Lettuce is one of the most important leafy vegetables in terms of production, and its global production is steadily on the rise (Shatilov et al., 2019; Ridder, 2022; Trenda, 2022). It is important not only due to its wide use in the food industry but also as a source of many bioactive compounds that improve the health of the population. These include polyphenols, carotenoids and chlorophylls (Shi et al., 2022).
Mineral wool is the world’s most used medium for growing fruit vegetables in greenhouses because of its advantages such as uniformity, inertness, easy handling and good physical parameters (Gruda et al., 2016). However, in the case of mineral wool, handling the used material as waste appears to be problematic. There have been efforts to reuse mineral wool mats for growing cucumbers. Unfortunately, the yield reduced when used the second time, so reusing it for growing is not recommended (Łaźny et al., 2021). The amount of mineral wool that is reused is very low, at approximately 10% (Bussell and McKennie, 2004). For these reasons, researchers are trying to find an alternative substrate that could provide good conditions for obtaining yields comparable to those using mineral wool while making the management of the resulting waste easier. Another commonly used substrate is perlite – aluminium silicate, which is produced from volcanic origin. Its advantages are low bulk density, high porosity, inertness and the fact that it does not undergo rapid decomposition (Awad et al., 2017). Unfortunately, according to the findings of Vinci and Rapa (2019), who investigated the impact of substrates (perlite, rockwool, coconut fibre, peat, etc.) on human health, ecosystems and resources, perlite was found to have worst outcomes. It is therefore necessary to find other alternatives.
When searching for an alternative substrate for hydroponic crops, some authors focussed on waste materials such as compost from vegetable waste or groceries (Mazuela et al., 2005; Moschou et al., 2022), recycled plastics and almond shells (Kennard et al., 2020), sheep wool (Böhme et al., 2008; Dannehl et al., 2015; Jug, 2018) or rice hulls (Sambo et al., 2008; Buck and Evans, 2010). Other authors used substrate materials from renewable sources such as hemp (Dannehl et al., 2015; Li et al., 2021), wood materials as fibre, bark or sawdust (Allaire et al., 2004; Muro et al., 2004; Dorais et al., 2007; Maboko and Modise, 2018; Rahman et al., 2018) or coconut husk, which is presented in the literature as coir, coco fibre or coco peat (Böhme et al., 2008; Suvo et al., 2017). In the present study, three organic substrates, namely, coco peat, wood fibre and sheep wool, were used for testing in the hydroponic system. Coconut husks are used as a material to produce a substrate for hydroponic cultivation with a porosity similar to peat. Due to its high lignin content, it resists decomposition and is thus suitable for long-term cultures. The high water-holding capacity represents an ideal source of water for plants for a longer period than other substrates. The advantage from an environmental perspective is that coco peat is created as a co-product of the food and clothing industries, is compostable and is a renewable resource, unlike peat (Noguera et al., 2000; Ali, 2011). On the contrary, coconut shells naturally contain a high amount of salt due to their place of origin. Many buffer solutions, such as calcium nitrate and water, are needed for this purpose (Peano et al., 2012). During the soaking process of coconut husks, a large amount of harmful substances such as pectin, pentosan, tannins and polyphenols are produced, which are released into the sea. Due to this, the content of various chemical substances increases and the oxygen content decreases in the sea, which results in the death of many marine organisms (Narayanan, 1999). Wood fibre is produced by mechanically crushing wood or extrusion using hot steam (Carlile et al., 2015). It has been used in horticultural substrates for a long time as a substitute for peat to limit its extraction. The use of wood fibre in hydroponic cultivation, which functions similar to coco peat, and thus the need to import materials from other parts of the world would be eliminated, thereby reducing the carbon footprint and total impact on the global ecosystem (Vinci and Rapa, 2019). According to Gruda and Schnitzler (2004), wood fibre-based substrates possess favourable physical parameters such as total pore space, air content or water capacity. Using wood