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Article

Disinfection Efficacy and Eventual Harmful Effect of Chemical Peracetic Acid (PAA) and Probiotic Phaeobacter inhibens Tested on Isochrisys galbana (var. T-ISO) Cultures

by
Elia Casoni
1,
Gloria Contis
1,
Leonardo Aguiari
2,
Michele Mistri
1,* and
Cristina Munari
1
1
Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Via Luigi Borsari, 46, 44121 Ferrara, Italy
2
Naturedulis s.r.l., Piazzale Leo Scarpa, 45, 44020 Goro, Italy
*
Author to whom correspondence should be addressed.
Water 2024, 16(16), 2257; https://doi.org/10.3390/w16162257
Submission received: 15 July 2024 / Revised: 3 August 2024 / Accepted: 8 August 2024 / Published: 10 August 2024
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Abstract

:
One of the main threats to aquaculture is represented by microbial pathogens, causing mass mortality episodes in hatcheries, which result in huge economic losses. Among the many disinfection methods applied to reduce this issue, the use of chemicals and beneficial microorganisms (probiotics) seems to be the most efficient. The aim of this study is to test the efficacy of two of them: a chemical, peracetic acid (PAA), and a probiotic, Phaeobacter inhibens. Tests were run on microalgae of the species Isochrysis galbana (var T-ISO). For both remedies, the microalgae survival rate and final cell concentration (cell/mL) were monitored. PAA analysis tested six different concentrations of the chemical: 7.5 µg, 10 µg/L, 20 µg/L, 30 µg/L, 40 µg/L, and 60 µg/L. Meanwhile, P. inhibens was tested with a concentration of 104 CFU/mL. Analysis for both the remedies was conducted on a laboratory scale using glass flasks, and on an industrial scale inside photobioreactors (PBRs). Among all the treatments, the one with PAA dosed with a concentration of 60 µg/L gave the best results, as the culture reached a final density of 8.61 × 106 cell/mL. However, none of the remedies involved in the experiment harmed microalgae or their growth. The results match perfectly with the condition requested for the tested remedies: to obtain an optimal breakdown of pathogens without interfering with culture growth. These features make PAA and P. inhibens good candidates for disinfection methods in aquaculture facilities.

1. Introduction

As mollusks represent around 17% of the total aquaculture production for the European Union (EU) [1], bivalve species like clams, oysters, and others are considered as important bred species, and their global farming maintains a continuous annual growth rate [2], supporting a significant portion of aquaculture worldwide [1]. Despite the high demand and the increasing global rate of aquaculture production, in some countries the growth has stagnated during the last two decades [3].
Issues related to mollusk farming, mostly larvae, are multiple and due to several factors: low water quality, high seeding density, sub-optimal temperature, pathogens, and algal metabolites. The majority of mortality events are associated with an elevated bacterial charge. For this reason, high water quality is considered the main precaution to avoid illnesses and pathologies (FAO, Food and Agricultural Organization, United Nations, 1990).
Usually, water disinfection is one of the most useful tools in hatcheries to exclude specific pathologies, or as a precautionary measure to avoid mortality events. In mollusk farming this precaution also has to be taken in microalgae culture, used to feed every life stage of the reared animals. Even though they should be axenic cultures, tanks or photobioreactors (PBRs), where they are usually inoculated and grown, may contain pathogens.
Some of the most common disinfection methods include chemical reagents. Among these, peracetic acid (PAA) is considered as a strong disinfectant with a wide antimicrobial spectrum of activity [4,5]. Moreover, it requires few hours to degrade in water and releases harmless, neutral residuals such as acetic acid and hydrogen peroxide [6].
PAA has already been studied as a long-term prophylactic method to reduce the total bacterial charge, or specific bacteria, like Aeromonas salmonicida or Flavobacterium sp. Choosing this remedy can reduce the antibiotics usage in aquaculture [7]. Short-term treatments with a high concentration of PAA seem to be effective, reducing bacterial concentration and preventing stable biofilm formation [8].
A more recently developed method for water disinfection is the use of probiotic organisms. According to FAO, a probiotic is considered as a “living organism that can grant a beneficial effect to the host, if dosed in the right amount”. Most of the studies related to probiotics have focused on the intestinal microbiota of fish and invertebrates [9,10,11] but their use could be extended to hatcheries and breeding environments [12,13,14].
Phaeobacter inhibens is a well-studied, Gram-negative bacterium, already known for its probiotic activity in aquaculture. It is often considered as a heterotrophic marine model organism, and is useful in investigating interaction mechanisms with higher organisms [15]. Most studies conducted on this microorganism have investigated its efficacy against bacteria of the genus Vibrio (e.g., Vibrio sp., Vibrio anguillarum, Vibrio vulnificus), but its probiotic activity has also been observed against other pathogens [16].
Its wide-spectrum efficacy is mostly due to the production of secondary metabolite tropodithietic acid (TDA) [17]. In addition, as a member of the widespread Roseobacter clade, P. inhibens is known to be an excellent colonizer of environmental surfaces [16], and is often already present in rearing environments. These features make P. inhibens a good alternative solution during the water disinfection phase, or in the early larval stages of reared animals [18].
Based on these features, the two remedies seem to represent good candidates as disinfectant methods in aquaculture facilities. This study tried to confirm this hypothesis, evaluating their efficacy on microalgae cultures of the species Isocrysis galbana (var. T-ISO) grown in the Naturedulis s.r.l. hatchery, a company involved in rearing some of the most important mollusk species in Italy, like clams and oysters.
To be considered as good candidates, the tested remedies must fulfill two conditions: (1) obtaining an optimal breakdown of pathogen charge, (2) without interfering in algae growth.

