Category: hydroponic agriculture

However, Al in the shoots exhibited no significant difference among rice varieties and the lowest contribution of varietal effect, likely due to the low phyto availability of Al colloidal precipitations in the slightly acidic hydroponic system, indicated that environmental factors also showed a marked influence on the rice ionomes. In contrast, the variance contributions of varietal effect to ionomic variations in Mg, Mn and Cu were even more than 60% in both shoots and roots. With large contributions of genotype effects in the shoots and roots, Mg, Mn and Cu were robust to environmental perturbations. In the present study, variations of Cr concentration in the shoots among all the subspecies were the largest, which was consistent with a previous study. Although the transport of Cr has been demonstrated to be associated with S transporters,such a significant difference among the rice genotypes indicated a complex underlying transport mechanism.The CVs of macro-nutrients excluding P in the roots in all rice subspecies were less than that of trace elements and heavy metals, indicating that the variations in macro-elements were stable among the rice varieties. Meanwhile, the lower CVs of nonessential elements in japonica demonstrating that micro-elements and heavy metal uptake in the roots showed greater genetic diversity in aus and indica than in japonica, which was consistent with previous reports that japonica subspecies exhibits less genetic and transcriptional diversity than indica and aus. The ionomic variation in rice varieties was mainly dependent on specific chemical element properties and genotype effects, as well as limitedly on subspecies. One of the most important values of ionomics is in determining the interactions between elements. The occurrence of antagonism and synergism between elements on uptake and translocation in plants has been reported in many studies. Correlation analysis showed that the rice subspecies and organs exhibited diverse strategies in establishing connections between elements, but many interactions were similar. 

Ca was always significantly and positively correlated with Ba and Sr, and there was also positive correlation between Ba and Sr,vertical rack system indicating a significant positive correlation among divalent cations, possibly explained by their sharing the non-selective cation channel transporter-protein super families, such as ZIP, heavy metal ATPases and yellow stripe-like in the xylem due to their similar chemical properties and identical ionic valences. Significant positive correlations between P-As, P-Se and As-Se were detected in both shoots and roots among the subspecies. It has been reported that P and Se share the phosphate transporter OsPT2 gene. Moreover, Cao et al  reported reduced As uptake in rice via a P transporter, OsPT4 gene knockout. In general, P application can activate the expression of OsPT2 and OsPT4 genes to improve As and Se uptakes in rice. These elemental interactions can provide a strategy to screen multi-element accumulation rice genotypes to breed rice varieties with abundant nutrients and that are safe to consume. Moreover, combined with the results of PCA on correlation coefficient, it was clear that the significantly positive correlations between minerals in the shoots of japonica were more obviously than those in indica and aus, while the patterns of ionomic correlations in the roots among the subspecies were similar. The elemental correlations in rice shoots mainly derive from ionomic transport, whereas in roots, it is due to element uptake, confirming that the ionomic differences were primarily caused by different transport mechanisms among the subspecies, consistent with previous studies. In rice domestication and breeding history, farmers have preferred to plant rice varieties adapted to the local agro-climatic conditions, with higher yield and better taste, usually not considering the micro-elements content,therefore the separation of elements in PCA mainly determined by different origins rather than subspecies. This finding further indicated that the genetic differences involving in ionomes in subspecies can vary with environmental changes. For example, the elemental differences in rice varieties from Japan mainly explained by most nonessential and toxic elements such as As and Cd, that can be related to the history of wide-ranging incidence of agricultural soil contamination in Japan. 

Therefore, considering the geographical and historical distributions of rice varieties associated with the subspecies effects might be informative. Thus, due to the large genetic variation in different rice vareties, it is worthwhile to screen for rice varieties with higher nutrient concentrations, lower levels of toxic elements and healthier food values for use in bio-fortification strategies. The transportation of elements by the root-to-shoot process has been considered a rate-limitation factor in the shoot-tograin system. Consequently, identification of element accumulation in shoots is positively correlated with that in grains and determines the distribution of elements in grains. Additionally, owing to the numerous correlations between elements, the elemental covariation effects in shoots should be identified to determine the nutritional values and safety of rice varieties. In the present study, we identified many varieties accumulated a high concentration of multi-metallic elements. Higher concentrations of essential metal or metalloid elements in rice are important in bio-fortification to promote the synthesis of the coenzymes or proteins required for human health. Meanwhile, consistenting with the results of ionomic correlations in the shoots,multi-metal accumulation is likely involved in the non-selective metal cation channel transporter-protein super families. However, nonessential and toxic metals can be also indiscriminately accumulated in these varieties due to similar chemical properties, and potentially resulting in health risks to humans. JRC06  accumulated high concentrations of B, As, Co and Cr, while a lower concentration of K. As a macro-nutrient, K plays a highly significant role in alleviating abiotic stress in plants,explaining why metal uptake is likely suppressed, leading to a higher concentration of heavy metals. Both high K and Cs concentrations in the shoots of some rice varieties can be explained by the positive correlation between K and Cs,but it seemed contrary to previous studies. Ishikawa et al  reported that the Cs concentration in rice is reduced by applying K, and Rai et al  found that the expression of K transporter OsHAK1 in rice roots is the main route for Cs to accumulate in rice plants under a low K status. The plants in this study with a higher K concentration likely had a stronger affinity for Cs due to their similar chemical properties. However, the multi-element accumulation in almost all the aus and indica subspecies was lower than that in the japonica subspecies,indicating that the elemental covariations in the shoots showed subspecies differences and that the correlation of elements was stronger in japonica than in indica and aus, which was consistent with the results shown in Fig. 3.

The indica variety WRC11  showed high Zn and low Cd concentrations, while the aus variety WRC26  showed high Zn and low Cr concentrations. It has been reported that the OsHMA2 transporter in rice is associated with the co-transportation of Zn and Cd from roots to shoots, and that Zn competes with Cd by sharing the same ZIP transporter. Understanding and screening for rice varieties with significant correlations and potential for high nutritional value and safety are essential for rice breeding. In conclusion, this study revealed the ionomic responses to the genetic effects of rice varieties and subspecies under a strictly controlled environment. The ionomic differences among the subspecies were within the pre-framework of the genus Oryza, i.e., the concentrations of macro-nutrients were greater than micro-nutrients, and the micro-nutrients and anions were mainly restricted to the roots. The variations in the rice ionomes primarily depended on genetic factors and specific element chemical properties, and to a lesser extent, on subspecies factors and the geographical and historical distributions of the varieties. Moreover, the rice varieties screened for higher nutrient concentrations and beneficial elements in rice showed a higher value in variety breeding for human health. The potential health risks can be reduced by growing the varieties screened for low heavy metal accumulation even on heavy metalcontaminated soils. Therefore, the identification of rice varieties for bio-fortification and safe breeding should be closely related to the local edaphic conditions. Furthermore, the ionomic datasets of the rice genotypes in this study can provide a theoretical basis for transcriptional expression analyses of elementrelated proteins and transporters.Recirculating aquaponic systems,which grow both fish and plants, contain either a dedicated bio-filter  and a hydroponic component for the plants, or a hydroponic component only that has the ability to act as a bio-filter. If balances between the biomass of fish grown  and the nutrient removal ability of the plants are matched, the amounts of buffer required in aquaponic systems should be lower than that of fish-only systems. This is because plants are known to release negatively charged, alkalising ions  when they are actively taking up nutrient ions such as nitrate. This is generally a one-to-one exchange mechanism  since its primary function is to balance the homeostatic charge status within the plant root. However, the bacterial mediated nitrification of one ion of ammonia to one ion of nitrate releases two hydrogen ions ; therefore, the release of alkalising ions during active plant assimilation of nitrate cannot completely counteract the acidification caused by the nitrification of soluble fish waste ammonia. Therefore, as in fish-only RAS, mobile grow rack aquaponic systems often require the addition of a basic buffer, however due to the activity of the plants, this usually require less buffer additions than equivalent Fish-only RAS. 

When buffer is added to a system,it is only the negative ion component that is used for pH buffering. Buffers are universally added as salts, as these are easier to acquire and handle. Hence, when buffers are added to aquatic systems, the positive ion component is unused and begins to accumulate within the system water. In fish-only systems, when buffered with sodium bicarbonate, sodium begins to accumulate. Sodium ion accumulation within fish-only systems is dealt with in the same manner as the build-up of other deleterious ions ; ionic concentrations are diluted by water exchange. One of the key advantages of recirculating aquaponic systems is that the addition of plants counteracts nutrient and other ionic accumulations, so that the reliance on water exchange to reduce ion accumulation is either lowered or negated completely. Therefore, aquaponic researchers have advocated the use of buffer salts to maintain pH that are based upon positive ions that are essential to plant growth and health, and therefore will not accumulate because the plants are actively using them in metabolic processes. Because fish feeds are generally lacking in potassium and calcium required for good plant growth, buffers based upon potassium and calcium salts are the most appropriate for aquaponic systems,as they provide the additional inputs of potassium and calcium that are essential for normal hydroponic plant metabolism. In previous experiments,sodium bicarbonate was used as the buffer species since it was the most common buffer used in fish-only, RAS culture. Whilst good plant growth was achieved using sodium bicarbonate as the buffer in this previous experiment, the current experiment was devised to test the suitability of both potassium-based and calcium-based buffers as alternatives, and to determine whether either of these buffers  conferred advantages. The fish species used was the Australian native, freshwater fish Murray Cod, Maccullochella peelii, shown to be well adapted to aquaponic systems in previous experiments  and the hydroponic vegetable was lettuce, Lactuca sativa. The system treatments were compared for fish growth, plant growth, buffer usage rate, nutrient accumulations,dissolved oxygen, pH, conductivity and water replacement rate.The experimental set-up was located, and all research was performed, within the Aquatic Culturing Laboratory Annex, Building 223, RMIT Bundoora Campus. The experimental, recirculating aquaponic system array consisted of 12 individual, identical aquaponic units, allowing replication of experimental treatments. Each aquaponic unit consisted of a fish holding tank, an associated bio-filter and a hydroponic component. The fish tank of each aquaponic unit was a round 100 L, opaque, white plastic tank. As well as fish, the tank contained an airlift pipe,a submersible water pump  and a 100 W thermostatically controlled electrical resistance aquarium heater.

Furthermore, the reduced need for filters in the DFP system compared to DAPS also indicates that FLOCponics might bring economic advantages for the producers. From an environmental point of view, the dependence on feeds is an aquaculture issue. Reducing the dietary CP level may mitigate the negative impact of feed on aquaculture sustainability due to the lower need for protein-rich ingredients and lower concentration of N excreted into the natural environment. With respect to the question of whether DFP might enable lettuce production in comparable yields to DAPS and traditional hydroponics,this study found no differences among the treatments for the growth parameters in the seedling  and final production phase. Interestingly, to achieve these similar yields, less commercial fertilizer was required in the DFP-32 compared to the other treatments. In Cycle 1, the volume of fertilizer added to a DFP-32 plant tank was approximately 51.9% and 6.4% lower than in HP and DAPS, respectively. In Cycle 2, these differences between DFP-32 to HP and DAPS dropped to 16.3% and 1.3%, respectively. Another important finding was that reducing the amount of N in the fish diet in DFP systems did not affect lettuce growth in either cycle. The higher volume of fertilizer added to the DFP-24 and DFP-28 compared to DPF-32 seems to have compensated for the reduction in the amount of N. Nevertheless, in both treatments, the volumes of fertilizer were lower or similar to those added to the DAPS plant tanks. The use of conventional dosages of a commercial fertilizer in the hydroponics subsystem could have hindered lettuce growth, as a result of nutrient imbalances in the water. However, the nutritional management employed in this study seemed to have facilitated lettuce production in both cycles and in all treatments. In spite of this, knowing the specific nutrients that need to be supplemented in the hydroponics subsystems, based on the profile of nutrients in the BFT effluents and on the requirements of the crop, might result in less fertilization dependence,vertical grow rack and possibly even greater plant production.