fibre as a substrate still has its pitfalls and several unanswered questions, especially in terms of phytotoxicity (Gruda et al., 2009). The problem of the phytotoxicity of wood materials used as a substrate is still being solved and warrants different methods of heat treatment during production (Yang et al., 2022). Sheep wool is a good fertiliser for plants (Zheljazkov, 2005; Komorowska et al., 2022). It contains some beneficial elements for plant nutrition, including calcium, magnesium, potassium, sodium, phosphorus and iron, and due to the structure of keratin, it slowly releases nitrogen. Furthermore, sheep wool fibres are hydrophilic and thus can retain water, which, in turn, is available to plants for a longer period (Petek and Logar, 2021). The number of sheep in the world shows a 20-year upward trend. The world population of sheep was 1.266 billion in 2021, with 59.55 million in Europe. The production of greasy sheep wool and clean sheep wool was 1.950 mkg and 1.0336 mkg, respectively (Eurostat, 2021; IWTO Market Information, 2022). Several studies focussed on using sheep wool as a growing medium or as a component of substrates for vegetables cultivated in greenhouses (Böhme et al., 2008; Górecki and Górecki, 2010; Dannehl et al., 2015; Jug, 2018). However, wool as a material for substrate production has not yet received enough attention to warrant further research and exploit the potential in the quantity in which wool is produced. Therefore, it was included among the substrates in the present study. The greatest challenge in terms of finding alternative substrates is to find one substrate that will meet the criteria of ideal physical and chemical properties and thus be a suitable medium for growing plants. The substrates should provide plants with sufficiently suitable conditions in terms of the availability of water, air and nutrients. Moreover, they should not possess characteristics that they many pose as obstacles to plant growth, such as phytotoxicity, immobilisation of nutrients from the solution or heavy metal content. Many of such deficiencies can be detected in substrate testing by monitoring stress factors.
The aim of this study is to conduct an experiment with a model lettuce crop grown in different substrates (rockwool, perlite, wood fibre, sheep wool and coco peat) and determine their yield and quality assumptions when growing lettuce hydroponically using the flood-and-drain method and compare biological and analytical parameters of lettuce. It is assumed that the use of individual substrates affects the growth parameters of lettuce, which will be reflected in the monitored stress indicators. A positive effect is expected from the physical and chemical properties of substrates of organic origin, which could thus replace mineral wool or perlite.
The experiment was carried out in the spring of 2022 in the greenhouse in Krakow (Poland). A closed flood-and-drain growing system was used, and 21 lettuce plants (7 plants for one repetition) were grown in each of five different substrates (perlite, mineral wool, spruce wood fibre, raw sheep wool and fine fraction coco peat). The physical and chemical properties of the substrate are detailed in Table 1.
Physical and chemical parameters of substrates.
Substrate | Bulk density | Total pore space | Air capacity | Total water-holding capacity | Chlorides in drain | EC | pH |
---|---|---|---|---|---|---|---|
Unit | kg · L–1 | % | % | L · m–3 | g · L–1 | mS · cm–1 | |
Mineral wool | 0.064 | 97.13 | 15.6 | 815 | 0.006 | 0.247 | 6.6 |
Perlite | 0.11 | 59.97 | 30 | 300 | 0.008 | 0.0828 | 7.99 |
Coco peat | 0.081 | 77.48 | 4.4 | 730 | 0.029 | 0.528 | 4.85 |
Wood fibre | 0.064 | 76 | 12.8 | 632 | 0.011 | 0.248 | 6.25 |
Sheep wool | 0.048 | 96.77 | 77 | 198 | 0.094 | 6.24 | 7.97 |
The pH values and electrical conductivity were measured in soil:distilled water (1:2.5 = v:v) suspension by using an electrometric method. Mohr’s method was used to determine the chloride ion concentration of a solution by titration with silver nitrate.
The content of selected elements in the drain from substrates was analysed using the ICP-OES method. As the sample for the analysis, 5 mL from the drain was mineralised in the Anton Paar Multiwave 3000 microwave system. After that, it was digested in a mixture of HNO3 and H2O2 in a ratio of 5:1 vol. The measurement of the number of elements was performed by inductively coupled plasma atomic emission spectrometry using an Optima7600 instrument (Perkin Elmer, Akron, USA).