2. Materials and Methods

The experiment was set up preparing three different trials to investigate the effect of the two candidate disinfection remedies, peracetic acid (PAA) and a probiotic, on microalgae of the species Isochrysis galbana (var. T-ISO): (1) PAA trial on a laboratory scale, (2) probiotic (Phaeobacter inhibens strain DSM17395) trial on a laboratory scale, (3) photobioreactor (PBR) trial involving both the chemical and probiotic, to observe their effects on a larger scale.
Microalgae involved in all trials were taken randomly from axenic stock culture, grown in sterile sea water (SSW), and maintained in Naturedulis facilities. Environmental parameters were constantly monitored, setting the temperature at 24 ± 1 °C, salinity of the culture medium around 30 psu, and pH at 8.0 ± 0.1. Constant aeration was obtained through an insufflation system, equipped with a 0.22 µm filter, and illumination was maintained at 100 µeinstein (µE).
All materials (except for PBRs) that had contact with algae were previously autoclaved or air sterilized.

2.1. Laboratory PAA Trial

A total of 150 mL of the algae stock culture was put in 500 mL Erlenmeyer flasks and enriched with Walne medium (see Table S1 for composition), dosed with a concentration of 1 mL/L.
Microalgae were maintained in controlled environmental conditions, as described above. Cultures had been treated with PAA dosed with different concentrations, to observe if an increasing concentration inhibited algal growth.
Six treatments were set up, adding the chemical obtained from a stock solution of PAA 15%: 7.5 µg/L, 10 µg/L, 20 µg/L, 30 µg/L, 40 µg/L, and 60 µg/L. An additional treatment was prepared without PAA, as a control group. All treatments were run in triplicate. The average microalgae concentration at the beginning of the analysis (T0) was 1.63 × 106 cell/mL. The experiment took two days, with PAA added at T0. Cell density was monitored daily through a Burker cell counting chamber, together with pH (measured with a pH-meter).

