May 07, 2024

Public workspaceWATER PRODUCTION FOR AWARE (Mesophilic Bacteria in PCA (Plate Count Agar)) V.3

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l22xxxl1y/v3
WATER PRODUCTION FOR AWARE (Mesophilic Bacteria in PCA (Plate Count Agar))
  • Celia Manaia1
  • 1Universidade Católica Portuguesa
AWARE Project
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l22xxxl1y/v3
Protocol Citation: Celia Manaia 2024. WATER PRODUCTION FOR AWARE (Mesophilic Bacteria in PCA (Plate Count Agar)). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22xxxl1y/v3Version created by AWARE Project
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 06, 2024
Last Modified: May 07, 2024
Protocol Integer ID: 99287
Keywords: water sampling, water processing, water analysis, waste water treatment, advanced tertiary treatment SOP, water production for aware, mesophilic bacteria in pca, water production, standard operating procedure, pca, procedure, optimization of advanced tertiary treatment, plate count agar, sop, mesophilic bacteria, advanced tertiary treatment
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:

According to drinking water EU legislation
Materials

ABCDEFGH
ParameterV (mL) x R SProcessingAnalytical methodResultLOD / LOQGoal value
Mesophilic Bacteria in PCA (Plate Count Agar)0.1 x 3NoSpread method Spread plate; 48 h incubation at 37 °C Number of Colony Forming Units (CFU/mL) 100 CFU/mL
Table 1: Samples, Processing and Analysis of the different parameters analysed.
V, volume; R, Replicates; S, Shipment conditions; LOD / LOQ, Limit of Detection / Quantification

Materials: Culture medium Plate Count Agar (PCA); Microbiological incubator; other microbiology consumables.
Safety warnings
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WATER PRODUCTION FOR AQUAPONICS
The water production for AWARE main activities includes three stages – disinfection by ultraviolet C radiation (UVC), storage forDuration12:00:00 -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.

Figure 1. Treatment and storage of municipal treated wastewater used for integrated aquaponics and an indication of the comprehensive quality and risk assessment.

1d 12h
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 atTemperature-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 dose, e.g., Amount5 mg O3) - MITO3X technology - (G)
- Effluent of the vacuum UV oxidation (VUV) (H)
- Effluent of reactive storage / Influent of the recirculation aquaculture system (RAS) (I)

Figure 2. Diagram representing the wastewater treatment plant (WWTP), advanced treatment and sampling points.

9h
Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
5d
Mesophilic Bacteria in PCA (Plate Count Agar):  
Analysis: Enumeration of culturable mesophilic bacteria atTemperature37 °C

Method:
Spread plate; unitsDuration48:00:00 a incubation atTemperature37 °C ; emuneration of colony forming (AmountCFU/ mL ):
2d
Observations: Samples processed and analysed withinDuration12:00:00 after collection.
12h
Parameters framed by Legal and Regulatory Requirements:
9h
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/100 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
- American Public Health Association (APHA). (2009). Standard Methods for the Examination of Water and Wastewaters. APHA – AWWA - WPCF (Eds.), Pennsylvania, Washington.
- ISO 4833:2003 - Microbiology of food and animal feeding stuff − Horizontal method for enumeration microorganisms – Colony count technique atTemperature30 °C . 
- EN ISO 11133:2014 - Microbiology of food, animal feed and water.  Preparation, production, storage and performance testing of culture media.