For the purpose of developing a specific fertilizer scheme for DFP systems, efforts to constantly characterize the profile of macro- and micro-nutrients in BFT water and to adjust the formulations of the fertilizer according to the dynamics of the BFT will be needed. The curves for lettuce growth presented in Figs. 5 and 6 may be used to predict production in the hydroponic subsystem according to the experimental conditions employed. A notable finding from these curves is the tendency of the seedlings’ growth rate in Cycle 1 to decrease. Possible explanations for this decrease might be that the nutritional management employed in Cycle 1 was sub-optimal, leading to growth limitations. Optimal nutritional management could have been adopted.The dataset presented in this article  provides details on the growth of 18 acclimatized and 11 non-acclimatized rice varieties grown hydroponically. The data from each variety were collected under three conditions: control, salinity, and salinity after acclimation. Growth measurements were taken at the end of 36 days of germination. The first dataset provides the shoot dry weight and length of acclimatized and non-acclimatized rice varieties, whereas the second dataset represents the root dry weight and length. In both cases, the first column is a variety name, and the next six columns are the measured traits of plants grown under the three different treatment conditions. Each column indicates the average of four samples along with the standard error of the measured trait. The same letter indicates no significant differences among the three treatments at p < 0.05. Table 1 presents the list of rice varieties with their taxonomic classification and country of origin. Fig.1 represents the  average shoot and root dry weight, and  average shoot and root lengths of both acclimatized and non-acclimatized varieties. Tables 2 and 3 represent the original data sets of dry weight and length of shoots and roots of both acclimatized and non-acclimatized varieties.The experiment was conducted in a glasshouse at Hiroshima University, Japan.

The conditions of the glass house were 55% humidity, 19–27 °C day/15–20 °C night temperature and natural sunlight. Seeds of twenty-nine rice varieties selected from the World Rice Core Collection were initially heat-sterilized at 60 °C for 10 min in a water bath, then surfacesterilized using 5%  sodium hypochlorite solution for 30 min, and finally rinsed thoroughly with distilled water. The seed germination process, seed transfer to Kimura-B nutrient solution, and the composition of the Kimura-B solution are provided in a related research article. The nutrient solution was changed every 3 days, and the pH was maintained daily between 5.0–5.5. Three sets of four seedlings from each variety were maintained throughout the experiment. One set received only the Kimura-B nutrient solution. In the second set, 1-week-old seedlings grown in the Kimura-B nutrient solution were acclimated with 1 mM NaCl  for 2 weeks and then exposed to 50 mM NaCl for the next 2 weeks. In the third set, hydroponically grown plants were directly subjected to 50 mM NaCl  during the third week of growth and maintained for the next 2 weeks. The seedlings were harvested at the end of the salinity treatment. After harvest, the roots were thoroughly rinsed with distilled water and then gently dried with a paper towel.For dry weight determination, each seedling was divided into leaves, sheaths, and roots, which were then oven-dried at 70 °C for 3 days before being weighed. Shoot dry weight was calculated by combining the dry weight of the leaves and sheaths.Climate change influences crop production worldwide due to longer and more unpredictable periods of drought stress. Drought stress occurs because of reduced water availability in the soil and enhanced water loss through evapotranspiration processes caused by atmospheric conditions. It is one of the most severe factors affecting plant growth and yield in agricultural crops. During periods of stress, plants are influenced at different scales ranging from phenological to morphological and molecular levels. 

In such cases, overall plant growth is reduced, where shoots are more inhibited in growth compared to roots. Moreover, photosynthetic processes are affected by impaired assimilate transport of sugars and amino acids to the plant parts where they are needed,leading to an accumulation of these osmotically active solutes in plant tissues. An increase in organic or inorganic solutes lowers the osmotic potential, improving cell hydration to maintain several metabolic processes under drought stress conditions. However, the severity of drought stress depends on the intensity and duration of the stress event and which plant species are affected. Potatoes are known to be sensitive to drought stress and water deficit due to their poorly developed root system. According to Hijmans,the global yield potential of potatoes could decrease by 18–32% between 2040 and 2069 due to drought stress. However, drought stress effects differ in developmental stages during plant growth. Tuber initiation and bulking are the most sensitive stages, while plants at maturity level are more tolerant to drought stress. During tuber formation, optimal temperatures between 15–20 ◦C and soil moisture above 65% favour tuber initiation while higher temperatures and lower soil moisture can reduce tuber formation and yield. Consequently, regulating plants’ water supply is particularly important, which can be improved especially by an adequate supply of potassium. K is the most abundant inorganic cation in plants  and is well described as an osmotic substance for maintaining cell turgor during drought stress. It regulates stomatal opening and closing, and therefore, water loss through transpiration. In addition, sufficient K supply positively affects phloem loading and consequently assimilate transport, e.g., sugars, from source to sink. For most crops, tissue concentrations of K ranging from 5 to 40 mg g− 1 DM are considered adequate. According to Sharma and Arora,the critical K content in leaves of potatoes varies between 3.69 and 5.15%, which is necessary to achieve 95–100% of the yield maximum and refers to the fourth leaf from the top. However, this range depends on the leaves harvested and the maturity of the plants.

The availability of K in plant cells strongly influences a wide range of enzymes for primary metabolism which are related to protein and carbohydrate synthesis. Furthermore, K is important for plant growth and improves crop productivity by increasing yield. However, the functions of K in osmotic regulation can be replaced by organic acids, amino acids or sugars, whose concentrations increase under K deficiency. Nevertheless, such production of compatible solutes is more energy-intensive compared to the accumulation of K in plant tissues. Adaption mechanisms to low K supply include a wide set of mechanisms, e.g., the reduction of plant growth for maintaining cell functions due to adequate tissue K concentrations or the redistribution of K to developing plant part. However, even under low K supply, plants can survive because of specialised high-affinity K transporters which were first identified and described for plants in the model plant Arabidopsis. Maintenance of cellular K homeostasis is the most important function of the high affinity K+/K+ uptake/K+ transporter  family,and they are triggered under low K supply. Several transporters are described in Arabidopsis, but only limited information is available for potato plants. In particular, vertical grow tables the expression level of these genes under drought stress or K supply is still scarce and needs to be further explored. The positive effect of K on plants exposed to drought stress affects many metabolic processes in plant development. It has been shown on different plant species that K addition increased biomass partitioning to roots, and enhances leaf membrane stability. Moreover, K regulates the photo-assimilation and translocation processes of carbohydrates together with related enzyme activities. In addition, K increases the abscisic acid  concentration in the leaves, thus reducing the transpiration of plants. Measuring the effects of drought stress on plants with differing fertilisation regimes on a field scale is challenging. For this reason, controlled systems offer a suitable approach to exclude external factors and provide detailed insights into physiological and metabolic processes within a plant.

To simulate drought stress, in hydroponic systems, polyethylene glycol  can be used to induce osmotic stress conditions in plants by promoting water deficiency and imitating soil drying. The hypothesis that sufficient K supply to the plant under drought stress conditions induced by PEG mitigates the adverse effects by triggering various adaption mechanisms on several plant levels such as on phenological, morphological and molecular level was the basis for this study. The specific objectives were to investigate the effects of osmotic stress, induced by PEG, on biomass production, water consumption, mineral and sugar allocation in potato plants under low and sufficient K supply,  to evaluate changes in metabolites and gene expression levels in leaflets before, during, and after drought stress simulated by PEG-induced osmotic stress under low and sufficient K supply, and  to determine indicators of adaption strategies by assessed parameters. The investigations were carried out using a hydroponic experiment with PEG 6000 to induce osmotic stress, with the two cultivars, Agria as tolerant and Milva as susceptible to drought stress. Leaf and whole plant analyses were performed to obtain insights into the stress responses of the two cultivars and to gain further information on adaptation mechanisms.Medium-early potato  cultivars Milva and Agria  were selected according to their drought stress tolerance. Agria is specified as a tolerant cultivar,whereas Milva is described to be more sensitive to drought stress. At first, seedlings of potato tubers were grown in 3 L pots filled with quartz sand at a sufficient K supply  and low K supply. All other nutrients were added in adequate amounts. At 28 days after planting,the plants were transferred to 5.5 L pots filled with nutrient solutions,according to Koch et al.. After transferring the plants to the hydroponic system, the nutrient concentration was gradually  increased every 3 days until the desired nutrient level was reached at 35 dap. The nutrient solution was constantly aerated. In addition, K was applied at low levels  and sufficient levels. Twenty biological replicates of each cultivar were grown, using 10 plants for each of the two different K supply regimes. Nutrient solutions were changed twice a week until 53 dap. During the last weeks of the experiment,nutrient solutions were changed once a week. At 60 dap, osmotic stress was induced by adding 8%  polyethylene glycol  6000 to half of the pots for each cultivar and K supply to simulate drought stress. 

The operation of both MAS was identical until the water temperature started dropping below 20 ◦C. At that moment,two different strategies were employed. In one of the MAS,the water was heated by means of a selfconstructed thermo-solar panel coupled with the system, in order to avoid water temperatures below 13 ◦C. If temperatures outside the greenhouse were close to zero degrees, two submersible heaters were placed in the fish tank, which were in operation for 8− 10 hours at night. The second MAS stopped working as an aquaponic, given that the fishes were removed to avoid heating the water. Therefore, it was transformed into a hydroponic installation in which only the grow bed tank, the NFT and the sump were functioning, with a total volume of 180 L. In order to provide the nutrients required for the hydroponic production of the plants, a bio-fertiliser obtained from a small vermicompost facility installed next to the greenhouse was employed. For the monitoring of the production of the different crops, information about the exact date of harvest, number of vegetables or fruits obtained and their weight was recorded. The fish biomass was also monitored by weighing the fish in each tank. An initial weighing was performed to obtain the baseline and then ten more measurements took place, distributed during the year. The fish were also weighed when they were harvested. Calcium hydroxide was used to increase the pH when it dropped below 6. Venturi devices and air compressors helped to maintain optimal oxygen levels. Simple and inexpensive water test kits that employ colorimetric methods were used for the measurement of the water chemical parameters. 

