of the Council of 16 December 2008 on environmental quality standards in the field of water policy.

#Step2.7: Organic Contaminants:
Directive 2008/105/EC of the European Parliament and of the Council of 16 December 2008 on environmental quality standards in the field of water policy.

[STEPS]
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2. Mesophilic Bacteria in PCA (Plate Count Agar):  
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.1. Analysis: Enumeration of culturable mesophilic bacteria at37 °C

Materials: Culture medium Plate Count Agar (PCA); Microbiological incubator; other microbiology consumables.

Method:
Spread plate; units2880 mina incubation at37 °C; emuneration of colony forming (CFU/ mL): 

Observations: Samples processed and analysed within720 minafter collection.
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.2. Total Coliforms, Fecal Enterococci and Escherichia coli: 

Analysis: Enumeration of fecal contamination indicators.

Material:  Sterile bottles, IDEEX kit, Colilert-18 reagent, Microbiological incubator.

Method:   
 
Reference:  
− UNI EN ISO 9308-2:2014 Water quality - Count of Escherichia coli and coliform bacteria - Part 2: Most probable number method. 
− EN ISO 11133:2014 - Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media. 

 
Reference:  
− UNI EN ISO 7899-1:2001 - Water quality - Research and enumeration of intestinal enterococci - Miniaturized method (Most Probable Number) for surface and wastewater. 
− EN ISO 11133:2014 - Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media. 

Observations: Samples were processed and analysed within720 minafter collection.
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.3. Total Bacteria and Antibiotic Resistance Genes: 

Analysis: Culture-independent detection and/or quantification of bacteria and bacterial contaminants.

Material: Membrane filtration (nitrocellulose 0.22 µm pore); DNA extraction (QIAGEN, Power Water); Quantitative PCR of genes such as 16S rRNA gene (total bacteria), uidA, marA (bacterial contaminant indicators), crassphage (human fecal contamination), intI1 / incF / sul1 / qacEΔ1 / tetX / ermB / mefC / ermF / aph(3´´)-ib (antibiotic resistance indicators); metagenomics analysis of selected samples.  

Observations: Samples were filtered within360 minafter collection the filtering membranes were immediately frozen and stored at-80 °Ctill shipping in dry ice to the respective partner who proceeded for DNA extraction. 

SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.4. Virus: 

Analysis: Detection and enumeration of virus.

Material: Rexeed 25A filtration units (Asahi Kasei Medical); other molecular biology consumables.

Method: Virus capture by filtration with Rexeed dialysis units, concentration with aluminium chloride, nucleic acid extraction and detection by RT-qPCR.  

Observations: Samples were filtered within360 minafter collection the filtering membranes were immediately frozen and stored at-80 °Ctill shipping in dry ice to the respective partner who proceeded for DNA extraction. 
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.5. Parasites: 

Analysis: Detection of parasites.

Material: Filtration ramp; DNA extraction kit, PCR equipment; fluorimeter for DNA quantification; outsourcing of sequencing service; molecular biology consumables. 

Method: 
Processing of Water samples 

1. Filtration.
3 Lof water were filtered through 0.45 µm pore size diameter Nitrocellulose / polycarbonate sterile membranes, using a membrane filter holder ( ) apparatus. For every approx.250 mL of water, a new sterile membrane was used to avoid clogging of membranes. Filters corresponding to each litre of water were separated, inserted into well-sealed tubes (50 mL) stored a -80 °Cand then transported in dry ice. 
2. Centrifugation.(1 L of water was concentrated by centrifugation at2.500 gfor15 minusing sterilized tubes. Pellets obtained were resuspended using of1 mLof fformaldehyde solution and maintained for1440 min. Pellets resuspended in formaldehyde solution were centrifugated at 2.500 gfor  , the supernatant was discarded and all pellets were pooled in1 mLof f ethanol.  

3.  DNA Isolation, Amplification and sequencing. 
DNA extraction was carried out using a commercially available kit (Powerwater kit, Qiagen) in accordance with the manufacturer's guidelines. The V4 and V9 of the 18S rRNA gene hypervariable regions of the eukaryote 18S rRNA gene were amplified using universal primers. Subsequently, PCR products were purified and subjected to external sequencing using Illumina MiSeq platform 300bp paired-end sequencing.  

4.  Bioinformatic analysis. 
Raw sequence data were processed using QIIME2 software 2023.9. Pairedend reads were subjected to quality filtering (FastQC) and denoising (DADA2 plugin). Subsequently, the taxonomic assignment was conducted using the reference database SILVA 18S release 138 with a clustering threshold of  similarity. Alpha rarefaction analysis, Shannon diversity index, richness estimation, and relative abundance calculations were performed using the Vegan R package to assess microbial community diversity and composition particularly focusing on the detection of genetic material from potential pathogens. 

