May 07, 2024

Public workspaceWATER PRODUCTION FOR AWARE (Total Bacteria and Antibiotic Resistance Genes):

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxyzodl8j/v1
WATER PRODUCTION FOR AWARE (Total Bacteria and Antibiotic Resistance Genes):
  • Celia Manaia1
  • 1Universidade Católica Portuguesa
AWARE Project
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.kxygxyzodl8j/v1
Protocol Citation: Celia Manaia 2024. WATER PRODUCTION FOR AWARE (Total Bacteria and Antibiotic Resistance Genes):. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxyzodl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 07, 2024
Last Modified: May 07, 2024
Protocol Integer ID: 99380
Keywords: water sampling, water processing, water analysis, waste water treatment, advanced tertiary treatment SOP, water production for aware, water production, standard operating procedure, procedure, optimization of advanced tertiary treatment, sop, total bacteria, antibiotic resistance gene, advanced tertiary treatment, water
Funders Acknowledgements:
Horizon Europe
Grant ID: 101084245
Abstract
The protocol summarises the procedures used for analytical control. The protocol describes the Standard Operating Procedure (SOP) for the optimization of advanced tertiary treatment of water, based on a comprehensive quality and risk assessment.
Guidelines
RECOMMENDED/ACCEPTED VALUE:

No legal recommendation available.
16S rRNA geneAmount<3 log units/ mL ; other faecal and antibiotic resistance genes < limit of detection (LOD) inAmount2000 mL
Materials

ABCDEFGH
ParameterV (mL) x R SProcessingAnalytical methodResultLOD / LOQGoal value
Total bacteria and antibiotic resistance genes2000 x 3On iceMembrane filtration 0.22 µM polycarbonate membranesDNA-based analysis quantitative PCR (e.g. 16S rRNA;intI1, uidA, sul1, qacE1, tetX, ermB, crassphage, mefC, ermF, aph(3´´)-ib Gene abundance per volume of water (Log-unit / mL) Removal values (if adequate) (Log-unit)Total bacteria: 0.7 Other genes: intI1 – 0.01 uidA – 0.01 sul1 – 1.22 qacEΔ1 – 0.2 tetX – 0.2 ermB – 0.2 crassphage – 1.0 mefC – 0.1 ermF – 0.01 aph(3´´)-ib – 1.20Total bacteria (3 log-units / mL) Other genes (
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.
Troubleshooting
Safety warnings
---
Total Bacteria and Antibiotic Resistance Genes):
The water production for AWARE main activities includes three stages – disinfection by ultraviolet C radiation (UVC), storage forDuration24:00:00 -Duration24:00:00 (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.



2d
Sampling, Processing, and Analyses

Water samples are collected (see Figure 2) and processed within aDuration06:00:00 interval, before being shipped for the partner responsible for the analyses (Table 1). In case no processing is needed, samples are frozen and stored atTemperatureTemperature-80 °C withinDuration03:00:00 . 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 dos, O3) - Amount5 mg MITO3X technology - (G)
- Effluent of the vacuum UV oxidation (VUV) (H)
- Effluent of reactive storage / Influent of the recirculation aquaculture system (RAS) (I)



9h
Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
6h
Total Bacteria and Antibiotic Resistance Genes:
Analysis: Culture-independent detection and/or quantification of bacteria and bacterial contaminants.
Observations: Samples were filtered withinDuration06:00:00 after collection the filtering membranes were immediately frozen and stored atTemperature-8 °C till shipping in dry ice to the respective partner who proceeded for DNA extraction.

6h
Parameters framed by Legal and Regulatory Requirements:
Using the EU Drinking Water Directive:
Mesophilic Bacteria in PCA (Plate Count Agar) – 0 CFU/Amount100 mL
Total coliforms and Escherichia coli –Number /Amount100 mL (0 MPN/Amount100 mL )
Fecal enterococci –Number/Amount100 mL (0 MPN/Amount100 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.
Protocol references