Three complete water analyses were performed in both MAS during the experiment. To do so, water samples were taken from the sump and analysed by spectrophotometry. When nitrate concentrations in the water exceeded 80 ppm, flower pots for sale new plants were put into the system. Foliar applications of K2SO4 and MnSO4 were performed when required. An EDDHA Sequestrene 138 Fe chelated iron solution  was directly added to the water. Seaweed fertiliser  at 10 mL L− 1 was used only for 3 weeks, sprayed on leaves, at the beginning of the operation of the aquaponic facilities, between April and May, because the fish biomass being still low, the nutrient level for plants in water was also low. FERRAMOL  at 5 g m-2 was used against the snails and slugs that appeared during the autumn. Nettle slurry  at 15 mL L− 1 was used as a foliar application against the white fly that affected lettuces in autumn. Other routines periodically performed were: observations of the health status of fish and plants, the control of the water levels  and flows,and maintenance and cleaning of equipment. Potassium soap  at 20 mL L− 1 mixed with Neem oil  at 1 g L-1 was used against aphids  from July to November. Slaked lime  was added in the sumps to raise the pH from 6–6.2 to 6.4–6.5. Micronised sulfur  was applied foliarly at 0.3 g L-1 against fungal pathogens  and occasionally against red spiders that appeared in a very small quantity in strawberry plants.The total horticultural production, number of plants and fruits, average production per plant and average weight of fruits or leaves in the different hydroponic sub-systems are shown in Tables 2 and 3. Both MAS had a similar annual total production per cropping area: 38.96 and 38.31 kg m− 2 for MAS1 and MAS2, respectively.

NFT was the most productive hydroponic sub-system, mainly due to the lettuce production. However, GB was the sub-system with the highest production per cropping area,followed by NFT  and DWC. The NFT subsystem had a higher density of plant species, with 20 plants m− 2. The plant density in the GB sub-system was 11.7 plants m− 2,the values being lower in the DWC. Cucumber and broccoli were the only plants that were cultivated in the three sub-systems, the GB being the one with the highest production for both species. The most productive species were courgette, cucumber and Roma tomato in the GB; lettuce, chard, Italian frying pepper and Roma tomato in the NFT; and pumpkin in the DWC. The plants that took, on average, the least time to be ready for harvest in MAS 1 were stevia,courgette  and cucumber. For MAS2, they were stevia,lettuce  and basil. On the other hand, the plants that took the longest to be produced in MAS1 were strawberry,water melon  and goat horn pepper. For MAS2, they were pumpkin,water melon  and goat horn pepper. The distribution of the production during the year  for all the species cultivated is also detailed in the Supplementary Information. Lettuce was the only plant species grown year-round in the GB and NFT subsystems, and therefore had the highest annual production with 68.7  and 64.5 kg. Also, cucumber and courgette had a high production. On the contrary, onion and cabbage were the species which contributed less to total production. The presence of aphids slightly affected the chards and cucumber; however, they were able to grow and give satisfactory production. The strawberry had a low production due to late planting, outside its natural season. The large-fruited and typical summer plants in Andalusia such as water melon, pumpkin and melon produced very few fruits in September and October. The water melon and melon plants were also very affected by the attack of the aphids, although they did bear fruit. The months with more production were April, July and August, accounting for 46 % of the total in the whole year, May and February being those with a lower production. In the case of May, this was because it was the system’s first month of operation, when the fish biomass was still low.

The total tilapia production was 33.5 kg in MAS1 and 29.28 kg in MAS2. The fish harvests occurred in August,September, October and April. The evolution of the fish biomass is shown in Fig. 5. The largest fish harvest was carried out in the fifth weighing: 43 fishes were extracted from MAS1 and 41 fishes from MAS2 as high fish biomass,plus high temperatures reduced dissolved oxygen levels in water to critical levels. In the seventh weighing, 16 fish were extracted from MAS1 and in MAS2 all the remaining fish  were harvested, so it became a completely hydroponic system. The mean weight per harvested fish was 317 g for MAS1 and 302 g for MAS2. Among the 31 fish finally harvested in MAS1 at the end of April 2019 after 1 year of growth, 14 of them weighed below 350 g. There was no predation of small fish; however, a total of 5 fishes died in MAS1 and 9 fishes in MAS2. Table 4 shows the average values for the growth indicators in each MAS. Fig. 6 shows the evolution over time of the plants harvest, fish biomass, nitrates and amount of fish feed.One of the main challenges of aquaponic production is maintaining an adequate water quality so the plants, fish and microorganisms can function correctly. Therefore, the key to a successful aquaponic system, especially if it is a micro-scale one, is to be able to keep the physicochemical water parameters within the limits that those three populations need. This is not easy in coupled systems, because the quality of the water circulating in the hydroponic subsystem is the same as the aquaculture subsystem. However, opting for decoupled systems on a small scale is not feasible due to their complex maintenance  which is not suitable for a single family but on a large scale such as a commercial environment. For instance, the pH is one of the most difficult factors to control as the optimal values for fish and plant production are different. During our experiment, the pH was maintained with values of 6.45 ± 0.47  and 6.62 ± 0.51,given that with values below 6, bacterial nitrification is considerably reduced. Hence, when the pH decreased, calcium hydroxide had to be added. At the time of transforming MAS2 from aquaponic to hydroponic,the pH, NO3 − and EC levels fluctuated rapidly. The pH quickly rose from 6.8-7 to 7.5 in the first hours, so hydrochloric acid was added to lower it to the previous levels. In aquaponic systems, tower garden the balance between the fish biomass and the plant production is very important in order to both maximise the production and to maintain an adequate quality of the water. Also, plant nutrients uptake depends, among other factors, on the pH.

If it is not optimal, the nutrients can be accumulated in the water. This is a drawback in coupled aquaponic systems. In decoupled systems, pH values can be better adapted without affecting fish and bacteria. Not only pH levels but also dissolved oxygen is important for the nutrients balance. Wongkiew et al.  stated that low levels of dissolved oxygen can lead to nitrogen loss mainly via nitrifier denitrification. They considered values of 3.8 mg L− 1 as low levels, while those around 7 mg L− 1 where high. In our experiment, dissolved oxygen ranged between 5 and 11 mg L− 1,being below 5 mg L− 1 only in August. The relation between the plants harvest, fish biomass, nitrates and amount of fish feed is clear. For instance, in MAS1, the maximum nitrate levels were 155 mg L− 1,corresponding with a peak in fish feed  and biomass. Indicating that the rate of nitrogen input to the system  was higher than the rate of assimilation by plants. The same was reported by  in aquaponic systems based on lettuce, pak choi and chives. This suggests that a greater surface could be dedicated to plant cultivation during summer. As an alternative, more fruiting vegetables could be introduced, these having a higher demand for nutrients than green leafy vegetables and also producing more kg at harvest. This is important because it would mean an increase in the annual plant production. In contrast, since the beginning of December 2018, the nitrate levels dropped, though 50 mg L− 1 were maintained with a low feed consumption  until the end of February 2019, when a minimum of 21 mg L− 1 was reached with a fish biomass of 8.34 kg and 24.9 g of feed. The decrease of nitrates was not so fast because of the expanded clay used in the GB sub-system, which could act as a “nutrient sponge” where a significant amount of solids accumulated and continued to be mineralised in well-oxygenated areas due to the emptying and filling effect of the bell-syphon.The total production during the study period accounting both for tilapia and plant production was nearly 211 kg in MAS1 and 204 kg in MAS2, employing a maximum cropping area of 4.56m2 and a fish tank volume of 0.95 m3,occupying a surface of 1.2 m2. Therefore, though the vegetal production per cropping area was slightly above 38 kg m− 2,the yields per production unit area  were around 36 kg m− 2. Still, in order to compare the yields per unit area obtained with other methods of production, the entire surface required for the production should be considered. Including service corridors and the area devoted to the clarifier and other equipment, the surface to be considered for each MAS was 16 m2. In our study the total production  was 415 kg of produce in a 45 m2 greenhouse,which means 9.2 kg m− 2. This value more than doubles that of the average yield per unit area in the social urban vegetable gardens in Seville. Sommerville et al.  estimated that an FAO-type MAS could produce 360 lettuce heads, 54 kg of tomatoes and 30 kg of fish during a year with a cropping area of 3 m2. Although these authors do not specify the average weight per lettuce head, if we estimate 250 g per head, the total production would be 90 kg of lettuces that, added to the tomatoes and fish, would result in 174 kg year− 1,slightly lower than the amount obtained in our study, though the fish production was very similar. The fact that our work involved 22 different species of vegetables, herbs and fruits makes it difficult to establish comparisons in terms of total plant production with other research works related with aquaponics in which only one crop was produced.

By managing the fertilizer supplementation and adjustable combination of fresh water intake and discharge of HPS water the solution can be kept within plant specific ranges . Transpiration of the crops is the main driver of water transfer between RAS and HPS, as the water level in the HPS is kept constant. Given the fact that also the amount of water in the RAS is kept constant, the variation in transpiration results in a varying freshwater intake in the RAS. In addition to the three basic elements, as shown in Figure 1, a reverse osmosis filtration system can be implemented to further increase the difference in nutrient concentration between the RAS and HPS . As a result of the filtration process, a nutrient-rich brine is added to the HPS, while the remaining water with largely reduced nutrient concentrations is fed back to the RAS. In this study, the system has a fixed hourly flow but is deactivated when either the nitrate or phosphorus concentration in the HPS nears its upper limit, to prevent the need for dilution. The RO filtration system was not used in most of the scenarios in this study and it is specifically stated in which cases it was used. Limited seasonal variation solar radiation climate was found to be beneficial for a balanced and efficient aquaponics system by Goddek & Körner . In seasonally varied climates, a mismatch can arise between the stable nutrient flow from the RAS to the HPS and the varied nutrient demand of the HPS . Two additional strategies were tested to achieve the benefits of stable climates, using water-buffering tanks as shown in Figure 2. Both options aim to better match the supply and demand of nutrients in the HPS throughout the year.For ease of access, the model created in this study was implemented in Microsoft Excel TM and consists of various sub-models, as shown in Figure 3.

While discussed only briefly in the following sections, each is described in detail in Supplementary Materials SM 1. For more advanced aquaponics model implementations we refer to Karimanzira et al and Reyes Lastiri et al . For clarity in categorizing parameters and performance indicators, a distinction was made between the aquaponics system and the greenhouse system . Unless stated otherwise, potted blueberries the parameter values used in this study are presented in Appendix 1. Figure 3. The four sub-models of the aquaponics model and the dynamic variables that are exchanged between them. As the nutrient solution is kept within suitable bounds for the crop, it is assumed that the Water & nutrients sub-model has no further influence on the Plants sub-model. The balance equations of the four sub-models are solved numerically using the Euler forward method. A time-step of one day was found to be sufficiently accurate for the Fish production and Water & Nutrients submodels as variation in the nutrient concentration throughout the day was not considered in this study. For the Greenhouse climate and Plants sub-models, five minutes was chosen as the smallest acceptable time step in terms of computational requirements. The growth of fish in the rearing tanks is calculated using the equations from Timmons & Ebeling , as implemented by Dijkgraaf et al. . Assuming the growing environment is optimal, in terms of e.g. pH, dissolved oxygen and fish feed, the weight gain of the fish is calculated using only water temperature and several fish-specific parameters. Besides being used to calculate the fish yield, the sub-model is used to determine the feed requirements throughout the production cycle. For this, a feed conversion rate is used, which describes the ratio between feed uptake and weight gain, as explained by Dijkgraaf et al. . Staggered production of fish provides a smoother nutrient supply . As this study specifically estimates the performance over one year of operation, the start-up phase of the RAS is omitted. The Water & Nutrients sub-model calculates the flows of nutrients and water throughout the system. It is based on the work of Dijkgraaf et al. , with several corrections . The model considers nitrogen and phosphorus, for their importance in crop growth and dynamics of RAS and AD. Potassium was not considered as it makes up an insufficient fraction of the fish feed to influence the system .

