3.2. Physical and Chemical Parameters of Drainage Waters
The type of substrate affected the physical and chemical parameters of the drainage waters. In a closed system of soilless cultivation, the drainage waters are reused by including them in the nutrient solution recirculation system. However, the application of drainage waters in the nutrient solution recirculation system depends on their quality, including such parameters as pH, mineral content, turbidity, microbial load and the presence of pathogens. The reuse of drainage waters for plant fertigation allows one to save water and mineral fertilizers, but only if the contamination of the leachates is not high (low turbidity and microorganisms density) and allows for their effective disinfection. Therefore, when choosing growing substrates for a closed system of soilless cultivation, attention should be paid to the characteristics of the leachates.
In our studies, the pH of the leachates from Carbomat was generally similar to the pH of the nutrient solution, which had an average level of pH 5.6 (
Figure 1), except for the first two weeks of cultivation, where this value was about one unit higher.
The drainage waters from rockwool and coir substrate presented pH levels of 6.2 and 6.1, respectively, both values being higher than for Carbomat. Biopot substrate, at the beginning of tomato cultivation, strongly alkalized the medium (pH nearly 8.0) in the root zone. However, after one month, the pH of the leachates from Biopot gradually decreased, and was similar to the pH the leachates from the rockwool and coir substrate. Similar results were obtained by Breś and Ruprik [
16] and Breś [
29]: the pH of the drainage waters from tomato cultivation on rockwool and coir substrate increased by one unit in relation to the dosed medium. The low pH in the Carbomat substrate was also observed by Dyśko et al. [
25] in their earlier experiments. The optimum pH in the root zone for most crops in soilless cultivation ranges from 5.5 to 6.5 [
31]. However, according to Adams [
32], pH values 5.0–5.5 and 6.5–7.0 still are not harmful for most plants, but pH >7 or <5 reduces their growth [
33].
The concentrations of minerals in drainage waters leaking from the cultivation mats, expressed as EC, were significantly higher than in the nutrient solution used for plant fertigation, during all vegetative seasons (
Figure 2).
This was also reported by Moya et al. [
34], who grew tomato plants in coconut mats. They had used nutrient solution with different EC values (EC 2.2, 3.5 and 4.5 mS.cm
−1), and in drainage waters the EC increased proportionally to the initial concentration of nutrients (EC 3.3, 5.6 and 7.7 mS.cm
−1). The higher EC of drainage waters is the result of nutrients concentration caused by increased transpiration during sunny weather in relation to their uptake by plants. In our studies, the highest content of elements was in the mats from the rockwool and coir substrate (EC 5.3 mS.cm
−1 and EC 5.1 mS.cm
−1, respectively), while the lowest concentration of mineral components was characteristic of leachates from Carbomat and Biopot, due to the biological and chemical sorption of these substrates (EC 4.5 mS.cm
−1 and EC 4.9 mS.cm
−1, respectively).
Hydroponic wastewater is particularly rich in nitrogen and phosphorus. The excess amount of runoff contains nitrate in quantities ranging 150–500 mg L
−1 and phosphorus in quantities ranging 30–100 mg L
−1 [
35]. Such a high concentration of the nutrients in hydroponic effluents discharged into the environment is dangerous. In a natural water system, high levels of N and P contribute to algal bloom [
36]. In the present studies, the content of N-NO
3 in the leachates from rockwool, Carbomat and coir substrate was at a similar level (
Figure 3). The average concentration of N-NO
3 in these leachates ranged 312–347 mg L
−1 (
Table 3). A significantly lower content of nitrate nitrogen was found in the drainage water from Biopot. Its average concentration was about 40% lower than in the other leachates. The lowest content of nitrate nitrogen in Biopot drainage waters was detected at the beginning of tomato growth. According to Dyśko and Kaniszewski [
24], Biotop is characterized by the biological sorption of nitrogen, especially in the initial period of cultivation.
The concentration of phosphorus was the highest for the leachates from the coir substrate and rockwool (
Figure 4), with a mean values of 69 and 79 mg L
−1, respectively (
Table 3). The concentration of this element in Carbomat drainage waters was close to nutrient solution. The lowest content of phosphorus was found in the drainage waters from Biopot (
Figure 4). The average value of this nutrient in Biopot leachates was 18 mg L
−1, and it was significantly lower than in the drainage waters leaking from other substrates (
Table 3).
Drainage waters also showed an increased concentration of potassium compared to the dripper (
Figure 5). However, the type of substrate only slightly differentiated K content in the initial period of tomato cultivation. During the first two months of tomato cultivation, the highest potassium concentration was found in the drainage waters from coir substrate, while the lowest was from Carbomat. Generally, the substrate type did not influence potassium content in the drainage waters (
Table 3).
The concentration of calcium and magnesium was twice as high as in the standard nutrient solution used for fertigation. The highest content of these minerals was found in rockwool leachates, although the differences generally were not significant comparing to other substrates. The substrate type had no influence on the amount of N-NH4.