Observations: Samples were filtered within720 minafter collection the filtering membranes were immediately frozen and stored a-80 °Ctill shipping in dry ice to the respective partner for further analysis. 
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.6. Metals: 

Analysis: Detection and quantification of metals 

Material: Metal-free centrifuge tubes 

Method: Acidified water samples were analyzed for several metals (e.g. Al, Cu, Ni, Co, Pb, Zn, Cd, Pt, Sb, Sn, U, As, Ag, Hg, REEs) by means of ICP-MS (7900 Agilent) using matrix-matched external calibration. 

Observations: Samples were filtered/centrifuged within 720 minafter collection, and then acidified to 0.15M HNO3.
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.7. Organic Contaminants: 

Analysis:  Screening of Organic Contaminants in Water.

Material:    

Methods:  Solid-phase extraction 

1. Sample filtration (0.45um PVDF). 

2. Collect 200 mL of the filtered water sample (e.g. volumetric flask or beaker) and spike with 50 µL of an internal standard solution. Produce two 200 mL replicates per sample. Mix well after the internal standard is added.  
  
3. Cleaning/Conditioning of the cartridges (OASIS HLB 200 mg) 
 
3.1. Pass 5 mL of MeOH 

3.2. Pass 5 mL of ultrapure water 

4. Pass the 200 mL of sample 

5. Rinse the volumetric flask or beaker that contained the sample with 2x 10 mL of ultrapure water, which are then passed through the cartridege 

6. Drying the cartridge resin (e.g. N2 flow for 30 min) 

7. Store the cartridges in a freezer (-20 °C) 

All samples are to be processed in duplicate.

For the cartridge blanks (n=2): start with step 3, and then spike directly on the cartridge the same amount of internal standard as for samples (50 µL mix de 1 ug mL).  Then go directly to step 6.
For the ultrapure water blanks (n=2), the same type of sampling flasks are filled with ultrapure water 1440 minbefore sampling. Then they are treated using the same protocols as for samples.

Elution of the loaded cartridges was carried out by gravity using 10 mL of methanol followed by 10 mL of dichloromethane. The eluates were concentrated approximately to ca. 0.5 mL using a Turbovap II concentrator (Zymark, Hopkinton, MA, USA), then to dryness under a gentle nitrogen stream and finally reconstituted in 500 µL of methanol and filtered with a 0.2 μm   GHP‱   syringe filter (Pall Corporation). The extracts were injected in an Agilent 1290 LC coupled with and Agilent 550 QTOF system. 
SECTION: Parameters framed by Legal and Regulatory Requirements:
3. Using the EU Drinking Water Directive:  

Mesophilic Bacteria in PCA (Plate Count Agar) – 0 CFU/100 mL

Total coliforms and Escherichia coli –Number /100 mL (0 MPN/100 mL) 

Fecal enterococci –Number/100 mL (0 MPN/100 mL)  

Viral concentration - There are no legal requirements for viruses. They are not included in any regulation now. 

Parasite  - EU legislation (2020/741)  

Metals - DIRECTIVE 2008/105/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 December 2008 on environmental quality standards in the field of water policy 

Organic contaminants - DIRECTIVE 2008/105/EC OF THE EUROPEAN PARLIAMENT AND THE 
COUNCIL of 16 December 2008 on environmental quality standards in the field of water policy.  

1. The water production for AWARE main activities includes three stages – disinfection by ultraviolet C radiation (UVC), storage for720 min-1440 min(according to water load and season) and ozonation. The water quality is monitored at these three stages, for the parameters indicated in Figure 1 below.

 

1.1. Sampling, Processing, and Analyses 

Water samples are collected (see Figure 2) and processed within a360 mininterval, before being shipped for the partner responsible for the analyses (Table 1). In case no processing is needed, samples are frozen and stored at-80 °Cwithin180 min.
For each sampling event, the date, day of the week and hour; the temperature and rain. Sampling points, indicated in Figure 2 were designated from A to I:
- Influent of primary treatment (A) 
- Influent of biological treatment (activated sludge) (B) 
- Treated secondary effluent (C) 
- Sand filter effluent (D) 
- UVC effluent (E) 
- Storage for reuse tank effluent (F) 
- Ozonation effluent (1 dose, e.g., 5 mgO3) - MITO3X technology - (G) 
- Effluent of the vacuum UV oxidation (VUV) (H) 
- Effluent of reactive storage / Influent of the recirculation aquaculture system (RAS) (I)