In the HPS, represented by the sub-models Greenhouse climate and Plants, the concentrations of N and P were determined by the incoming flow and the nutrient uptake of the crops. Given the very complex process of nutrient uptake by plants and given the overall aim of the study to study the operation and design of a full aquaponics system, the basic assumption that the nutrient uptake by the crops is equal to the product of the crop transpiration and nutrient concentration, as in the studies of , was made. Nutrient supplementation or dilution of the HPS water was used to keep the concentrations within the suitable range for the crop. The ranges used in this study are 140 – 180 mg/L nitrate and 40 – 60 mg/L phosphate , following Resh . The main function of the Plants sub-model is to use data from the greenhouse climate to determine the transpiration of the crop, according to Stanghellini and de Jong and . Crop transpiration depends on plant-specific parameters and environment variables. In this study, the growth of the crops was not affffected by the Water & Nutrients or Fish production sub-model, as nutrient concentrations were kept within optimal bounds. In the Greenhouse climate sub-model, the temperature, humidity and light inside the greenhouse are simulated, representing the growing environment of the crops. The greenhouse climate is described by physics based, dynamic balance equations for uniform temperature and absolute humidity of the greenhouse, as done by Van Beveren et al. , see Supplementary Materials SM 1. All variables and parameters are also explained in SM 1. While the model created for this study is of relatively low complexity, the outcomes fifit within those found in the literature, as shown in Supplementary Materials SM 1.5. To ensure a suitable temperature and humidity for the crops, management and control strategies have been modelled, as well. For instance, the energy screen is deployed when solar radiation is below a threshold, as described in Kempkes & Janse . The shade screen is deployed when solar radiation exceeds a maximum or when outside temperatures are close to the maximum allowed greenhouse temperature. Artificial lighting, if used, is activated between 8 am and 8 pm if solar radiation is below a threshold and is disabled from May to October to save energy. Temperature and humidity are controlled based on the energy and vapor fluxes of the greenhouse in four steps, as displayed in Supplementary Materials SM 1.The total transpiration of the greenhouse is directly proportional to the HPS floor area. As it is assumed that nutrient uptake is proportional to the crop transpiration and the concentrations in the nutrient solution, an increase in the HPS area will increase the nutrient demand.

In general, a system with a high HPS area will require nutrient supplementation, while dilution is required for a smaller HPS, as is shown in Figure 4. While nutrients can be supplemented individually, dilution results in the loss of all nutrients in the diluted volume. At a low HPS area, the dilution resulting from one nutrient may lead to the need to increase supplementation of the other, as is the case for N with an HPS area smaller than 1500 m2. The chosen irrigation technique determines the volume of water in the HPS. While nutrient supply and demand are initially unchanged by this, the buffering capacity of high-volume systems can prevent shocks in the nutrient concentrations. Overall, a larger volume results in an increase in performance, as shown in Figure 5, with most benefits for medium and large-sized HPS areas . The discontinuity at around an HPS area of 2700 m2 for DWC is a result of the absence of any need for dilution. The remaining inefficiency is due to fixed losses from the nutrients in the waste sludge of the digester. The ratio between the phosphorus and nitrogen content in the fish feed is an important factor in balancing the nutrient supply to the crop demand. Determining the optimal ratio is complicated by the different ways through which N and P are supplied to the HPS. Most of the nitrogen flow is directly from the RAS and is linked to the transpiration rate, which varies greatly throughout the season. Phosphorus, on the other hand, mainly enters the HPS through digester effluent , with only a slight oscillation resulting from the staggered fish production . With the reference settings, the ratio between the nutrients was optimal for a low P content between 0.65% and 0.8%. However, this could be insufficient for proper fish growth. While Sugiura et al. reported no difference in the growth of tilapia between 1% and 1.4%, a decrease of 28% in overall growth was found for a P content of 0.5% by Eding et al. . The reverse osmosis system results in an additional transfer of nutrients from the RAS to the HPS, square plastic pot which also lowers the nitrate concentration in the RAS. Besides increasing the HPS area, adding RO is practically the only way of reducing NO3 in the RAS, which is especially important for more sensitive fish species. The RO system was temporarily disabled when the concentration of either N or P in the HPS was at 95% of their respective maximum, as it could otherwise result directly in dilution. This puts a limit on the desalination flow and decreased additional benefits at higher flow capacities, especially for smaller HPS areas. As the N content in the RAS and HPS are closely linked, the RO system is mainly able to transfer nutrients when demand is low, resulting in only a minor increase in performance. When demand is high in summer, the low nutrient concentrations of the RAS results in only a small additional transfer from the RO.

As shown in Figure 3, the Greenhouse climate and Plant sub-models can only influence the AP system through the transpiration rate. A large variation in crop transpiration can result in a mismatch between the relatively constant supply of nutrients and the transpiration-dependent demand. This large variation can also lead to the requirement of both supplementation and dilution of a nutrient within the same year, as is also more likely in the low-volume NFT system. The same effects of variation can be seen for the nitrate concentration in the RAS. While low variation results in lower peaks in the cold months, the higher concentration in summer also means that more nitrate is transferred to the HPS, lowering supplementation requirements.The objective of this research was to investigate and demonstrate the effect of changes in various physical and operational parameters on an on-demand coupled aquaponics system with anaerobic digester and potential use of RO filtration system. The research focused on changes that could result in energy savings, as it is one of the crucial aspects of sustainable food production in northern latitudes. While there are studies that compare the system performance at different locations, the specific influence of physical and operational parameters was not yet investigated for aquaponics systems.Goddek & Körner suggested that a plant production system with a constant nutrient demand , would result in a balanced system with better performance. This observation resulted in our hypothesized correlation between the variation in transpiration and the performance of the AP system. It has been found that the coefficient of variation of the transpiration rate, as representative for the different scenarios , was an appropriate factor that highly correlates with the chosen KPIs of the AP system , given that the HPS area is resized as shown in Appendix 3. This strong correlation also holds for the average KPI, as was demonstrated in Figure 10. While increasing variation generally results in decreasing efficiency, the sensitivity to it was not the same for different settings of the aquaponics system.

We therefore speculate that Ev1.1 may have first gained the mutation in rsiX and was selected for owing to increased eps expression, whereas later on the increased matrix production was reverted by the mutations in gtaB, resulting in a non-functional protein and thereby reduced precursors for EPS production, which was selected for owing to the reduced cost. In accordance with the mutations observed in this study, a non-synonymous mutation in sinR in two isolates forming Snow-type colonies and increased in root colonization as well as several mutations in gtaB in two isolates with a Smooth colony morphology were observed in our recent study on diversification of B. subtilis during adaptation to A. thaliana roots.To get a more general insight into the mutations arising in B. subtilis during EE on A. thaliana roots, the seven endpoint populations were also sequenced. This revealed mutations within genes related to biofilm formation , flagellar motility , and cell wall metabolism  across independent populations. Moreover, there is limited data available on the impact of Fe plaque formation on Cd accumulation by different rice varieties usually grown in Australia as significant genetic variations were reported among rice varieties in the accumulation of metals in rice grain . Thus, it is important to investigate the performance of Fe plaque formation by different rice varieties and subsequent Cd accumulation. Consequently, the objectives of this present study was to: explore the protective function of Fe plaque in reducing Cd accumulation and translocation in different varieties and comprehend the interaction effect of Fe and Cd in rice seedlings. Cadmium is a highly toxic heavy metal and it does not have any beneficial physiological functions in plants when it is accumulated, affects all aspects of growth and development .

Cd toxicity stress causes the reduction in the rice growth and biomass yield . Consistent with these observations, the dry biomass of rice cultivar’s roots and shoots were significantly reduced with increasing Cd . In the absence of Fe additions to the nutrient solution, chicken fodder system Cd0.5 and Cd1.0 significantly decreased the dry weight of roots and shoots compared with the control Cd0. Our findings are in good agreement with a recent hydroponic study conducted by Hussain et al. who demonstrated that roots and shoots biomass reduced with increasing Cd levels in solutions. Liu et al. also found that rice plants treated with Cd had smaller dry weights of roots and shoots compared with plants grown with no added Cd under hydroponic conditions. It has been shown that a reducing photosynthetic activity attributed with decremental rice biomass production under Cd stress . In this study, substantial increases in the biomass of roots and shoots were observed when Fe was added to the nutrient solutions with added Cd, and this is most likely due to the Fe plaque deposition, which alleviated the effects of Cd toxicity on the growth of rice plants. Our results differ from those obtained with other toxic trace elements. According to Liu et al. , Fe plaque or Fe supplement with As had no advantageous effects on the rice seedling’s growth raised in culture suspension. Ye et al. also demonstrated that the Fe addition or root plaque did not influence the yield of common cattail seedlings. However, our findings are consistent with those of others who observed that seedlings with high Fe plaque formation tended to greater root and shoot biomass production compared to those not having Fe plaque. The results also showed that CBD-Fe and Feroots and Feshoots of rice seedlings increased significantly when the Fe concentration of the nutrient solution increased, irrespective of Cd supply. Exogenous Fe application or Fe plaque have been shown to elevate the Fe content of plants , which is consistent with this study’s findings. Similar findings were reported by Sebastian and Prasad who found that greater Fe plaque deposition on the rice seedling’s root surface after FeSO4 application.

This also led to an increase in the Fe content of rice plants, which protected the rice plants from Cd-induced Fe deficiency and metal toxicity. The amount of Fe in Fe plaque are in agreement with numerous researchers who reported results for hydroponically grown rice seedlings with concentrations of Fe . Excessive Fe plaque coating on the root was reported when additional Fe was supplied to the growth medium . The presence of large quantities of Fe plaque on the root tends to extensive Cd adsorption and sequestration and this was evident by the elevated Cd concentration in CBD extractant solution of rice seedlings when subjected to exogenous Fe . Several reports showed a positive correlation between concentration of Fe and metals such as As, Cd etc. in root plaque . In this present investigation, the Cd concentration in Fe plaque increased with the iron supplementation which implied that Fe plaque demonstrated strong Cd sequestration capacity in terms of Cd immobilization and translocation by rice seedlings. Liu et al. also observed that higher CBD-Cd and root Cd concentration substantially reduced the Cd concentration in the shoots of rice seedlings. The root plaque deposition could alter and diminish the translocation and accumulation of heavy metals in the upper portions of rice plants but opposite results were reported . This inconsistency could possibly be ascribed to variations in the amounts of Fe plaque being formed, growth medium, pH, metal ion type, metal ion content, different plant types and cultivars. In this study, the use of Fe50 and Fe100 concentrations significantly decreased the concentration of Cdshoots of the rice seedlings compared with control Fe0 at both Cd supply concentrations . This observation is consistent with Liu et al. who found Cd concentrations in shoots were reduced by Fe plaque produced from 100 mg Fe L− 1 and with 0.1 mg L− 1 Cd in nutrient solutions. It was further confirmed in a recent hydroponic study conducted by Hussain et al. who observed that Cd concentration in rice seedling’s roots and shoots were reduced under sufficient Fe supplementation. Literature shows that the accumulation of Cd among various rice genotypes varied significantly . A study reported that the three key transport processes responsible for Cd accumulation such as root uptake, shoot transport through xylem and finally grain loading via the phloem .

