Phase change materials for portable isothermal incubators as a solution to shorten effective analysis times in microbiological quality control of drinking water

The microbiological quality control of water for human consumption of parameters relevant as E.coli and total coliforms does not start on the field despite the existence of test methods that could make it possible. One of the things that makes this difficult is the possibility of initiating an effective and reliable incubation at the sampling site. The appearance of isothermal media with phase change materials solves this limitation. When phase change materials combine a relatively high melting heat with a suitable melting temperature adapted to the application temperature, they become excellent materials for thermal protection and for thermal energy storage. Starting the test at the same sampling point means that the effective times to obtain a result are shorter, improving water quality control. On the other hand, operationally, it also allows longer sampling routes. Both aspects are essential for managers responsible for controlling water quality for human consumption. In this work, the evidence that demonstrates the feasibility of this approach is presented.


Introduction
The control of the microbiological quality of water for human consumption mainly focuses on using different bacterial indicators related to faecal contamination and bacterial regrowth (Saxena et al., 2015).E. coli is not the only faecal pollution indicator, but it is the primary reference.On the other hand, the total coliform level is a standard reference as a process indicator.
The absence of E. coli and negligible or very low levels of total coliforms are associated with good water quality and proper operation of the distribution system.These two parameters, especially the first one, are transposed in all regulations on water quality for human consumption and are of particular interest to all operators since their result determines whether water quality can be acceptable for human consumption.
Although molecular techniques on the horizon could allow obtaining results in a few hours, their detection limits are still too high, and therefore, cannot be applied in potable water (Yuan et al., 2018).On the other hand, at present, regulatory testing protocols, at least in Europe, are based on culture according to ISO 9308 standards (ISO, 2018).Therefore, at least 18 h of incubation are required today to get results.
In daily practice, drinking water suppliers structure their self-monitoring program according to daily Vol:.( 1234567890) sampling from different points.The context of each sampling point determines the frequency of controls; a reservoir or a representative point of a network can be sampled daily, and other points instead can be sampled weekly or at low frequency.In any case, in the water industry, the analysis times of parameters such as E. coli should be as short as possible because in the event of faecal contamination, people's health can be compromised and rapid actions would be required.
Each laboratory plans its sample collection routes according to the priorities defined above, but very often, 4-6 h can elapse between sampling and the start of the analysis.Therefore, between sampling and detection of a risk event for human health, at least 20-24 h can elapse easily.
Starting the test at the same time as sampling would allow, on the one hand, shortening the detection times of adverse situations as much as possible while allowing much longer sampling routes without the need for interruptions to deliver samples to the laboratory.On the other hand, it has been shown that for untreated water, it is advisable to analyse the samples as soon as possible and before 8 h (Pope et al., 2003).
For this to be possible, it would be necessary to have a test method that allows work in situ and a field incubator that guarantees a temperature of 34-38 °C effectively and constantly.In the first case, the ISO 9308-2 standard (ISO, 2012), which is based on the Colilert-18 method (IDEXX, Maine, USA), allows the reagent to be quickly inoculated into the sample container (100 mL) and to perform a qualitative and simultaneous test of E. coli and total coliforms.
There are portable incubators on the market that can be connected to a car's electrical supply, and there are also developments for equipment with lithium batteries (Deep et al., 2021).For practical purposes, these types of incubators present operational problems associated with their permanent handling in work vehicles, also being conditioned by the state of the batteries or the electrical capacity of the vehicle's 12-volt connector.In summary, although it is technically possible to carry out this approach, as far as we know, it is a rare practice.
The advances in phase change materials (PCMs) have made possible to develop latent heat incubators with sufficient energy storage capacity to build isothermal media capable of maintaining specific temperature conditions during significant periods.
PMCs can be used to store thermal energy as latent heat, providing a greater density of energy storage with a smaller temperature difference between storing and releasing heat than the sensible heat storage method (Pielichowska & Pielichowski, 2014).The transition from solid material to a liquid state allows storing of energy that is released constantly during the melting (Fig. 1).
The devices based on PMCs have a low cost and much higher resistance than a conventional incubator.In this paper, we evaluate the potential of these devices for testing according to ISO 9308-2 in the operational context of quality control in a drinking water company.