As for micronutrients, their concentration increased in the drainage waters (
Table 4), especially in the case of boron, which increased sevenfold compared to the nutrient solution (0.20 mg L
−1 for nutrient solution and on average 1.3 mg L
−1 for the leachates). Moreover, a high chlorine content was determined in the overflow from tomato cultivation on Biopot and coir substrate mats (308 mg L
−1 and 163 mg L
−1, respectively). Kleiber [
17] pointed out that less environmental contamination is generated in the case of cultivation on organic substrates. However, this has not been confirmed in these studies, comparing rockwool (mineral substrate) and other organic mats (coir substrate, Carbomat, Biopot), except for the lower N-NO
3 and P amounts in Biopot leachates.
Turbidity is one of the most important indicators of water quality. It is caused by suspended and colloidal matter, such as clay, silt, fine particles of organic and inorganic matter, plankton and microorganisms, as well as by colored organic and inorganic compounds [
37]. Turbidity can be used as a measure of sediment amount [
38], and in some cases may indicate the microbial contamination of water [
37]. In soilless plant cultivation, high amounts of sediments may block fertigation equipment, and in the closed system with nutrient solution recirculation, the reuse of drainage waters may be restricted due to the low efficiency of disinfection. High turbidity caused by fine matter particles deteriorates the quality of disinfection by filtration or UV radiation [
35,
39].
In these studies, the most polluted drainage waters were found after the cultivation of tomato on Biopot (
Figure 6). This was caused by the leaching of organic particles from this substrate. For five weeks the turbidity in the drainage waters from Biotop exceeded 350 NTU. The mean value of turbidity determined by the nephelometric method was 185 NTU. The microbial analyses of drainage waters, presented below, indicate that the high turbidity in Biopot leachates was not related to the multiplication of microorganisms. The microbial density in all types of drainage water was comparable (
Table 5 and
Table 6). However, these results indicate that drainage waters from Biopot are not suitable for recirculation. The drainage waters from tomato cultivation on rockwool, Carbomat and coir substrate were characterized by low turbidity (10.4, 8.5 and 6.1 NTU, respectively), with the coir substrate leachate having a brown color for almost two months.
3.3. Microbial Analyses of Drainage Waters
A higher number of total bacteria and fungi was determined in nutrient solutions taken from the root zone of tomato plants, grown in different types of substrates, compared to the drainage waters (
Table 5 and
Table 6). However, the differences were not significant. These results are confirmed by the studies of Koohakan et al. [
40], who were detecting more bacteria and fungi on roots than in nutrient solution, in various types of tomato soilless production systems. The higher number of microorganisms in the root zone is the result of the passive and active leakage of root exudates, which serve as nutrient source for these organisms [
41,
42].
Bacteria are the predominant microorganisms in soilless culture. The number of bacteria on tomato roots can reach 10 log
10 cfu g
−1 for fresh roots, while in nutrient solution this can be 5–6 log
10 cfu mL
−1 [
1,
43]. The fungal population on roots, estimated by Koohakan et al. [
40], were 4–5.5 log
10 cfu g
−1. In this study, the number of bacteria ranged from 4.7 to 6.2 log
10 cfu mL
−1 in drainage water and from about 5.5 to 6.9 log
10 cfu mL
−1 in the solution from the root zone (
Table 5). For fungi, this was from 2.0 to 4.1 log
10 cfu mL
−1 in drainage water and from 2.3 to 5.1 log
10 cfu mL
−1 in the solution from the root zone (
Table 6).
The size of the bacterial population was stabilized during cultivation season, in agreement with the observation of Koohakan et al. [
40]. In turn, the number of fungi decreased in the middle of cultivation, and remained at a similar level until the end of the experiment (
Table 6). This is contrary to the results reported by other authors, who have found that the number and diversity of fungi in the soilless cultivation of tomatoes grew over time [
40,
44]. However, it was related to the increase in the number of pathogenic fungi (
Pythium spp.,
Fusarium spp.) at the end of the vegetation of the plants, whereas, in our studies, the numbers of fungi genera
Pythium spp.,
Phythophthora spp. and
Fusarium spp., evaluated on the selective media, were very low. In most studied samples these fungi were not detected.
Pythium spp. And
Phythophthora spp. fungi were not isolated from rockwool, Carbomat and coir substrate (drainage waters and root zone) in the whole experimental period. In the case of Biopot, these fungi were isolated only from the root zone, in average quantities (for three years) of 0.44 and 0.78 cfu mL
−1 of the solution, respectively, for
Pythium and
Phytophthora.
Fusarium spp. was isolated from all kinds of tested leachates (but not from all samples) during tomato vegetation. The numbers of these fungi were low—0.6, 1.7, 1.3, and 13.6 cfu mL
−1 of the drainage waters from rockwool, Carbomat, coir substrate and Biopot, respectively. No tests have been carried out to recognize whether these were pathogenic or saprophytic strains. Symptoms of fusariosis have been observed on the plants.