2.2. Probiotic Trial on a Laboratory Scale

The bacterial strain Phaeobacter inhibens DSM17395 [19] was obtained from the Leibniz institute (DSMZ—German Collection of Microorganisms and Cell Cultures GmbH), Braunschweig—Sud, Germany. A stock culture was grown and maintained in Marine Broth (Millipore—Sigma-Aldrich) at 24 ± 1 °C, with gentle shaking. A Marine Broth culture medium was prepared adding 37.4 g of powder to 1 L of distilled water.
Before measuring the bacterial density, 50 mL from the bacterial stock culture was transferred in a Falcon tube and centrifuged at 4500× g for 3 min to harvest cells. Then, cells were washed twice in SSW and the pellet was resuspended using a vortex mixer.
Bacterial cell density was calculated counting the colony forming units (cfu). The washed culture was serially 1:10 diluted, and the optical density at 550 nm (OD550) was measured with a spectrophotometer. In addition, 100 µL for each dilution was plated on Marine Agar and incubated at 24 ± 1 °C for 48 h. OD550 values were compared with the corresponding number of cfu on the plates, finding that a unit of absorbance was equal to 109 cfu/mL.
An analysis of microalgae was set up in 500 mL Erlenmeyer glass flasks filled with 150 mL from T-ISO stock culture and Walne medium (1 mL/L), with the same environmental conditions as the PAA trial.
Two treatments were set up: one control group with only T-ISO, and one experimental group containing T-ISO treated with P. inhibens strain DSM17395, dosed with a concentration of 104 cfu/mL at the beginning of the experiment (T0). Both were run in triplicate. The experiment lasted for three days. The initial concentration of microalgae at T0 was around 1.20 × 106 cell/mL, then was monitored daily with a Burker cell counting chamber.

2.3. Photobioreactor Trial

Both the PAA and probiotic treatments were also tested on industrial scale, in photobioreactors located in the Naturedulis s.r.l. hatchery. Cultures in PBR had a total volume of 150 L: around 18 L of T-ISO culture, with the addition of filtered natural sea water (NSW) to reach the final volume. A Walne culture medium was added with a concentration of 1 mL/L. Environmental conditions were constantly monitored. The temperature was set at 24 ± 1 °C, salinity around 30 psu, and constant illumination to 100 µE. Aeration was granted through an insufflation system with variable CO2 levels, to maintain the pH level around 8.0 ± 1 point, monitored with a Mettler-Toledo InPro 3250i sensor (Mettler-Toledo, Milano, Itlay).
The experiment lasted two days, with both PAA and P. inhibens treatments administered at T0, with a concentration of 60 µg/L and 104 cfu/mL, respectively. An additional PBR was left without treatments as a control group. The average T-ISO concentration at T0 was around 1.32 × 106 cell/mL. Cell growth and eventual pH variation were monitored daily as in laboratory-scale trials.
In addition, at the end of the analysis, rapid efficacy evaluation tests were carried out to investigate the presence/absence of Vibrio bacteria in control and treated PBRs. To do so, an aliquot of 100 µL from each of the three PBRs involved was plated on TCBS agar (OXOID—TermoFisher Scientific), prepared adding 88 g TCBS powder to 1 L of distilled water. The plates were incubated overnight at 24 ± 1 °C, to observe the eventual growth of Vibrio colonies.

2.4. Statistical Analysis

To compare the effects of the different treatments, the average specific growth rate (µ) has been calculated for every culture involved, through the following edited equation from the Alga Growth Inhibition Test (OECD, 1984):
μ = lnN n − lnN 0 T n − T 0
where:
  • N0 = measured number of cells/mL at time T0.
  • Nn = measured number of cells/mL at time Tn.
  • T0 = time of the first measurement after the beginning of the test.
  • Tn = time of the nth measurement after the beginning of the test.
All the statistical analyses were run on RStudio version 2024.04.2+764. One-way analysis of variance (ANOVA) was carried out through a “car” package. Pairwise differences among treatments were observed through Tukey’s HSD test, run with a “multcompView” package. Related graphs were created with a “ggplot2” package.
All the analyses were considered significant with a 95% level of confidence (p-value < 0.05).