The fresh leaf biomass, number of leaves and stem diameter were measured. The stem diameter was measured after cutting the plant using a caliper. The leaf area was measured 1 day before the end of the experiment using the LeafScan mobile application (by Carlos Anderson), which is a non-destructive method of measuring the leaf area of the entire head of lettuce.
The analysis of content of nitrates, antioxidant activity (AA), ascorbate peroxidase (APX), total phenol content (TPC) and glutathione (GSH) were performed. The nitrate content in the lettuce was assayed following the method by Cataldo et al. (1975).
A total of 2 g of dry matter was put in 10 mL (4 × 2.5 mL) of hot water (90–95 °C). The extracts were then placed in a water bath for 30 min at 80 °C. After that, the solution was mixed for 3 min at 200 rpm and cooled to room temperature (RT). Then, the samples were centrifuged for 10 min at 4,500 rpm. A total of 0.2 mL of the extract was mixed with 0.8 mL of 0.5% salicylic acid (5 g in 100 mL 96% sulfuric acid), and then 19 mL of 0.5 M NaOH was added. After cooling to RT, the absorbance was measured at 420 nm on a spectrophotometer (UV/VIS Helios Beta).
An analysis of total AA was performed following the method by Molyneux (2004) using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The extract was prepared from 2 g of ground samples with 10 mL of 80% methanol in four portions and centrifuged (4,500 ×
The APX analysis was conducted following the method of Nakano and Asada (1981) with some modification. A measure of 2 g of leaf sample was homogenised in 50 mM potassium phosphate buffer (pH 7.0) containing 1mM ethylene diamine tetra acetic acid (EDTA), 1% soluble polyvinyl pyrrolidone (PVPP), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM ascorbic acid (AsA). All extraction steps were carried out in ice at 4 °C. APX was measured according to the oxidation of ascorbate. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate and 0.1 mM hydrogen peroxide and 0.15 mL of the enzyme extract. The oxidation of AsA with hydrogen peroxide was measured continuously for 5 min by decreasing absorbance at 290 nm assuming an absorption coefficient of 2.8 mM · cm–1. The APX activity was expressed as μg AsA · min–1 · g–1 of fresh weight.
The TPC was determined using the Folin–Ciocalteu method according to Djeridane et al. (2006). A measure of 2 g of the fresh sample was homogenised with 10 mL of 80% methanol in four portions (4 × 2.5 mL). The mixture was centrifuged (4,500 ×
The GSH analysis was performed using the method by Guri (1983) with some modifications. A measure of 2 g of the fresh sample was homogenised with 10 mL of 0.5 mM EDTA and 1% trichloroacetic acid (TCA) in an ice bath (4°C). After centrifugation (13,986 ×
The experiment was performed in three repetitions for each variant of the substrate, and the data were presented as the mean supplemented with standard deviation. Differences in the biological parameters of the lettuce fresh leaves’ biomass, number of leaves, stem diameter and leaf area, as well as the analytical parameters of the dry matter, nitrate content, TPC, AA, APX and GSH, were evaluated using an analysis of variance with Statistica 12 (TIBCO Software Inc., Palo Alto, USA). Significant differences were calculated using Scheffé’s test at a significance level of
The substrates can be ranked according to the total content of all measured elements in the drain of the substrate in descending order, starting from sheep wool and followed by coco peat, mineral wool, perlite and wood fibre. There was no statistical difference in the total elements content in the drains from perlite and from wood fibre. Looking at the individual elements, the content of all monitored elements was significantly higher in the drain from sheep wool (see Table 2). The most represented elements in general were potassium (12–2,996 mg · L–1), sodium (5–58 mg · L–1) and calcium (5–127 mg · L–1). The least represented element in the drain was zinc (0–1.2 mg · L–1) for each substrate. The order of individual elements in the drain of substrates was as follows:
Perlite K > Na > Ca > Fe > Mg > S > P > Zn
Mineral wool S > Na > Ca > K > Mg > Fe > P > Zn
Wood fibre K > Ca > Na > P > Mg > S > Fe > Zn
Sheep wool K > Ca > S > Na > Mg > P > Fe > Zn
Coco peat K > Na > Ca > Mg > S > P > Fe > Zn
Elements content in the drains from the substrates (mg · L–1).