The kind of growing medium in soilless system has an impact on the microorganisms that inhabit this medium. Gunert et al. [
45] found that the type of medium makes a more significant contribution in the differentiation of microbial communities than time of sampling. Using high throughput sequencing analysis combined with molecular techniques, they found stable microbial community structures in the organic growing medium, while the mineral medium (rockwool) displayed high variability, increasing with plant growth. In these studies, Biopot (the mixture of recycled wool residues and organic wastes) contained the highest numbers of bacteria. In the three other substrates, the numbers of these microorganisms did not differ significantly (
Table 5). The total number of filamentous fungi was the highest for coir substrate and Biopot (
Table 6). Lower numbers of fungi were observed for Carbomat, but the differences were not always significant compared with coir substrate and Biopot. The lowest number of filamentous fungi was obtained in rockwool. Carbomat and Biopot were characterized by the presence of fungi genera
Trichoderma. The highest number of these fungi was found in Carbomat, in both the root zone and in drainage water (
Table 7). In this substrate,
Trichoderma spp. represented on average as much as 89% of the fungal population. In Biopot, this average value was 56%. In both substrates the density of
Trichoderma increased during plant growth.
Trichoderma spp. are cosmopolitan fungi that occupy a variety of habitats, and are widely used for the biocontrol of numerous plant pathogens [
46]. These fungi are known not only as biocontrol agents, but they also support plant growth and root development and induce plant defense mechanisms [
47]. There are numerous reports in the literature about the beneficial effects of
Trichoderma on tomato growth and yielding, for example Uddin et al. [
48] or Herrera-Téllez et al. [
49]. However, in the presented studies, no positive effect on the yield of tomato was found in the substrates highly colonized by these fungi (Table 10). Apparently, the strains of
Trichoderma inhabiting Carbomat and Biopot did not have beneficial properties. However, these results suggest that both of these substrates may create an environment conducive to colonization by these fungi. This can be useful information when considering substrates for artificial inoculation with biocontrol
Trichoderma preparations.
The specific genera of bacteria, studied in this experiment, were fluorescent pseudomonads (
Table 8). They are especially well adapted to grow and multiply in the plant rhizosphere, where these bacteria utilizing root exudates may produce a wide spectrum of bioactive metabolites and growth-promoting substances, and they compete successfully with other microorganisms [
50,
51].
Pseudomonas spp. are widely known for their plant growth-promoting properties, their induction of systemic resistance in plants and their suppression of plant pathogenic microorganisms [
52]. Numerous studies indicate that
Pseudomonas represents the core of plant growth-promoting rhizobacteria for many crops, including tomato [
53,
54]. The dynamic of the fluorescent pseudomonads’ population in soilless cultures of tomato plants was investigated Koohakan et al. [
40]. They found the highest population of these bacteria in tomato roots at the beginning of the experiment. Then, the population decreased and tended to stabilize at the same amount. Our studies confirm this trend. The highest number of fluorescent pseudomonads was observed in the samples analyzed in May, at the beginning of the vegetative season (
Table 8). The number of bacteria ranged from 1.6 to 4.1 log
10 cfu mL
−1; however, there were no significant differences in pseudomonads density between nutrient solutions taken from the root zone and the drainage waters. The type of substrate also had no effect on the concentration of these bacteria. In July, the number of pseudomonads decreased, with the exception of Biopot, where the amount of these bacteria was at a comparable level throughout the entire vegetation period. The mean number of fluorescent pseudomonads in the tomato root zone, after stabilization, was about 2 log
10 cfu mL
−1. This represented about 30% of the total number of isolated bacteria. The higher number of these bacteria in the soilless cultivation represents the better growth and health of the plants. Déniel et al. [
55] found that high populations of
Pseudomonas in the filters in slow filtration systems increased the elimination of pathogenic fungus
Fusarium oxysporum.
The other specific genera of bacteria, studied in this experiment, were coliforms (
Table 9). These bacteria may indicate the presence of fecal contamination in water [
56]. The sanitary quality of vegetables is of great importance. Although soilless cultivation in a greenhouse ought to reduce the risk of human pathogen contamination of the produce, by eliminating contact with animals and manure fertilizers, there is a potential risk of pathogen transfer with water and nutrient solution [
57,
58]. The other risk in soilless production may be related to the applied organic substrates, alternatives to rockwool and peat, that may also potentially serve as transmitters of pathogenic microorganisms. The presence of pathogens in substrates or nutrient solution brings about the risk of cultured vegetable contamination, but also contamination of the leachates, which can get into the environment or can be recirculated. The limit to the number of coliforms for drinking water is defined. Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption includes coliforms as an indicator parameter [
59]. The Directive sets the parametric value for it at 0 cfu in 100 mL of water. However, for irrigation water, the parameters are not such strictly defined. The different guidelines generally set the limit at ≤10
3 coliforms in 100 of water or at ≤10
5 coliforms in 100 of water for restricted irrigation [
60,
61].
In the presented experiments, the mean number of coliforms in the nutrient solutions and in drainage waters was 1.4 log
10 cfu mL
−1, and did not exceed ≤2.0 log
10 cfu mL
−1 for all studied substrates (
Table 9). The density of this bacterial group was consistent throughout the vegetation period. This means that the amount of coli bacteria did not exceed the limit, and the type of substrate had no significant effect on the concentration of these bacteria.