3. Results

3.1. Peracetic Acid Trial

Six different peracetic acid (PAA) concentrations were tested on I. galbana (var. T-ISO), on a laboratory scale, to observe if one or more of them inhibited culture growth. T-ISO concentrations at T0 ranged from 1.39 ± 0.129 × 106 cell/mL (7.5 µg/L PAA group) to 1.97 ± 0.081 × 106 cell/mL (30 µg/L PAA group).
None of the treatments showed signs of inhibiting algal growth for the entire duration of the experiment. After 48 h (T2) culture concentration reached a mean value of 6.19 ± 1.299 × 106 cell/mL. The highest value was registered in the 60 µg/L treatment, where cultures reached a density of 8.64 ± 3.532 × 106 cell/mL. The lowest final cell density was measured in the 30 µg/L treatment, with 4.62 ± 2.305 × 106 cell/mL, making it the only group with a final cell density lower than the control (Figure 1a–c). Despite the gap between the highest and the lowest cell concentration values, an analysis of variances showed no significance differences (1-way ANOVA; p-value > 0.05). Also, Tukey’s test detected no pairwise statistical significance differences, with the only exception being the control group and the 60 µg/L PAA group after 24 h (T1) (p-value < 0.05).
The average specific growth rate value (µ) for PAA trials reflected previous results, as the highest value (0.033 × 106 cell/mL/h) was observed in the 60 µg/L treatment and the lowest (0.15 × 106 cell/mL/h) in the 30 µg/L treatment (Figure 2a). Here no statistical differences were detected through ANOVA or Tukey’s HSD test (p-value > 0.05).
pH ranged in a very small interwall enclosed between 8.00 and 8.60 for all the treatments, during the entire duration of the experiment. The highest value corresponded to the 60 µg/L group, reaching 8.61 at T2.

3.2. P. inhibens Trial

The results from the analysis carried out involving P. inhibens strain DSM17395 as an antibacterial remedy reveal that the probiotic is not harmful to T-ISO growth when dosed with a concentration of 104 cfu/mL. The initial concentration at T0, for both the experimental and control group, was 1.20 × 106 cell/mL. Cell density for the group treated with probiotic was always higher during the entire duration of the experiment, reaching a mean value of 5.07 ± 0.310 × 106 cell/mL after 72 h (T3). The control group registered a cell density of 4.46 ± 0.465 × 106 cell/mL (Figure 3). However, both analysis of variances (1-way ANOVA) and Tukey’s HSD test showed that the difference between the two values was not statistically significant (p-value > 0.05). The same results have been observed for average specific growth rate values. The culture treated with the probiotic registered a µ value of 0.020 × 106 cell/mL/h while the control group was slightly lower, with 0.018 × 106 cell/mL/h (Figure 2b). The resulting differences were not statistically significant (1-way ANOVA, Tukey’s test; p-value > 0.05).

3.3. Photobioreactor Trial

The trial conducted on photobioreactors (PBRs) located at the Naturedulis hatchery also showed the efficacy of both PAA and P. inhibens treatments on industrial scale, as none of the cultures showed signs of growth inhibition. PAA treatment cultures, with a final concentration of 4.15 ± 0.085 × 106 cell/mL, gave the highest results in terms of cell growth, after two days. PBR treated with the probiotic showed a final concentration of 3.55 ± 0.250 × 106 cell/mL. However, at T2, both treated PBRs’ cell density resulted as being higher than control group (3.46 ± 0.032 × 106 cell/mL) (Figure 4). Despite the similar T2 results, statistical analysis showed a significant difference among the three groups (1-way ANOVA; p-value < 0.05). In fact, pairwise Tukey’s HSD tests revealed a significant difference when both control and probiotic groups were compared with the treatment group (p-value < 0.05).
The average specific growth rate for every culture was calculated, and PAA-treated and probiotic-treated PBRs showed a µ-value of 0.030 × 106 cell/mL/h and 0.026 × 106 cell/mL/h, respectively. The control group instead registered a µ-value of 0.012 × 106 cell/mL/h (Figure 2c). One-way ANOVA showed a statistically significant difference among the three PBRs. When analyzed through Tukey’s HSD test only the difference between the PAA and control groups were significant (p-value < 0.05).
pH values ranged in a very small interwall of values, between 7.98 and 8.54, for the entire duration of the analysis. Finally, the rapid efficacy evaluation tests on PBR through plating on TCBS agar revealed the presence of Vibrio colonies only in the control group photobioreactor (Figure 5).