Materials and methods
The isothermal incubator is a prototype designed and made by LVM Thermal Solutions (Sant Esteve d'en Bas, Spain).It consists of a stainless-steel cubic cuvette (10 L of volume) with a filling jacket containing a phase change material duly chosen as a heat source.This cuvette and an airtight lid form a watertight incubation set placed inside an insulating box of expanded polypropylene (PPE).The insulated box has an external dimensions of 390 × 330 × 281 mm, and a thickness of 30 mm (Fig. 2).
PCMs with high latent transition heat and temperature adapted to the application temperature have been used by LVM Thermal Solutions to ensure a constant temperature.In this case, the choice was solid-to-liquid and a liquid-to-solid transition (melting and solidification).The PCM is a mixture of organic materials, with a melting temperature of 36 °C and a head capacity storage of 100 kJ/kg.
During the melting process (Fig. 1a), the PCM stores a certain amount of energy as latent heat; this energy is restored during the solidification process (Fig. 1b).The duration of the energy storage process and/or the energy restitution process depends on the usage conditions like the amount of PCM and the difference between external and application temperature.
To start up the incubator, the cuvette must be activated according to the following protocol: First, 2 h at 120 ± 2 °C to allow the phase change of the filling material, followed by 55 ± 2 °C for at least 12 h so that the material settles thermally stabilise.The 55 °C Vol.: (0123456789) was chosen as the stabilization temperature because, at this level, the PMC still does not start to work.
From this moment, the incubator is ready to start working on collecting samples and on ensuring a temperature between 34 and 38 °C for more than 18 h.
The temperature control has been carried out with a Testo 175 T2 probe (Lenzkirch, DE).The microbial tests have been carried out in accordance with ISO 9308-2, using IDEXX (Maine, USA) reagents and consumables in a laboratory with the test accredited according to ISO 17025 (ISO, 2017) by ENAC, the Spanish accreditation body.The in situ trial and incubation methods presented in this paper are not part of the current scope of accreditation.
The methodology has been verified in 7 days of testing carried out in the laboratory as an initial verification with samples of continental waters with natural contamination diluted with different volumes of tap water samples.Subsequently, it was carried out in 5 field days, starting the test at the same sampling point; in this case, the samples were not spiked.
Samples were collected according to standard methods in a single bottle (ISO, 2006).At each point, 250 mL of sample were taken in a sterile Pyrex bottle, with 0.25 mL of 3% thiosulfate.Immediately after sampling, an aliquot of 100 mL was transferred to a sterile PET container (Idexx ref 98.09221.00)and the Colilert-18 reagent (Idexx, reference 98.08877.00)was added, following the manufacturer's protocol.Before starting incubation, the sample was shaken manually for 5 s.

Results and discussion
Table 1 shows the results of the tests carried out in the laboratory, where the ambient temperature is regulated at 23 °C.A total of 35 samples were processed in 7 independent days, with contamination levels throughout the range of interest.In general, a precise equivalence of results can be observed.Formally, only three pairs of discordant results can be observed in samples 11, 15 and 34, but a review of inoculum levels concerning the NMP result shows that they are in an analytical range of 1-3 in which the possibility of one of the pairs of results being 0 is greater than 50% (ISO, 2018).For this reason in this qualitative range (1-3), it is not surprising that the differences between pair of samples.The results show one positive sample in the alternative incubator that becomes negative in the conventional incubator (sample 11) and the opposite (samples 15 and 34).
Regarding the field analysis of the 25 network samples taken in 5 independent days, all results did not detect E. coli nor total coliform.
Concerning the temperature of the sample, in all cases, it was achieved that they remained within the intervals 18 h, 34-38 °C.While it is true that during the winter periods, the time and temperature ranges were lower, the opposite occurred in summer.Our geographical area has mild winters and hot summers with temperatures ranging from 10 °C to 35 °C, respectively.
Graphic 1 depicts the temperature profile of 3 field samplings carried out in the months of February, June and November 2020.With average daily temperatures of 13 °C, 21 °C and 15 °C, respectively (data obtained from www. meteo mataro.com).The data demonstrates that external environmental conditions affect the capacity of the evaluated isothermal medium.Based on several initial tests (data not shown) in a scenario below 10 °C, a complete incubation of 18 h cannot be achieved, but it is close.An improvement in insulation or a higher temperature activation protocol would indeed allow charging to transfer enough energy to achieve a total incubation time.
Even in these non-favourable conditions, it is possible to guarantee a correct incubation during a long sampling plan of 7 h.In this situation, the samples can be transferred to a conventional incubator once they arrive in the laboratory.As stated before, starting the test at the same sampling point means that the effective times to obtain a result are shorter than those needed in the conventional practice.As an example of this approach, in our context, the samples collected at 14:00 will have analytical results at 8:00 the next day instead of midmorning.In case of non-compliance, the application of corrective measures will be much more efficient.

Conclusions
The incorporation of phase change materials in the isothermal media used to transport samples allows it to start incubating the samples immediately after the sampling.In this scenario, the E. coli detection assay initiates immediately.In practice, this allows obtaining the analytical results as fast as possible.This fact allows more effective control of water quality and allows managers to deploy corrective actions a few hours earlier than usual.A second advantage for laboratories will be the possibility of optimising their sampling routes, not making it necessary that, in some cases, they are interrupted by the convenience of starting a test as soon as possible.

Fig. 1 Fig. 2
Fig. 1 Diagram about PCMs behaviour.These compounds can absorb (a) or release (b) much latent heat while transforming physical properties.The solid line shows the temperature profile of PCMs.During the transition to solid-liquid or liquid-solid (grey shadow area), the temperature remains stable with a different thermal profile than a conventional material (dashed line)

Table 1
Results of the verification in the laboratory.Alternative incubation refers to the results obtained with the autonomous incubator with the qualitative method.Conventional incubation refers to the counting method with NMP Q-tray incubated in a standard incubator Vol.: (0123456789)