It has been found that the acquisition of Cd in rice plant is mediated by the translocation from root to shoot through xylem, which is the main pathway of Cd entry in shoots and grain of rice plants . Another study revealed that application of Fe and Cd in nutrient solution increased relative expression of OsNRAMP1, OsNRAMP5, OsIRT1, CAL1and OsNAAT1 in the roots of rice plants and reduced the expression of OsHMA3, which lead to increase Cd content in rice plants nurtured in solution culture . Our results demonstrated that the concentration of Cdroots was higher than the CBD extracts and in shoots of rice seedlings. The percentage distribution of Cdroots in rice seedlings accounted for approximately 64.6–93% of the total Cd in the rice seedlings, which was greater than that in CBD extracts and in shoots . The higher proportional distribution of Cdroots indicated that root systems absorbed a lot of the Cd and serve as an effective barrier to hinder Cd delivery in rice plants. This is essentially in agreement with Ye et al. who indicated that root cells were exhibited the stronger barriers to suppress the accumulation and transportation of Cd and Pb in rice plants. In hydroponics experiments, Liu et al. revealed that Cd roots displayed approximately 65% of the total Cd concentration and in shoots and CBD extracts, the concentrations ranged from 9 to 40%. Exogenous Fe application can increase the Cd compartmentation and detoxification in the root cells which needs further investigations. The world population is expected to increase to 9.5 billion people in the next 40 years. This calls for an increase of over 60% in food production worldwide at least by 2050 to combat the crisis faced by the continuously increasing population . Unfortunately, natural resources such as: land meant to sustain food production and meet the demands of such an expected population increase are diminishing coupled with the high cost of the limited existing land . The high rates of urbanization and environmental degradation caused in the last decade have negatively impacted on the quality and quantity of food production . Besides the above challenges, there is a problem of nutrient depleted soils and water scarcity across the globe and these are expected to exacerbate in the face of the increasing population especially in urban areas .

We therefore speculate that Ev1.1 may have first gained the mutation in rsiX and was selected for owing to increased eps expression, whereas later on the increased matrix production was reverted by the mutations in gtaB, resulting in a non-functional protein and thereby reduced precursors for EPS production, which was selected for owing to the reduced cost. In accordance with the mutations observed in this study, a non-synonymous mutation in sinR in two isolates forming Snow-type colonies and increased in root colonization as well as several mutations in gtaB in two isolates with a Smooth colony morphology were observed in our recent study on diversification of B. subtilis during adaptation to A. thaliana roots . To get a more general insight into the mutations arising in B. subtilis during EE on A. thaliana roots, the seven endpoint populations were also sequenced. This revealed mutations within genes related to biofilm formation , flagellar motility , and cell wall metabolism  across independent populations.  Moreover, there is limited data available on the impact of Fe plaque formation on Cd accumulation by different rice varieties usually grown in Australia as significant genetic variations were reported among rice varieties in the accumulation of metals in rice grain . Thus, it is important to investigate the performance of Fe plaque formation by different rice varieties and subsequent Cd accumulation. Consequently, the objectives of this present study was to: explore the protective function of Fe plaque in reducing Cd accumulation and translocation in different varieties and comprehend the interaction effect of Fe and Cd in rice seedlings.Cadmium is a highly toxic heavy metal and it does not have any beneficial physiological functions in plants when it is accumulated, affects all aspects of growth and development.Consistent with these observations, the dry biomass of rice cultivar’s roots and shoots were significantly reduced with increasing Cd . In the absence of Fe additions to the nutrient solution, Cd0.5 and Cd1.0 significantly decreased the dry weight of roots and shoots compared with the control Cd0. Our findings are in good agreement with a recent hydroponic study conducted by Hussain et al. who demonstrated that roots and shoots biomass reduced with increasing Cd levels in solutions. Liu et al. also found that rice plants treated with Cd had smaller dry weights of roots and shoots compared with plants grown with no added Cd under hydroponic conditions.

It has been shown that a reducing photosynthetic activity attributed with decremental rice biomass production under Cd stress . In this study, substantial increases in the biomass of roots and shoots were observed when Fe was added to the nutrient solutions with added Cd, and this is most likely due to the Fe plaque deposition, which alleviated the effects of Cd toxicity on the growth of rice plants. Our results differ from those obtained with other toxic trace elements. According to Liu et al. , Fe plaque or Fe supplement with As had no advantageous effects on the rice seedling’s growth raised in culture suspension. Ye et al. also demonstrated that the Fe addition or root plaque did not influence the yield of common cattail seedlings. However, our findings are consistent with those of others who observed that seedlings with high Fe plaque formation tended to greater root and shoot biomass production compared to those not having Fe plaque. The results also showed that CBD-Fe and Feroots and Feshoots of rice seedlings increased significantly when the Fe concentration of the nutrient solution increased, irrespective of Cd supply. Exogenous Fe application or Fe plaque have been shown to elevate the Fe content of plants , which is consistent with this study’s findings. Similar findings were reported by Sebastian and Prasad who found that greater Fe plaque deposition on the rice seedling’s root surface after FeSO4 application. This also led to an increase in the Fe content of rice plants, which protected the rice plants from Cd-induced Fe deficiency and metal toxicity. The amount of Fe in Fe plaque are in agreement with numerous researchers who reported results for hydroponically grown rice seedlings with concentrations of Fe . Excessive Fe plaque coating on the root was reported when additional Fe was supplied to the growth medium . The presence of large quantities of Fe plaque on the root tends to extensive Cd adsorption and sequestration and this was evident by the elevated Cd concentration in CBD extractant solution of rice seedlings when subjected to exogenous Fe . Several reports showed a positive correlation between concentration of Fe and metals such as As, Cd etc. in root plaque . In this present investigation, the Cd concentration in Fe plaque increased with the iron supplementation which implied that Fe plaque demonstrated strong Cd sequestration capacity in terms of Cd immobilization and translocation by rice seedlings. Liu et al. also observed that higher CBD-Cd and root Cd concentration substantially reduced the Cd concentration in the shoots of rice seedlings.

The root plaque deposition could alter and diminish the translocation and accumulation of heavy metals in the upper portions of rice plants but opposite results were reported . This inconsistency could possibly be ascribed to variations in the amounts of Fe plaque being formed, growth medium, pH, fodder systems for cattle metal ion type, metal ion content, different plant types and cultivars. In this study, the use of Fe50 and Fe100 concentrations significantly decreased the concentration of Cdshoots of the rice seedlings compared with control Fe0 at both Cd supply concentrations . This observation is consistent with Liu et al. who found Cd concentrations in shoots were reduced by Fe plaque produced from 100 mg Fe L− 1 and with 0.1 mg L− 1 Cd in nutrient solutions. It was further confirmed in a recent hydroponic study conducted by Hussain et al. who observed that Cd concentration in rice seedling’s roots and shoots were reduced under sufficient Fe supplementation. Literature shows that the accumulation of Cd among various rice genotypes varied significantly . A study reported that the three key transport processes responsible for Cd accumulation such as root uptake, shoot transport through xylem and finally grain loading via the phloem . It has been found that the acquisition of Cd in rice plant is mediated by the translocation from root to shoot through xylem, which is the main pathway of Cd entry in shoots and grain of rice plants . Another study revealed that application of Fe and Cd in nutrient solution increased relative expression of OsNRAMP1, OsNRAMP5, OsIRT1, CAL1and OsNAAT1 in the roots of rice plants and reduced the expression of OsHMA3, which lead to increase Cd content in rice plants nurtured in solution culture . Our results demonstrated that the concentration of Cdroots was higher than the CBD extracts and in shoots of rice seedlings. The percentage distribution of Cdroots in rice seedlings accounted for approximately 64.6–93% of the total Cd in the rice seedlings, which was greater than that in CBD extracts and in shoots . The higher proportional distribution of Cdroots indicated that root systems absorbed a lot of the Cd and serve as an effective barrier to hinder Cd delivery in rice plants. This is essentially in agreement with Ye et al. who indicated that root cells were exhibited the stronger barriers to suppress the accumulation and transportation of Cd and Pb in rice plants. In hydroponics experiments, Liu et al. revealed that Cd roots displayed approximately 65% of the total Cd concentration and in shoots and CBD extracts, the concentrations ranged from 9 to 40%. Exogenous Fe application can increase the Cd compartmentation and detoxification in the root cells which needs further investigations. The world population is expected to increase to 9.5 billion people in the next 40 years. This calls for an increase of over 60% in food production worldwide at least by 2050 to combat the crisis faced by the continuously increasing population . Unfortunately, natural resources such as: land meant to sustain food production and meet the demands of such an expected population increase are diminishing coupled with the high cost of the limited existing land . The high rates of urbanization and environmental degradation caused in the last decade have negatively impacted on the quality and quantity of food production . Besides the above challenges, there is a problem of nutrient depleted soils and water scarcity across the globe and these are expected to exacerbate in the face of the increasing population especially in urban areas . Traditional farming is generally faced with problems of weather changes, water pollution, soil degradation and soil infertility . Africa alone continues to fight the problem of food insecurity where improved yield and sustainability in the agriculture sector can best be achieved through climate smart agriculture . CSA has been defined as an intervention vital for maintainace of global food security and nutrition through changing and readjusting agricultural practices within the new era of climate change . In order to conserve sustainable crop production systems, there is need to utilize spaces like: non-arable fields that do not support crop cultivation and develop alternative cultivation methods . This justifies the increasing use of various smart agricultural technologies to meet these rising levels of food insecurity.