4. Discussion

The aim of this work was to investigate the efficacy of two disinfection methods, to minimize production losses in aquaculture facilities. The major cause of these losses, which mainly affect larvae, is due to pathogens typically present in this environment [20]. Vibrio bacteria are one of the most present groups, showing high colonization potential in marine and brackish environments, thanks to their metabolic versatility and genetic variability [21]. To minimize their presence, prophylactic methods require disinfectants that are effective against pathogens but harmless to animals and microalgae [22]. The candidate methods selected in this study were chemical peracetic acid (PAA) and bacterial probiotic Phaeobacter inhibens. Both were tested on microalgae cultures of Isochrysis galbana (var. T-ISO). This species is one of the most commonly employed microalgae strains in mollusk farming. Its biochemical profile, rich in fatty acids and ascorbic acid, makes it a good fit to feed mollusk larvae [23]. In addition, its ability to adapt to a wide range of environmental conditions (e.g., temperature or photon flux) make it a good choice for mass culture [24].
The average microalgae concentration at the beginning of each trial (T0) was set under 2.0 × 106 cell/mL, to ensure that the culture had not yet reached the exponential phase. This way, any potential slowdown in growth would have been observable.
However, the two remedies resulted in no negative influence on microalgae growth, and almost every treatment tested showed better results than the control groups.
PAA has been used for decades, as a lot of studies documented its strong disinfectant action against viruses, bacteria, fungi, protozoa, and others [4,5,25,26], whilst degrading in a very small amount of time. Its decay is controlled by such abiotic parameters as organic matter (dissolved and particulate), temperature, light, pH, and salinity, and it has been observed to be four time faster in marine water than in fresh water [27]. In environments like these, PAA completely degrades within a few hours, producing acetic acid (CH3COOH), hydrogen peroxide (H2O2), and water [6]. The literature is poor in pre-existing studies regarding microalgae tolerance when treated with PAA (in particular T-ISO); thus, a preliminary laboratory trial has become necessary, testing different concentrations of the chemical. It has been carried out involving six different PAA concentrations: 7.5 µg/L, 10 µg/L, 20 µg/L, 30 µg/L, 40 µg/L, and 60 µg/L. As observed in the results, none of the treatments showed signs of inhibition in culture growth, and, after 48 h (T2), some of them are even higher than the control group, in terms of cell/mL.
pH variation was monitored along the entire duration of the experiment and the results ranged around the interval of 8.0–8.5 points, optimal for T-ISO, without inhibiting its growth [28]. Even the highest PAA concentration tested was not enough to provoke a significant decrease in pH levels.
Observing these results, we can assume that (except for 60 µg/L treatment) there is no linear correlation between the increasing PAA concentration and microalgae growth, as a higher amount of the chemical does not necessarily result in a higher or lower final concentration of microalgae.
The same goes for average specific growth rate values (µ). Observing Figure 4, it is clear how, for every treatment, µ values follow the same trend as cell growth results. In addition, it can be seen that the treatments cluster within two groups, the first with µ values lower than 0.025 × 106 cell/mL/h, comprising 10 µg/L, 20 µg/L and 30 µg/L treatments; the second, conversely, in which µ values have been registered to range above 0.025 × 106 cell/mL/h, for the remaining four groups (7.5 µg/L, 40 µg/L, 60 µg/L, and the control group). These results support the hypothesis that, among the tested treatments, a higher amount of PAA does not necessarily result in a better growth rate, and vice versa. Considering the minimal variation of pH values in tested culture, the efficacy of the remedy is identified in another feature. According to [29], the antimicrobial property of PAA is due to the ability to alter the permeability of the cell membrane and interfere with protein synthesis, oxidizing the sulfhydryl and sulfur bonds in bacterial cells, causing death.
Also, probiotic treatment (both in the laboratory and PBR) showed no sign of inhibition on microalgae cultures. However, despite the positive growth of the culture, final concentration results were lower when compared with microalgae treated with PAA, with few exceptions (10 µg/L and 30 µg/L treatments). However, Tukey’s HSD test does not consider this difference as statistically significant (p-value > 0.05).
