3.1. Water Conditions for Aquaponic and Hydroponic Systems
The water quality parameters in the aquaponic and hydroponic systems are shown in
Table 2. The aquaponics water temperatures averaged 27.5 ± 1.4, 25.9 ± 1.2, and 27.5 ± 1.7 °C for lettuce, basil, and tomato, respectively, and the hydroponics averaged 20.3 ± 1.2 °C. Tilapia is a tropical fish that can grow in a wide range of temperature from 22 to 34 °C; however, the feed conversion ratio, fish weight gain in relation to feed consumption, and daily weight gain of tilapia are known to be better at temperatures between 26 and 30 °C [
37]. The pH values averaged 6.7 ± 0.5 and 5.7 ± 0.5 in the aquaponics and hydroponic systems, respectively (
Table 2). There were no significant differences between aquaponic and hydroponic systems in EC levels and nitrogen species concentrations, which averaged 1.2 ± 0.5 and 1.6 ± 0.1 and 72.9 and 76.6 mg/L, respectively. The average DO level was significantly lower in aquaponics (6 mg/L) than in hydroponics (10 mg/L), although it was maintained at full saturation in both systems.
It is well-documented that environmental factors, such as temperature, pH, nutrient availability, and DO, affect bacterial populations [
2,
3,
38,
39]. Bacterial pathogens survived for a longer time (up to 91 days) at cold (4−8 °C) and freezing (−4 °C) temperatures than at warmer (20−30 °C) temperatures (up to 84 days) when river water and sterilized soil were inoculated [
40].
E. coli,
L. monocytogenes, and
Salmonella sv. Typhimurium can survive in animal waste at 28 °C during anaerobic digestion [
41], and the population can be affected by the interaction between temperature and pH [
42]. Environmental factors in our study were similar between aquaponics and hydroponics, and, therefore, the differences in temperature and pH between aquaponic and hydroponic systems are not likely to have affected the population of bacteria between the systems. For example, the pH values in aquaponic and hydroponic systems were 6 and 7 which are in the optimal ranges of 6 to 9 for enteric pathogens [
43,
44,
45], and at pH 6, the population densities of
E. coli O157:H7 have been reported to be the same at 20 and 30 °C [
46].
EC is a common indicator of soluble salts dissolved in a nutrient solution because soluble salts carry an electrical charge, and their presence affects the EC of the solution. The availability of nutrients (e.g., nitrogen) and energy sources is a key factor affecting the survival of bacteria in the environment [
3]. It has been demonstrated that the viability of
E. coli O157:H7 is increased in nutrient-rich soils [
47]. Since the high level of nutrients in the hydroponic solution is also ideal for the growth of bacteria, the irrigation water containing a high concentration of nutrients poses the biggest contamination risk in soilless culture systems [
48]. In fact, it was reported that the nutrient reservoirs of hydroponic systems can be a source of contamination [
49,
50]. Enteric pathogens are facultative anaerobic bacteria [
44,
51]. The average DO levels observed in our study were sufficient to allow the growth of
E. coli O157:H7,
Listeria spp., and
Salmonella spp. The implication of these environmental conditions is that pathogenic bacteria can grow in greenhouse-based aquaponic and hydroponic systems if they are introduced by any means.
3.2. The occurrence of Shiga Toxin-Producing E. coli (STEC), Listeria Monocytogenes, and Salmonella spp.
We tested the presence of STECs in fish feces in the aquaponic and aquaculture systems and found STECs in fish feces and the water regardless of the system (
Table 3). The colonies were confirmed after incubation at 37 °C for 20 h and detected by PCR targeting of the
stx1 gene (
Figure S2). STEC were also detected on the root surfaces but were not found to internalize into the roots or the edible parts of lettuce, basil, and tomato grown in the aquaponic system (
Table 3;
Figure S2). These results indicated that water contaminated by fish feces is likely the primary source of root surface contamination.
Irrigation water is often a major source of contamination in outbreaks associated with bacterial pathogens, and this can be particularly true for the field-grown vegetables due to overhead irrigation with contaminated water, damaged roots and shoots by herbivores, and/or groundwater contamination by a plume of wastewater [
49,
52,
53,
54]. However, our study suggests that there is a potential risk associated with aquaponic produce even when the solutions are directly applied to the roots due to water contamination. Our separate aquaculture system confirmed that fish feces were a major source of contamination with STEC in the aquaponic system. These results indicate that introducing contaminated fish can be a source of foodborne pathogens in aquaponics. Previous work has shown that, when fish were reared in ponds where the concentration of coliforms was low, a small number of
E. coli O157:H7 cells were recovered from fish intestines but not fish muscle [
55,
56]. The bacterial intestinal flora of fish can survive up to 84 days in water at 20‒30 °C [
57,
58]. Therefore, Tilapia fish used in this study may have been contaminated before receipt, and the STEC could have been introduced by the fish to the tanks, contaminating the water in the aquaponic systems. Importantly, the contaminated water did not lead to the internalization of STEC into the roots and edible parts of lettuce, basil, and tomato grown in the aquaponic systems suggesting the main source of contamination during production would be from accidental splash of the water to the edible portions of the plant during harvest (
Table 3;
Figure S2).