Element | Perlite | Mineral wool | Wood fibre | Sheep wool | Coco peat |
---|---|---|---|---|---|
P | 0.4 ± 0.0 a | 0.2 ± 0.0 a | 2.6 ± 0.0 b | 22.5 ± 0.5 d | 4.3 ± 0.1 c |
K | 30.4 ± 1.1 c | 12.1 ± 0.2 a | 22.6 ± 0.7 b | 2,995.9 ± 1.5 e | 103.0 ± 3.0 d |
S | 0.9 ± 0.1 a | 45.3 ± 1.8 c | 1.6 ± 0.0 a | 69.3 ± 1.4 d | 10.8 ± 0.4 b |
Ca | 4.6 ± 0.5 a | 14.6 ± 0.5 b | 7.0 ± 0.5 a | 126.6 ± 1.2 d | 32.6 ± 1.4 c |
Na | 4.8 ± 0.3 a | 43.9 ± 1.9 c | 6.1 ± 0.2 a | 57.5 ± 2.0 d | 33.7 ± 0.8 b |
Fe | 1.7 ± 0.5 b | 0.3 ± 0.1 a | 0.2 ± 0.0 a | 10.9 ± 0.2 d | 4.2 ± 0.1 c |
Mg | 1.1 ± 0.2 a | 4.8 ± 0.0 a | 2.5 ± 0.1 a | 30.1 ± 2.6 c | 12.5 ± 0.2 b |
Zn | 0.0 ± 0.0 a | 0.0 ± 0.0 a | 0.1 ± 0.0 a | 1.2 ± 0.1 c | 0.2 ± 0.0 b |
Sum | 44.0 ± 0.8 a | 121.2 ± 3.5 b | 42.8 ± 1.8 a | 3,314.0 ± 7.8 d | 201.4 ± 5.4 c |
The table shows the average values with standard deviation.
Different letters for individual values indicate significant differences at
All monitored biological parameters were significantly affected. After a statistical analysis using Scheffé’s test (
Lettuce grown in coco peat had the highest leaf biomass, with an average of 75 g per plant (see Figure 1). The lowest biomass was recorded for sheep wool and wood fibre substrates. Leaf biomass measured for perlite and mineral wool was significantly lower than that for coco peat but significantly higher than that for sheep wool and wood fibre.
In the number of leaves (Figure 2), there was a similar trend to the leaf biomass. The average number of leaves for lettuce grown in coco peat was 16.9, which was significantly higher than that for all substrates that were used in the experiment. The lowest number of leaves per plant was recorded for wood fibre and sheep wool substrates at 10 and 11.6, respectively.
The largest diameter of the stem that was measured in the root neck was for lettuces from coco peat, with an average of 11.7 mm. No differences were found between the stem diameter of wood fibre and sheep wool and between that of perlite and mineral wool (see Figure 3). The smallest stem diameter was measured in sheep wool, with 7.3 mm on average.
The largest leaf area, as measured by using the non-destructive method focussing on whole heads of lettuce before harvest, was recorded for lettuce grown in coco peat, with an average leaf area of 836.2 cm2. The smallest leaf area was measured for lettuce grown in sheep wool at only 268.7 cm2 (Figure 4). Furthermore, a significant difference was found between the leaf area of lettuce grown in wood fibre and that grown in perlite. However, the leaf area of lettuce grown in mineral wool did not differ from that grown in wood fibre and perlite.
Significant differences were found in the parameters of dry matter, content of nitrates, TPC and AA, as shown in Table 3. Measurements of APX and GSH did not show significantly different values.
Analytical parameters of lettuce. The table shows average values with standard deviation.