An explanation for this result could be found in [30], which studied the interaction between different bacterial strains and some microalgae species used for farming purpose. In that work, the interaction between Phaeobacter inhibens and Isochrysis galbana (var. T-ISO) was also observed, and resulted in a slight drop in microalgae growth. This seems to be due to a competitive advantage in the bacterial–microalgae co-culture, as the bacteria outcompeted microalgae for nutrient intake [31,32]. In addition, the proliferation of bacterial cells and the release of their secondary metabolites contribute to decreasing the penetration of light, reducing the phototrophic activity of microalgae.
As already stated in many other studies that involve P. inhibens, there are two main features that make this organism a useful choice against Vibrio pathogens: tropodithietic acid (TDA) production and biofilm formation.
TDA is an antibacterial compound detected in members of Rhodobacteriaceae family and plays a key role in the colonization success of the probiotic [33]. Its mode of action consists of disrupting the membrane gradient of the pathogen modifying the proton transport inside and outside the cell, facilitating a one-to-one exchange of H+ for a charged metal ion [34].
In addition, the efficacy of this metabolite is enhanced by the difficulty of pathogens to develop resistance forms against it [35,36].
The second feature, biofilm formation, allows P. inhibens to physically occupy sites otherwise available for pathogens [37], and it works synergically with the TDA. In fact, it has been proven that the more extended the biofilm, the higher the production of this molecule [38].
The only trial that shows statistically significant differences among treatments was the one conducted in PBRs (1-way ANOVA; p-value < 0.05). In the control group, the final cell concentration and average specific growth rate were lower than the experimental groups, and Tukey’s HSD test detected a significant difference when the control group and PAA treatment were compared. In addition, when the cell/mL parameter is considered, the comparison between the PAA group and the probiotic group also showed a significant difference. All the data related to Tukey’s test result are shown in Table 1.
Finally, the practical effect of the two remedies has been revealed trough the rapid test on TCBS agar for the presence/absence of Vibrio bacteria. This genus in fact includes a large number of species often responsible for mortality events in aquaculture facilities [39].
Among the three plates prepared (one for each PBR involved in experiment), only the control one turned a yellowish color, and many colonies were visible on the surface (Figure 5a). The chemical composition of TCBS includes sucrose and bromothymol blue. The fermentation of sucrose operated by Vibrio bacteria provoked a lowering in pH level, which in turn induced bromothymol blue to turn yellow.
The other two plates displayed “clean” results instead, and the color of the medium remained green (Figure 5b,c)—a sign of a very low concentration, or absence, of Vibrio bacteria.
Even though both remedies revealed their efficacy as a disinfectant, PAA seems to be a better choice and more efficient than P. inhibens. In fact, cell growth and average growth rate are always higher when algae are treated with PAA. From this perspective, the use of this remedy is recommended in aquaculture facilities, as it is demonstrated to be a safe and environmentally friendly alternative when compared with other chemicals or antibiotics.
The promising results obtained from this study indicate that research in the aquaculture field is heading in the right direction. However, there are several aspects that need to be investigated.
The efficacy of probiotics has been largely studied in the aquaculture field, testing a wide number of species, but remains a topic of research. In fact, probiotics represent a valuable alternative to antibiotics and vaccines in the aquaculture field, and so, further studies on their efficacy are recommended. As an example, it has been observed that I. galbana shows antibacterial activity itself, increasing the synthesis of fatty acids in the presence of pathogens [40,41]. Polyunsaturated fatty acids (PFUAs), such as linoleic acid and gamma-linoleic acids, inhibit the growth of Vibrio bacteria but appear to be harmless for other heterotrophic species [42]. This aspect should be further investigated to observe if the co-culture with P. inhibens also results in increased production of these molecules and a consequent inhibition of probiotic growth.
Otherwise, few are the studies that focus on PAA as a disinfection remedy in aquaculture [6,7,27,43], and fewer are the ones that take microalgae as a target organism [22]. From this perspective, this study may represent a useful data source to set up future research. The efficacy should be tested on other harmful microorganisms, since Vibrio bacteria are not the only threat in aquaculture facilities [44,45]. Moreover, the sensitivity of microalgae species different from T-ISO can be investigated, to assess the usefulness of this remedy in different types of aquaculture.