STEC were also detected in the water of the hydroponic systems. The bacteria may have been introduced accidentally to the hydroponic systems during experimental setup or handling, possibly allowing the formation of biofilms on the surfaces of the hydroponic culture unit, nutrient reservoir, and/or irrigation tubing. A variety of factors, such as water temperature, pH, nutrient availability, solar radiation, the presence of other microorganisms, and the ability to form biofilms, influence both survival and proliferation of STEC in water [
57,
59,
60]. STEC concentrations in irrigation water are also affected by diurnal and seasonal variations [
61]. In fact, the biofilm of
E. coli O157:H7 can grow rapidly and have a high number of adherent cells, even at low nutrient availability and low temperature [
62,
63]. Once formed, it is hard to remove them from the system because the biofilm increases the survival rate of
E. coli O157:H7 even if exposed to hydrogen peroxide, quaternary ammonium sanitizer, or citric acid [
64]. Therefore, we speculate that the hydroponic systems in this study may have been contaminated with STEC due to incomplete sanitation before cultivating plants.
Human activities can also increase the risk of contamination. In a study to determine the source of contamination in hydroponic tomato greenhouses, work and personal shoes were identified as a vehicle for transmission of
E. coli O157:H7 [
65]. In this study, there is the possibility that the bacteria may have been introduced from the aquaponic system to the hydroponic systems from visitors to the greenhouse, during the feeding of fish, during sample collection, and/or by cross-contamination from other human activities [
15,
66]. Contrary to our results, very low levels of generic
E. coli and undetectable pathogenic
E. coli O157:H7 and
Salmonella spp. were found in water and plant samples of aquaponic produce originated from outdoor aquaponic farms located in a tropical climate [
67]. In their study, solar radiation and heat might have played a key role in controlling the bacterial levels in the system [
68,
69]. However, the results cannot be translated into the indoor aquaponic and hydroponic systems located in a temperate climate since solar radiation is often limited, and the environmental conditions are very different from the situation in a tropical climate.
Our results indicated that contamination with bacterial pathogens could likely be reduced in aquaponic and hydroponic systems if the entire systems were thoroughly sanitized before each use and pathogen-free fish were used for the operation. One of the major routes of enteric pathogen internalization is through sites of biological or physical damage and/or through natural openings on the plant surface, such as stomata, lenticels, and sites of lateral root emergence [
4]. The contaminated irrigation water in our study did not have direct contact with the edible aerial parts of the plants as there was a foam board between the water and the plants, and the plants were not disturbed until harvest. It should be noted that damaged roots during handling would create entry points for the bacterial pathogens, and, therefore, the risk of contamination can be avoided if the plant tissues, particularly the roots, are carefully handled during production and harvest. Similarly, previous reports found that foodborne illness was controlled in aquaponics when the following practices were performed: cleaning and sanitation of reusable plastic containers, environmental controls, handwashing, and the use of clean irrigation water [
70,
71].
E. coli can also be internalized if contaminated seeds or water are used during seed germination [
4,
72]. That no internalization was observed in the present study suggested that the seeds and seedlings were free of foodborne pathogens. Several studies demonstrated that bacterial pathogens can contaminate fresh produce in the greenhouse if it is not protected from wild animals or heavy rain causing a flood [
65]; however, this possibility can be eliminated as the source of contamination in this study because the plants were grown in a well-protected greenhouse.
While the optimal water temperature and the pH pose a potential contamination concern with
L. monocytogenes in aquaponic and hydroponic systems (
Table 1), our selective growth medium and colony PCR assay did not detect the occurrence of
L. monocytogenes in fish feces, recirculating water, and plants grown in the systems. These results suggest that there was no contamination source of
L. monocytogenes in either system (
Table 3;
Figure S2). One source of
L. monocytogenes is from the soil, and there was no soil in the greenhouse during this experiment. Other potential sources of
L. monocytogenes are human activities and contaminated seeds. In the event of pathogen introduction to the systems,
L. monocytogenes can internalize through natural openings on the plant surface or through the sites of biological or physical damage [
4]. Although the fish feces or human activities were demonstrated as potential sources of contamination with STEC, our results showed that these were not the major sources of contamination for
L. monocytogenes, and the production practices employed in this study were not associated with the risk of contamination with
L. monocytogenes in either system.
Similarly, various environmental factors, such as nutrient, pH, and water temperature, affect the population of
Salmonella spp. [
43]. The optimal pH range for the growth of
Salmonella spp. is between 6.5 and 7.5, and the temperature range is between 20 and 30 °C in the soil [
40]. Our results showed that there were no
Salmonella spp. in water samples collected from fish tanks, nutrient reservoirs, or hydroponic culture units, although the pH and temperature were within the optimal range for their growth and the nutrient level was sufficient to allow the growth of
Salmonella spp. in both systems (
Table 1). Further,
Salmonella spp. were not present in fish feces or the roots and edible portions of plants (
Table 3;
Figure S2). Outbreaks associated with fruits and vegetables contaminated with
Salmonella spp. have also been attributed to contaminated irrigation water sources [
73]. It should be noted that we used reverse osmosis water to fill and replenish the systems. It is apparent that the plants in the aquaponic and hydroponic systems did not have contact with contamination sources of
Salmonella spp. A previous study reported that tomato fruit was not contaminated with
Salmonella spp. even when the nutrient solution was inoculated with an avirulent strain of S. Typhimurium at a level of 10
5 colony-forming units (CFU)/mL in a hydroponic system [
50] and when the plants were irrigated with 350 mL of 10
7 CFU/mL of S. Montevideo every 2 weeks for 10 weeks [
74].