Emami et al. described smart agriculture as the use of technology that has the capability to increase food security if well streamlined to the domestic levels. On other hand, CSA synchronizes actions by researchers, policy maker, private institutions, societies and farmers to promote climate resilient systems, practices and technologies .Soilless culture is one of the growing smart agriculture technologies in East Africa which encompasses growing crops with or without a media or using a static/flowing nutrient solution . Media refers to an organic or inorganic solid material that is used in the place of soil either in single or mixed form to provide support to the plant, for example: perlite, vermiculite, rice hulls, saw dust, coco-peat . Soilless farming is largely used under controlled environment mainly for horticultural crops and gives the opportunity to cultivate in areas with un favorable agriculture conditions such as: poor soils and limited space among other benefits . Soilless farming has the capacity of solving some of prior challenges such as: limited water availability and soil degradation, reduced pests and diseases, while promoting sustainable agriculture . Soilless farming is divided into 3 main categories which are: hydroponics, aeroponics and aquaponics . Aquaponics is a soilless farming system where plants and fish are raised in an associated relationship as the water is recycled through the system and plants uptake nutrients acquired from recycled fish waste water . Aeroponics on the other hand is a technique where devices like foggers are used to supply plant roots with nutrients inform of a mist .Arshad Mahmood et al. described hydroponics as an agriculture system for growing crops in water composed of mineral nutrients supported by medium. This system which uses less water as compared to soil farming has successfully been used for cultivation of different vegetables like: lettuce, spinach, cucumbers, tomatoes among other crops as these respond well to hydroponics due to low nutrient demands and short growth period . New drifts in agriculture have shown hydroponics as one of the new innovative soilless farming systems to realize satisfactory outcomes and has the potential to produce more yields in minimal space and promote food security through production of food vertically thus should be considered as a better farming option for East Africa facing a quandary of challenges as earlier discussed. Hydroponic farming has different types which include: Nutrient Film Technique , Wick system, Drip system, Ebb and Flow and Deep water culture . Wick system is the simplest hydroponic method which uses wicks to draw nutrients from the reservoir without use of pumps or timer while NFT hydroponics is a method where shallow channels are used to supply the nutrient solution to the bare plant roots through re-circulation process . DWC is a method of hydroponics in which plant roots are suspended directly into the nutrient rich water solution while drip system uses micro emitters to drip the nutrient and water directly to the plant roots with the help of a pump .

Arshad Mahmood et al. described hydroponics as an agriculture system for growing crops in water composed of mineral nutrients supported by medium. This system which uses less water as compared to soil farming has successfully been used for cultivation of different vegetables like: lettuce, spinach, cucumbers, tomatoes among other crops as these respond well to hydroponics due to low nutrient demands and short growth period . New drifts in agriculture have shown hydroponics as one of the new innovative soilless farming systems to realize satisfactory outcomes and has the potential to produce more yields in minimal space and promote food security through production of food vertically thus should be considered as a better farming option for East Africa facing a quandary of challenges as earlier discussed. Hydroponic farming has different types which include: Nutrient Film Technique , Wick system, Drip system, Ebb and Flow and Deep water culture . Wick system is the simplest hydroponic method which uses wicks to draw nutrients from the reservoir without use of pumps or timer while NFT hydroponics is a method where shallow channels are used to supply the nutrient solution to the bare plant roots through re-circulation process . DWC is a method of hydroponics in which plant roots are suspended directly into the nutrient rich water solution while drip system uses micro emitters to drip the nutrient and water directly to the plant roots with the help of a pump.

The minimal medium used in the EE should render the bacteria dependent on root exudates and dead plant material to survive.To test this, we used a modified version of the minimal medium employed during the EE. Instead of 0.05% glycerol, the MSN medium was supplemented with 0.5% cellobiose . Cellobiose is a disaccharide and a product of partial hydrolysis of cellulose, found in plant cell walls . In addition, MSNc was supplemented with 0.5% xylan. The ancestor showed a growth profile typical of bacterial growth under planktonic conditions . In contrast, several evolved isolates displayed distinct growth profiles, including 3.2, 7.1, 7.2, and 7.3, which showed no decline phase, but instead displayed a pro-longed stationary phase. When analyzing the carrying capacity , several isolates showed significantly increased carrying capacity , whereas few isolates showed significantly decreased carrying capacity . While cellobiose and xylan do not completely represent the plant compounds present in the selective environment, these results suggest that adaptation to the plant root could also be facilitated through the altered utilization of plant compounds.During the EE, B. subtilis was adapted to the plant root alone – in the absence of other microbes. This selective environment is far from its natural habitat in the rhizosphere, where B. subtilis encounters other microbial residents. In fact, the ancestor DK1042 is a derivate of the wild strain NCIB 3610, originally isolated from hay infusion . To this end, we wondered how the pro-longed adaptation of B. subtilis to the plant root environment in the absence of other microbial species affected the ability to colonize the root in the presence of soil microbes. The ancestor and Ev7.3 were tested for their ability to colonize A. thaliana roots in the presence of a synthetic, soil-derived community . This community comprises four bacterial species, Pedobacter sp., Rhodococcus globerulus, Stenotrophomas indicatrix and Chryseobacterium sp. that were previously isolated from soil samples that also contained B. subtilis, thereby representing bacterial soil inhabitants that B. subtilis would normally encounter in nature.

The isolate Ev7.3 was chosen for this test since it was highly adapted to the selective environment, i.e. the isolate displayed significantly increased individual root colonization and out competed the ancestor during competition on the root, where it formed a robust biofilm along the root . To capture any potential difference in the establishment on the root,dutch bucket for tomatoes here defined as root colonization after 48 h, between B. subtilis ancestor and Ev7.3 in the presence of the community, the ancestor or Ev7.3 was co-inoculated with the community in four different ratios: 0.1:1, 1:1, 10.1, and 100:1 of B. subtilis and community, respectively. When B. subtilis was initially under-represented or highly in excess, i.e. inoculation ratio 0.1:1 and 100:1, respectively, no significant difference was observed in the establishment on the root between B. subtilis ancestor and Ev7.3 within the same inoculation ratio . In contrast, when co-inoculated with the community in intermediate ratios, i.e. 1:1 and 10:1, isolate Ev7.3 showed significantly enhanced establishment on the root compared with the ancestor. Since Ev7.3 displayed increased carrying capacity in MSNc + xylan in monoculture compared with the ancestor , we wondered whether the enhanced establishment on the root by Ev7.3 in the presence of the community could be partly attributed to improved utilization of plant compounds. Indeed, growth profiles in MSNc + xylan of the ancestor or Ev7.3 in co-culture with the community revealed that Ev7.3 displayed a significantly increased carrying capacity at inoculation ratio 1:1, 10:1, and 100:1 compared with the ancestor . Finally, in vitro confrontation assays on LB agar showed no major difference in the inhibition of the community members by Ev7.3 compared with the ancestor . Taken together, these results show that even though B. subtilis was adapted to the plant root alone, isolate Ev7.3 displayed increased root colonization also in the presence of a synthetic, soil-derived community under certain inoculation ratios, possibly mediated by robust biofilm formation on the root and enhanced utilization of plant compounds.

A recent study investigated the adaptive response of the PGPR Pseudomonas protegens to the A. thaliana rhizosphere in a sand system, which revealed mutations in genes encoding global regulators and genes related to motility and cell surface structure across independent populations and during such adaptation, the initially plant-antagonistic P. protegens bacterium evolved into mutualists . Furthermore, Lin et al. observed that adaptation of Bacillus thuringiensis to A. thaliana roots under hydroponic conditions led to the evolution of multicellular aggregating phenotypes, which, surprisingly, in certain lineages were accompanied by enhanced virulence against the Galleria mellonella larvae. Here, we employed experimental evolution to study the adaptation of B. subtilis to A. thaliana roots under hydroponic conditions. Our initial hypothesis was that B. subtilis would adapt to the plant roots by acquiring mutations that would provide the bacteria with a fitness advantage over the ancestor during root colonization. We could demonstrate that B. subtilis rapidly adapted to the plant roots, as observed by evolved isolates displaying improved root colonization relative to the ancestor already after 12 transfers and the detection of genetic changes in evolved isolates from transfer 12, 18, and 30. In addition, competition between the ancestor and two selected evolved isolates from the final transfer on the root revealed that both evolved isolates had a fitness advantage over the ancestor during root colonization, thereby confirming our hypothesis.Further phenotypic characterization of the evolved isolates from the final transfer revealed that most isolates across independent populations developed more robust biofilms in response to the plant polysaccharide xylan compared with the ancestor. Except for isolate Ev3.3, the robust biofilm formers tended to be increased in individual root colonization, indicating that robust biofilm formation is associated with adaptation to the plant root.

Motility represents an important trait for many bacteria as it allows them to explore the environment for nutrients and escape unfavorable conditions. Of relevance to the adaptation of B. subtilis to plant roots, motility has been shown to be important for root colonization of different plant species under different conditions. For example, a B. subtilis Dhag mutant was shown to be delayed or reduced in A. thaliana root colonization under hydroponic conditions as well as in tomato root colonization under vermiculites pot conditions . Yet, we found that five out of six isolates from two independent populations were impaired in both swimming and swarming motility, indicating that motility is not important for root colonization in the selective environment of the EE, i.e. under hydroponic, shaking conditions. Indeed, this was verifified in a competition experiment between a non-motile Dhag mutant and the WT, revealing that motility is not required for root colonization under shaking conditions. In contrast to our observations, Li et al. observed several evolved isolatesIn B. subtilis, motility and biofilm formation are incompatible processes: B. subtilis can exist as single, motile cells or in chains of sessile, matrix-producing cells which is regulated by an epigenetic switch involving SinR . The enhanced biofilm formation and impaired motility of isolates from populations 6 and 7 could thereby indicate a possible biofilm-motility trade-off. An inverse evolutionary trade-off between biofilm formation and motility was observed when the opportunistic pathogen Pseudomonas aeruginosa was subjected to repeated rounds of swarming that lead to the evolution of hyper-swarmers that were impaired in biofilm formation . Considering that the ability to form robust biofilm in vitro was shown to positively correlate with root colonization in B. subtilis , and the demonstration that motility is not important for root colonization under shaking conditions, a possible biofilm-motility trade-off could provide B. subtilis with enhanced fitness during root colonization in the selective environment. Indeed, isolate Ev7.3, which developed hyper-robust biofilms in LB + xylan and was impaired in motility, significantly out competed the ancestor during root colonization.

Re-sequencing of selected evolved isolates revealed that Ev7.1, Ev7.2, and Ev7.3 all harbored a single nucleotide polymorphism two base pairs upstream from the start codon of the sinR gene , encoding a transcriptional repressor of matrix genes . This SNP is located in the spacer region between the Shine Dalgarno sequence and the start codon in the ribosome binding site. Interestingly, the nucleotide composition of the spacer sequence has been shown to influence translation efficiency . The SNP upstream from sinR might thereby potentially affect the translation efficiency from the mRNA transcript, resulting in reduced levels of SinR. Reduced levels of SinR could in turn result in increased expression of matrix genes. This is supported by Richter et al. who demonstrated that a DsinR mutant shows increased matrix gene expression, and by Subramaniam et al. reporting that SinR translation and therefore protein level affects matrix gene expression. Potential increased matrix production caused by this mutation could contribute to the Snow-type colony morphology, as this colony morphology was exclusively observed for isolates harboring this mutation. Furthermore, in accordance with the robust biofilm formation and increased root colonization observed for Ev7.1, Ev7.2 and Ev7.3, a DsinR mutant was shown to form a hyper-robust biofilm in biofilm-inducing medium as well as on tomato roots , supporting the possible relevance of this mutation for the observed phenotypes of these isolates. Based on these previous studies, we therefore speculate that the mutation upstream from sinR results in increased matrix gene expression, which in turn enables more robust biofilm formation and increased root colonization as observed for the three isolates in population 7. These three isolates did not harbor mutations in motility-related genes. However, besides a possible effect of reduced SinR levels on the epigenetic switch that could lock the cells in a sessile, matrix-producing stage, a potential reduction in SinR levels leading to over-expression of the eps operon could possibly reduce motility owing to the EpsE clutch . Such mutation and the observed corresponding phenotypes could be responsible for the biofilm-motility trade-off,blueberry grow pot and be an example of antagonistic pleiotropy in which the same mutation is beneficial in one environment, i.e. during root colonization under shaking conditions, but disadvantageous in another, i.e. where motility is required for survival.