5. Conclusions

The suitability of remedies tested in this study has been confirmed as the analysis revealed how both PAA and P. inhibens fulfill the conditions requested, obtaining an optimal breakdown of pathogens (observed through TCBS testing) without interfering in the growth of microalgae culture, as shown in laboratory and PBR trials.
This is not the first study investigating the efficacy of these methods, but it could contribute to increasing knowledge in aquaculture sector, improving the farming condition in rearing facilities and consequently the quality of the final product.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w16162257/s1, Table S1: The composition of Walne nutrient medium.

Author Contributions

Conceptualization, E.C. and L.A.; methodology, E.C. and L.A.; validation, C.M., M.M. and L.A.; formal analysis, E.C. and G.C.; resources, C.M., M.M., L.A.; data curation, E.C.; writing—original draft preparation and revision, E.C., C.M., M.M. and L.A.; supervision, C.M., M.M. and L.A.; funding acquisition, E.C., C.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work has been supported by Italian National Operational Program (OPN) on research and innovation through Quota ricerca—Area Green—Sostenibilità Ambientale e Benessere—Casoni Elia; ID: 2021-DOTT-DM-1061_CE_01_RIC.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We acknowledge Naturedulis s.r.l. company and its staff for hosting and permitting the accomplishment of this study by providing facilities and equipment. We also thank the anonymous reviewers whose constructive criticism helped to improve the quality of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 16 02257 g001
Figure 1. I. galbana growth expressed as 106 cell/mL (±SD) (left graphs) and relative pH variation (±SD) (right graphs) for every treatment tested in the laboratory trial involving PAA. Each graph shows the results of two treatments and the control group (CTRL): (a) 7.5 µg/L and 10 µg/L treatments; (b) 20 µg/L and 30 µg/L treatments; (c) 40 µg/L and 60 µg/L treatments. The trial lasted from T0 (0 h) to T2 (48 h).
Figure 1. I. galbana growth expressed as 106 cell/mL (±SD) (left graphs) and relative pH variation (±SD) (right graphs) for every treatment tested in the laboratory trial involving PAA. Each graph shows the results of two treatments and the control group (CTRL): (a) 7.5 µg/L and 10 µg/L treatments; (b) 20 µg/L and 30 µg/L treatments; (c) 40 µg/L and 60 µg/L treatments. The trial lasted from T0 (0 h) to T2 (48 h).
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Figure 2. I. galbana average specific growth rate (µ) related to every treatment tested in each trial, expressed as 106 cell/mL/h. (a) PAA laboratory trial: the blue square contains the treatments with µ values grouping under the 0.025 × 106 cell/mL/h threshold (bold horizontal line); the red square contains the µ values grouping above the 0.025 × 106 cell/mL/h. No statistical differences were detected among treatments. (b) P. inhibens laboratory trial. No statistical differences were detected among treatments. (c) PBR trial: different letters (“x”, “y”) indicate a significant statistical difference, according to Tukey’s HSD test (p-value < 0.05).
Figure 2. I. galbana average specific growth rate (µ) related to every treatment tested in each trial, expressed as 106 cell/mL/h. (a) PAA laboratory trial: the blue square contains the treatments with µ values grouping under the 0.025 × 106 cell/mL/h threshold (bold horizontal line); the red square contains the µ values grouping above the 0.025 × 106 cell/mL/h. No statistical differences were detected among treatments. (b) P. inhibens laboratory trial. No statistical differences were detected among treatments. (c) PBR trial: different letters (“x”, “y”) indicate a significant statistical difference, according to Tukey’s HSD test (p-value < 0.05).
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Figure 3. I. galbana growth related to the laboratory trial involving P. inhibens strain DSM17395, expressed as 106 cell/mL (±SD). The trial lasted from T0 (0 h) to T3 (72 h).