Substrate Perlite | AA (% scavenging of DPPH) | GSH (mg · g–1 DM) | APX (AsA · g–1 FM · min–1) | TPC (mg GAE · g–1 FM) | Nitrates (mg · kg–1 FM) |
---|---|---|---|---|---|
Perlite | 21.6 ± 4.9 a | 44.0 ± 19.7 a | 108.8 ± 58.5 a | 43.3 ± 0.9 a | 604.2 ± 110.3 ab |
Rockwool | 32.6 ± 3.0 ab | 22.5 ± 12.1 a | 129.6 ± 25.6 a | 40.6 ± 1.6 a | 555.1 ± 58.1 ab |
Wood fibre | 46.1 ± 5.9 bc | 31.0 ± 4.9 a | 105.0 ± 32.2 a | 55.4 ± 7.9 b | 447.6 ± 38.2 a |
Sheep wool | 54.9 ± 11.5 c | 23.1 ± 5.2 a | 135.9 ± 24.5 a | 74.0 ± 1.3 c | 686.2 ± 86.5 b |
Coco peat | 40.3 ± 2.7 abc | 19.9 ± 2.5 a | 126.4 ± 25.5 a | 48.0 ± 1.6 ab | 552.6 ± 69.9 ab |
Different letters for individual values indicate significant differences at
AA, antioxidant activity; APX, ascorbate peroxidase; AsA, ascorbic acid; DM, dry mass; DPPH, 2,2-diphenyl-1-picrylhydrazyl; FM, fresh mass; GAE, gallic acid equivalents; GSH, glutathione; TPC, total phenol content expressed as GAE.
Within the measurement of total AA expressed as % scavenging of DPPH, conclusive differences were found. The lowest DPPH scavenging activity was recorded in lettuce growing in perlite, and the highest was recorded in sheep wool. Significant differences were observed between these two substrates. The rest of the evaluated substrates did not differ.
When comparing the content of GSH in lettuce of all varieties, certain tendencies were observed, but the difference was not significant.
The average APX activity ranged between 105 and 136 mg AsA · min–1 · kg–1 of fresh weight. The activity of APX in lettuce grown in perlite and wood fibre was lower by 14%–23% than that grown in mineral wool, sheep wool and coco peat, but the differences were not significant.
The content of total phenols, expressed as GAE, was significantly higher in the lettuce grown in sheep wool. The increase was 33%–82% compared with other substrates. Lettuce grown in perlite and mineral wool substrates showed a significantly lower TPC than lettuce grown in wood fibre but did not differ to lettuce from coco peat.
The level of nitrates varied between 448 and 686 mg · kg–1 of FM. The influence of the substrate on the content of nitrates in lettuce was confirmed; however, a significant difference was observed only between wood fibre and sheep wool, where sheep wool had the highest content of nitrates and wood fibre the lowest.
When testing alternative substrates in a hydroponic flood system, significant differences were found in all biological parameters of lettuce, which was used as a model crop. From the perspective of the values of the biological parameters measured for salads, the individual substrates can be ranked from the lowest value of sheep wool, followed by wood fibre, mineral wool, perlite and coco peat. Several factors could have affected the observed results, the most important of which are the physical and chemical parameters of the substrates. Many authors have studied the substrate parameters that would provide plants with ideal conditions for growth. The finding was that a suitable substrate should meet the following requirements: bulk density ≤0.4 kg · L–1, total pore space 75%–85%, air capacity 10%–30%, water-holding capacity 600–1,000 L · m–3, EC 0.75–3.49 mS and pH 5.2–6.3 (de Boodt and Verdonck, 1972; Boertje, 1983; Abad et al., 1993; Abad and Noguera, 2000). From the substrate parameters listed in Table 1, it is obvious that not all substrates meet the given requirements. The significantly highest leaf biomass, number of leaves, stem diameter and leaf area were achieved in lettuce grown in coco fibre. Sarkar et al. (2021) cultivated