In addition, this mutation affecting a biofilm regulator could possibly explain why Ev7.1, Ev7.2, and Ev7.3 show improved biofilm formation also in the absence of xylan. Isolate Ev6.1 and Ev6.3 harbored a non-synonymous point mutation in the fliM gene, which was not present in Ev6.2. This gene encodes a flagellar motor switch protein, part of the basal body C-ring controlling the direction of flagella rotation . Interestingly, Ev6.1 and Ev6.3 were impaired in both forms of motility, whereas Ev6.2 showed similar swimming as the ancestor and was less affected in swarming. We speculate that the R326I substitution affects the function of FliM and consequently the flagellar machinery, resulting in hampered motility in these two isolates. Since we showed that motility was not important for root colonization under shaking conditions, a mutation hampering motility could provide the bacterium a fitness advantage during the adaptation to A. thaliana roots owing to the reduced cost of this apparently redundant trait. However, we do not expect the mutation in fliM to result in reduced cost; it merely changes an amino acid in a protein part of the flagellar machinery. Other mutations in the population six isolates must explain the robust biofilm formation by Ev6.2 and Ev6.3 and the fitness advantage of Ev6.1 over the ancestor during root colonization. For example, isolate Ev6.1 and Ev6.3 harbor a mutation in kinA encoding a two-component sensor kinase which once activated initiates the phosphorelay leading to phosphorylation of the master regulator Spo0A . The isolate from population one at transfer 30 harbored a frame shift mutation in the rsiX locus, encoding an anti-sigma factor controlling the activity of SigX . Inconsistent with the smooth morphology and reduced root colonization of this isolate, an DrsiX mutant was shown to have increased eps expression . However, Ev1.1 additionally harbored several mutations in gtaB encoding a UTP-glucose-1-phosphate uridylyltransferase involved in the biosynthesis of a nucleotide sugar precursor for EPS biosynthesis . A study conducted by Reverdy et al. showed that acetylation of GtaB is important for biofilm formation of B. subtilis and that a gtaB mutant was reduced in pellicle formation. In addition, Xu et al. showed that a DgtaB mutant of Bacillus velezensis SQR9 was significantly decreased in colonization of cucumber roots compared with the WT, although the effect of gtaB on root colonization may be species-dependent.

Allium schoenoprasum, which is commonly known as chive, is a perennial plant that is used as a popular seasoning and is cultivated globally . Phytochemical analyses of A. schoenoprasum extracts revealed that its water extract contains flflavonoid compounds, glycosides, and saponins while the methanol and ethyl acetate extracts contain alkaloids, flavonoids, glycosides, and saponins . Chive leaves have great medicinal values, such as positive effects on the circulatory system by reducing blood pressure and antimicrobial effects . Chive leaf infusions, often rich in potassium, are used for treatment of kidney stone disease by dissolving calcium oxalate in kidneys . Kucekova et al. demonstrated that chive flower extract obtained by solid-liquid extraction have a great effect on human keratinocyte cell line by decreasing cell proliferation, perhaps, due to their phenolic compounds. It is worth-mentioning that chives may contain many of the bio-active phenolic compounds such as gallic acid, coumaric acid, ferulic acid, and rutin that are commonly found in other Allium spp. Hence, the objective of this study was to assess plant growth of chive and secondary metabolites of chive leaves and roots following cultivation in a plant growth medium inoculated with conidia of an endophytic entomopathogenic fungus under greenhouse conditions. This is the first study on the experimental inoculation of chive with an endophytic fungus, vertical hydroponic nft system and its subsequent effects on plant growth and production of secondary metabolites.An indigenous B. bassiana strain that was previously isolated from a soil sample collected from the Cape Winelands in South Africa was used in this study. This strain was identified using molecular and morphological techniques as described in Moloinyane and Nchu .

The strain is being maintained at Cape Peninsula University of Technology in Bellville, South Africa. The fungus was cultivated on half-strength potato dextrose agar ; 0.02 g/L of ampicillin , and 0.04 g/L streptomycin in 9 cm and 14 cm diameters petri dishes. Fungal cultures were incubated at 25 C in the dark for four weeks. Using a spatula, mature four-week-old B. bassiana conidia obtained from PDA plates were transferred to 2 L glass bottles containing sterile 0.01% Tween 80 and sterile water. Bottles were capped, mixed by shaking for 5 min and by using a magnetic stirrer to homogenous conidial suspensions. The conidia inoculum concentration was enumerated using a haemocytometer and observed with a light microscope at 400X magnification. In order to obtain the desired concentration , the volume of sterile 0.01% Tween was increased or conidia were added to the glass bottle. This was followed by 10-fold serial dilutions to obtain lower concentrations of conidial inoculum suspensions; 1 104 , 1 103 conidia mL 1 . A conidial germination test to determine conidial viability was carried out according to the method described by Inglis et al. and high spore germination of over 90% was obtained.Chive seedlings, were purchased from Stodels Nurseries Ltd in Bellville, Western Cape Province, South Africa. Plants were maintained in the greenhouse at Cape Peninsula University of Technology in Bellville, South Africa at 23–25 C, 60%–80% RH and 13/11 natural light/dark regime. Individual chive plants were separated from a clump and one plant was transplanted to each 10 cm diameter pot containing a substrate mixture of one-third of river sand, one-third of vermiculite, and one-third of perlite by volume. The substrate materials were sterilized using 1% sodium hypochlorite for 1h before rinsing with sterile distilled water. The plants were fed with water soluble, formulated hydroponic fertilizer, Nutrifeed . The fertilizer was dissolved in sterile distilled water at a concentration of 10 g/5 L and 100 mL of the mixture was added to each plant once a week. Each plant was watered with 100 mL reverse osmosis water once a week.Leaf length from the soil surface to the top of the highest leaf and leaf number of each plant were measured weekly for three weeks.

Leaf length increment was calculated as the difference between leaf length at weeks one and three and percentage growth was calculated as follows: 100 x the leaf length increment divided by week one leaf length. Similarly, leaf number was counted and leaf number increment and % increase were calculated. At the end of the experiment, three weeks post fungal inoculation, root length and fresh weights of plants were recorded.Dry weights of the plants were determined by placing plants in paper bags in a drying oven at 35 C for 7 days.To determine fungal colonization of leaf tissue, three sections of leaf as well as root sections were collected from each plant soon after harvesting. The excised material was surface sterilized by first dipping in 70% ethanol for 3 s and then rinsing in sterile distilled water for 1 min. Thereafter, the sterile leaf and root sections were placed on solid half-strength PDA, incubated in the dark at 25 C, and were checked for outgrowth of B. bassiana in root and leaf tissues under stereo microscope after one and two weeks. efficiency of surface sterilization was evaluated by placing drops of the previously used 70% ethanol and distilled water for surface sterilization on plates containing solid half-strength PDA, and then incubated and checked for fungal outgrowth.The spectroscopic method described by Fadhil and Reza was used to determine total alkaloids in the plant extracts. Briefly, 100 mg of the chive leaf and root materials were extracted separately with 10 mL of aqueous ethanol for 2 h, centrifuged and the supernatant was used in the assay. Two milliliters of the extract supernatant and atropine standard solutions were mixed with 5 mL sodium phosphate buffer and 12 mL bromocresol green solution. Thereafter, 12 mL of chloroform was added to the solution and the solution was mixed vigorously using a vortex mixer. The absorbance at 417 nm was determined and the concentration of mg atropine equivalent per g dry weight in the sample using a standard curve of atropine was calculated. The total polyphenol content of the aqueous ethanol extracts of dried leaf and root dried materials of the chives were determined by the FolinCiocalteu method . The method of Swain and Hills was adapted for the plate reader.

Using a 96-well microplate, 25 μL of the sample was mixed with 125 μL Folin-Ciocalteu reagent and diluted 1:10 with distilled water. After 5 min, 100 μL aqueous Na2CO3 was added to the well. The plates were incubated for 2 h at room temperature before the absorbance was read at 765 nm using a Multiskan plate reader . The standard curve was prepared using 0, 20, 50, 100, 250 and 500 mg/L gallic acid in 10% ethanol and the results were expressed as mg gallic acid equivalents per g dry weight . The flavonol content of the aqueous ethanol extracts of dried leaf and root materials of the chives were determined using quercetin 0, 5, 10, 20, 40, and 80 mg/L in 95% ethanol as standard. In the sample wells, 12.5 μL of the crude aqueous extracts were mixed with 12.5 μL 0.1% HCl in 95% ethanol, and 225 μL 2% HCl and incubated for 30 min at room temperature. The absorbance was read at 360 nm, at a temperature of 25 C . The results were expressed as mg quercetin equivalent per g dry weight .The B. bassiana isolate used in the present study was able to endophytically colonize chive plants. The re-isolation of the fungus from chive leaf samples showed that the fungus was systemic, i.e., inoculum was transferred from the growth medium to the leaves. This is the first record of successful experimental inoculation and colonization of B. bassiana in chives. Previous studies have reported the colonization of B. bassiana in other plant species with different inoculation methods . Inoculation with B. bassiana did not improve the growth of chives over the control treatment . In fact, the control plants had slightly higher biomass and root growth compared to the treated plants. These results differ from those reported in previous studies, which showed that B. bassiana promotes plant growth of cassava , faba bean and cotton . However, Lewis et al. reported no significant difference in the growth of maize plants exposed to seed treatments with B. bassiana and the corresponding control treatment. Jaber and Enkerli reported inconsistent endophyte-induced plant growth promotion across sampling dates following foliar inoculation of faba bean plants with B. bassiana , B. brongniartii and M. brunneum . The root lengths varied significantly among treatments, and the shortest length was observed in plants exposed to the highest concentration of fungal treatment, which may suggest that fungus might have had a negative effect on root length. In a more recent study, B. bassiana inoculation had a positive influence on plant growth parameters including root length of common beans .

Nevertheless, the colonization of plant tissues by fungal endophytes can be influenced by many factors, such as inoculation method, species and fungal strain . Fungal endophytes might affect the nutrient cycle and uptake of nutrients from the soil by plants . In this study, chemical analysis revealed that the leaves and roots of both B. bassiana-exposed and unexposed chive contained polyphenols, nft hydroponic system alkaloids and flavonols, and also that these secondary metabolites were significantly more concentrated in the leaves than in the roots. Chives and other Allium species, such as onion and garlic contain polyphenols, alkaloids, flavonoids, glycosides and organosulfur . Furthermore, higher alkaloid content was detected in the leaves of plants inoculated with B. bassiana than in the control treated plants . In a previous study, which involved the same fungal strain used in this study, drenching potted grapevine plants with its conidial suspension induced higher production of anti-insect volatile compounds including Naphthalene in the fungus-exposed plants compared to the control . The better yield of alkaloids in fungus-treated plants could be due to the synthesis of secondary metabolites by fungus in the plant tissues . Also, endophytes can potentially induce host plants to accumulate secondary metabolites . Zhang et al. reviewed a wide range of bio-active alkaloids that are produced by endophytic fungi. Lozano-Tovar et al. reported that B. bassiana produce secondary metabolites that can induce anti-fungal activity. B. bassiana produces several biological active metabolites of the class of alkaloids such as tennelin, bassianin, pyridovericin, and pyridomacrolidin .