Figure 3. I. galbana growth related to the laboratory trial involving P. inhibens strain DSM17395, expressed as 106 cell/mL (±SD). The trial lasted from T0 (0 h) to T3 (72 h).
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Figure 4. Growth curve and pH variation related to the PBR trial involving the 60 µg/L PAA treatment, P. inhibens treatment, and a control group. The results for cell growth are expressed as 106 cell/mL (±SD). The trial lasted from T0 (0 h) to T2 (48 h).
Figure 4. Growth curve and pH variation related to the PBR trial involving the 60 µg/L PAA treatment, P. inhibens treatment, and a control group. The results for cell growth are expressed as 106 cell/mL (±SD). The trial lasted from T0 (0 h) to T2 (48 h).
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Figure 5. Rapid efficacy evaluation test results, to assess the presence/absence of pathogen bacteria belonging to genus Vibrio in cultures analyzed during the PBR trial: (a) PBR control group; (b) PBR treated with PAA; (c) PBR treated with probiotic P. inhibens.
Figure 5. Rapid efficacy evaluation test results, to assess the presence/absence of pathogen bacteria belonging to genus Vibrio in cultures analyzed during the PBR trial: (a) PBR control group; (b) PBR treated with PAA; (c) PBR treated with probiotic P. inhibens.
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Table 1. Complete dataset of Tukey’s HSD p-values for pairwise comparison among treatments tested in each of the three trials. Values in red refer to final microalgae concentration comparison (cell/mL); values in blue refer to average specific growth rate comparison (µ). Asterisk (“*”) indicates a statistically significant difference (p-value < 0.05).
Table 1. Complete dataset of Tukey’s HSD p-values for pairwise comparison among treatments tested in each of the three trials. Values in red refer to final microalgae concentration comparison (cell/mL); values in blue refer to average specific growth rate comparison (µ). Asterisk (“*”) indicates a statistically significant difference (p-value < 0.05).
Tukey’s HSD Test Results for Treatments Pairwise Comparison
Trial PAAP. inhibensPBR
TreatmentCTRL7.5 µg/L10 µg/L20 µg/L30 µg/L40 µg/L60 µg/LCTRL104 cfu/mLCTRL60 µg/L104 cfu/mL
PAACTRL 0.9990.9970.9990.9880.9990.961
7.5 µg/L0.999 0.9930.9990.9780.9990.976
10 µg/L0.9930.973 0.9990.9990.9990.741
20 µg/L0.9300.8510.999 0.9970.9990.921
30 µg/L0.6630.6630.9840.999 0.9960.648
40 µg/L0.9990.9950.9990.9930.941 0.926
60 µg/L0.9990.9990.9300.7580.5490.979
P. inhibensCTRL 0.130
104 cfu/mL 0.135
PBRCTRL 0.7360.003 *
60 µg/L 0.022 * 0.007 *
104 cfu/mL 0.0550.731
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Casoni, E.; Contis, G.; Aguiari, L.; Mistri, M.; Munari, C. Disinfection Efficacy and Eventual Harmful Effect of Chemical Peracetic Acid (PAA) and Probiotic Phaeobacter inhibens Tested on Isochrisys galbana (var. T-ISO) Cultures. Water 2024, 16, 2257. https://doi.org/10.3390/w16162257

AMA Style

Casoni E, Contis G, Aguiari L, Mistri M, Munari C. Disinfection Efficacy and Eventual Harmful Effect of Chemical Peracetic Acid (PAA) and Probiotic Phaeobacter inhibens Tested on Isochrisys galbana (var. T-ISO) Cultures. Water. 2024; 16(16):2257. https://doi.org/10.3390/w16162257

Chicago/Turabian Style

Casoni, Elia, Gloria Contis, Leonardo Aguiari, Michele Mistri, and Cristina Munari. 2024. "Disinfection Efficacy and Eventual Harmful Effect of Chemical Peracetic Acid (PAA) and Probiotic Phaeobacter inhibens Tested on Isochrisys galbana (var. T-ISO) Cultures" Water 16, no. 16: 2257. https://doi.org/10.3390/w16162257

APA Style

Casoni, E., Contis, G., Aguiari, L., Mistri, M., & Munari, C. (2024). Disinfection Efficacy and Eventual Harmful Effect of Chemical Peracetic Acid (PAA) and Probiotic Phaeobacter inhibens Tested on Isochrisys galbana (var. T-ISO) Cultures. Water, 16(16), 2257. https://doi.org/10.3390/w16162257

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