The stress response of plants is generally due to increased AA (Oh et al., 2010; Malejane et al., 2017; Paim et al., 2020). The response of plants to drought stress is to reduce the rate of photosynthesis because it is not possible to process all the captured light (Chatterjee and Solankey, 2014). This is caused by the increased production of reactive oxygen species (ROS), which destroys chloroplasts, thus reducing carboxylation. The main sites of action of ROS are chloroplasts, mitochondria and peroxisomes. Carboxylation is also limited by a smaller leaf area (Bahadur et al., 2011). The defence reaction to the increased amount of ROS is the production of antioxidants, such as peroxidases, catalases, APX, GSH reductases and others (Mittler, 2002). The effect of excessive salinity on plant growth has two phases. In the first phase, when vacuoles are filled with Na+ and Cl-, the growth of leaves and roots is only partially affected, while in the second phase, when vacuoles are completely filled, the concentration of ions in cytoplasm increases, and the action of enzymes is limited (Munns, 2005; Parihar et al., 2015). Salinity stress on lettuce can manifest itself in a higher total content of phenols, anthocyanin and proline concentrations (Neocleous et al., 2014; Flores et al., 2022). GSH is concentrated in chloroplasts, cytosol, mitochondria, peroxisome and apoplasts. It occurs under the action of toxic substances or oxidative stress mainly caused by H2O2 (Meister and Anderson, 1983; Noctor and Foyer, 1998). GSH in the plant body can be divided into the following two categories: sulphur metabolism and a defence response against stress (Noctor and Foyer, 1998). If we consider the AA (% scavenging of DPPH) and compare it with the content of GSH (both in Table 3), it can be concluded that GSH did not have to play a major role in the stress response of lettuce to drought or salinity. Multiple regression analysis between the content of GSH and AA (% scavenging of DPPH) cannot be verified since the correlation was not statistically significant (
The nitrate content in lettuce ranged between 426 and 686 mg · kg–1 of FM and therefore did not exceed the limits set by commission regulation (EU) No. 1258/2011 for any substrate, where the nitrate limit is set at 4,000 mg · kg–1 of fresh weight. Lettuce grown in wood fibre showed the lowest nitrate values, but the significant difference was only comparable to lettuce from sheep wool. Regarding the nitrate content, none of the assessed substrates can be considered problematic at the fertilisation level of 1.5–2 mS in the present study. Kaniszewski and Sabat (2015) observed the effect of the fertilisation level on lettuce yield using different substrates. Their findings show that the higher the EC of the nutrient solution, the higher the nitrate content. The nitrate content found in the present study (see Table 3) was statistically the highest in lettuce grown in sheep wool. This substrate also had the highest EC (6.24 mS). Unfortunately, the nitrogen content in the substrate was not investigated in the present study, but other sources state that raw sheep wool is its rich source (Böhme et al., 2012; Dannehl et al., 2015; Pina et al., 2021). This could be the reason the highest nitrate content in sheep wool. In the aforementioned study by Kaniszewski and Sabat (2015), the nitrate content in the rockwool and coir substrates was compared. The authors concluded that lettuce grown in coir had a higher nitrate content in both growing seasons. The results of the present study do not confirm this trend because there was no difference in the nitrate content for these two substrates.
The results of this study showed that only coco peat can fully compete with the common substrates mineral wool and perlite in terms of biological parameters. Regarding the lettuce grown in coco peat, leaf biomass was 40% and 70% higher, respectively, than that grown in perlite and mineral wool. The leaf biomass of lettuce grown in wood fibre showed a reduction of 48% and 34% compared to that grown in perlite and mineral wool. For lettuce grown in sheep wool, the reduction was even higher at 73% than lettuce grown inperlite and 66% than lettuce grown in mineral wool. The deteriorated results of the biological parameters can be explained by the stress response of lettuce, which was confirmed by the increased AA (% scavenging of DPPH). Three stress–response indicators were monitored: GSH, APX and TPC. After the statistical analysis, it became clear that the main response to a stressful situation (increased AA) caused either by a lack of water due to the physical parameters of the substrates or by salinity stress due to high salinity was an increase in the production of phenolic substances (