In the current study, since the specific alkaloid compounds were not detected, it is not possible to establish with certainty whether the higher total alkaloid content detected in the fungus-treated plants in this study was due to the direct production of alkaloids by B. bassiana or the fungus physiologically influenced the plant cells to produce more alkaloids. It is worth mentioning that fungal endophytes can produce mycotoxins in their host that are potentially harmful to livestock and humans . In conclusion, this study demonstrated that endophytic entomopathogenic fungi could be used to improve the yield of alkaloids in medicinal plants. In order to further understand the influence of fungal endophytes on plant production of bio-active compounds, future studies involving detailed phytochemical elucidation of the bio-active constituents of fungus-treated plants are warranted.The accumulation of biosynthesised secondary metabolites in plants could enhance the value of plant-based medicinal materials . However, the physiological and morphological processes in plants are affected by ambient environmental factors. Water, for example, is an essential resource for plant growth and survival, and its availability influences plant physiological processes including biosynthesis of secondary metabolites and enzyme activities.Water has many essential roles in plants: it is used for translocation and distribution of nutrients and metabolites, it maintains rigidity of plant organs, it is a medium for chemical reactions, and it is an essential component of the photosynthetic process . Water deficit is the most important limiting factor of plant growth .Water deficits can affect photosynthesis through stomata closure and decreased CO2 diffusion to the chloroplast . Drought stress is also associated with the enhancement of accumulation of many classes of natural products in plants, such as terpenes, phenols, alkaloids and glucosinolates.Leaves of water-stressed Ctenanthe setosa tended to accumulate more carbohydrates of low molecular weight and phenolic acids .

They are smaller than baby greens and they are harvested later than sprouts. Due to their immaturity,they tend to have concentrated flavors, tender textures, vibrant color, and densely-packed nutrients. Various micro-greens, such as broccoli, kale, celery, opal basil,and red beet, are available in the market. Because of their potent  flavors and appealing sensory qualities,in the past few years,micro-greens have gained popularity and are often used by high-end chefs for garnishing soups and sandwiches. Their functional benefits have also drawn attention from nutrition researchers and have opened the door for use in the field of nutrition and health. This functionality is attributed to their high content of vitamins and minerals, as well as other bio-active compounds. It has been reported that many species of micro-greens are more saturated with micro-nutrients than the adult versions of the same plants. For instance, micro-greens have shown to be high in vitamins or their precursors, including carotenoids, ascorbic acid, to copherols and tocotrienols, phylloquinone and folate. Other phytochemicals found to be high in micro-greens include chlorophyll, phenolic compounds, anthocyanins, and glucosinolates. Results from Huang et al. showed that red cabbage micro-greens reduced high-fat diet induced weight gain and significantly lowered circulating LDL levels as well as expression of hepatic inflammatory cytokines in mice fed a high-fat diet.

Polyphenols, a class of phenolic compounds, were indicated to be a major contributor of the aforementioned effects of red cabbage micro-greens, partially owing to their antioxidant and anti-inflammation properties. Phenolic compounds, also seem to have an influence on sensory qualities of micro-greens. Xiao et al. reported that the total phenolic concentration was strongly correlated with the overall eating quality and several aspects of sensory qualities,ebb flow table including intensity of astringency, sourness, bitterness, and sweetness of micro-greens. Nowadays, products of micro-greens are available to consumers in both chain grocery stores and local farms. The growth environments and harvesting methods, however, are quite different. Farms grow micro-greens in soil or hydroponically. On the other hand, commercial micro-greens are usually hydroponically grown, which increases the productivity of micro-greens but may compromise their nutritional and sensory quality. In addition, micro-greens purchased from local farms are usually fresher than those from grocery stores due to shorter transportation time, which may further affect the nutrition and sensory properties of the plants. In spite of the increase of research on micro-greens, no study has evaluated the nutritional and sensory qualities of these specialty vegetables grown differently or from different sources. Such information is important for consumers in purchasing or for health professionals in conducting research or giving dietary advice. Therefore, the main objective of this study was to investigate the nutritional qualities and sensory properties of micro-greens grown and harvested in a commercial setting versus a local farm setting. The nutritional analysis will focus on chlorophyll content, phenolic compounds content, vitamin C content, and antioxidant capacity, which are known to contribute to the health benefits or sensory attributes of micro-greens. Since broccoli micro-greens is the only species available to researchers in both local grocery stores and local farms, it was selected for the comparison in nutritional and sensory qualities.

In addition, six other micro-greens that available in local farms were also analyzed for nutritional quality. We hypothesized that micro-greens grown in soil in a farm setting would possess higher nutritional qualities and produce better sensory properties as compared to those grown hydroponically and those from a commercial source.Commercial broccoli micro-greens samples which were hydroponically grown, were purchased from the Fresh Marketin Tuscaloosa, Alabama. These samples were designated as CH in this research. Farm grown broccoli micro-greens samples were purchased from the Alabama micro-greens . To elucidate the effects of growth environment on the qualities of micro-greens, micro-greens samples both grown in soil and hydroponics were obtained from the Alabama micro-greens. Six other micro-greens, including amaranth, kale, kohlrabi, pea, spicy broccoli, and wasabi, were also purchased from the Alabama micro-greens. The commercial micro-greens were packaged in plastic clamshell containers with unknown harvest date, while the farm samples were harvested on the same day of delivery and packaged in paper clamshell containers. Samples were used for laboratory analysis on the day of purchase and stored at 4 ◦C for the sensory study during following three days. The optical images of the three broccoli micro-greens samples are shown in Fig. 1.The sensory study was conducted on campus at the University of Alabama to evaluate the sensory qualities of micro-greens samples. Data collection was carried out in the student center during weekday lunch time . Participants were recruited by being asked whether they were interested in evaluating micro-greens and completing a survey. After being explained with the purpose of the study and showed the consent form, participants were provided the three samples of broccoli micro-greens in a random order, each in a small cup, for testing. Information about the source and growth conditions of the samples was blind to participants. Participants were asked to observe, smell, and taste each sample and evaluate their liking of smell, appearance, taste, and overall liking in a 7 Likert scale from very poor to excellent .

Participants were asked to drink water between samples. Upon completion of the survey, participants received a $3 Amazon gift card as an incentive. The study procedure and survey questionnaire were reviewed and approved by the Institutional Review Board at the University of Alabama.As shown in Fig. 2A, broccoli micro-greens samples from the local farm had significantly higher TCC than that from the commercial source . The TCC of hydroponically grown samples from the local farm was higher than that of the soil grown samples, but the difference did not reach statistical significance. Chlorophyll is a green pigment found in most plants and is essential for photosynthesis. A higher concentration of chlorophyll might indicate the production of more energy nutrients in the plants. Chlorophyll cannot be synthesized by animal tissues and must be obtained from plant foods. Recently, chlorophyll and chlorophyll-rich diets have been reported to play roles as cancer-preventive agent attributed to the ability of chlorophyll to form complexes with specific carcinogens, as well as its antioxidant and antimutagenic properties. Some other preventive or therapeutic properties of chlorophyll were also reported in literature, such as stimulating immune system, detoxifification of the liver, and normalizing blood pressure. Although more research is needed to elucidate the mechanisms under these functions of chlorophyll, it could be a valuable dietary compound for human nutrition and health. It was reported that mature broccoli contains about 0.02 mg/g of TCC, while the results showed that TCC in farm grown broccoli micro-greens was about 15 times higher. The TCC in those farm grown broccoli micro-greens was also higher than that of many other species of mature vegetables, such as celery, lettuce, and artichoke. The incorporation of these micro-greens in the diet can be a promising way to provide the health-beneficial chlorophyll and warrants more investigation. As compared to the high level of chlorophyll in farm grown broccoli micro-greens, commercial samples had a much lower TCC. Several factors might contribute to this difference.

The commercial samples might have been harvested before the development of the cotyledon leaves, where chlorophyll accumulates. The plant does not require light for the first few days of growth. The chlorophyll might also be degraded due to long supply chain and storage time of the commercial samples, deteriorating the freshness of the vegetable. The TCC could affect sensory quality of the micro-greens, especially their appearance and the impression of freshness.Results in Fig. 2B showed that there was no significant difference in the TPC between commercial and local farm samples. Furthermore, whether it was soil grown or hydroponically grown did not affect the TPC of the samples from the local farm. Phenolic compounds are a large class of plant secondary metabolites that comprise an aromatic ring with one or more hydroxyl substituents. These compounds are important for the quality of plants and are strongly associated with the flavor properties of vegetables, such as taste and color. For instance, phenolic compounds, e.g., tannins and phenolic acids, contribute to the astringency taste, i.e., the drying, puckering, and shrinking sensation in the oral cavity. Some phenolic compounds are colorless, while some others show various colors. Phenolic compounds show numerous bio-active properties, hydroponic grow table most well-known of which are their antioxidant and anti-inflammatory activities. Furthermore, a number of phenolic compounds demonstrate inhibitory effect on the activity of enzymes to digest starch in gastrointestinal tract, e.g., amylase and glucosidase, indicating a potential of these compounds in improving glucose homeostasis and metabolic conditions. The average TPC in broccoli micro-greens samples in this study was in the range from 10.71 to 11.88 mg/g. It is about 10 times higher than the TPC in a lot of species of mature vegetables, including broccoli, brussels sprout, and kailan, which are known to be excellent sources of phenolic compounds. Therefore, these micro-greens can be regarded as great sources of this class of phytochemicals. Future research is needed to analyze the profiles of specific phenolic compounds in micro-greens as well as their in vivo bioavailability, and to explore their beneficial effects in human health. Our results indicated that different growing and harvesting conditions did not affect the TPC of the micro-greens. The TPC of plants depends on the balance between its synthesis and oxidation. Phenolic compounds are produced through the phenylpropanoid metabolic pathway, which starts with the reaction of l-phenylalanine converted to trans-cinnamic acid, and other phenolics are produced via subsequent reactions. As antioxidants, phenolic compounds can be oxidized to quinone under oxidative stress. It was previously reported that light exposure or different packaging methods did not affect the TPC of radish micro-greens. Future research on the effects of growth environment, harvesting condition, and/or post-harvest interventions on the synthetic or oxidation pathway of phenolic compounds in plants will provide insights into the key factors that determine the TPC level of micro-greens.

Results on VCC indicated that broccoli micro-greens samples grown in soil from the local farm possessed significantly higher VCC than hydroponically grown ones from the farm and the commercial ones also grown hydroponically. Vitamin C is a co-factor required by many enzymatic reactions and a critical antioxidant. Previous studies showed that higher circulating vitamin C concentrations are associated with lower risks of hypertension, coronary heart disease, and stroke. Mature raw broccoli, known as an excellent source of vitamin C, contains an average of 0.89 mg/g of total ascorbic acid according to the USDA National Nutrient Database for Standard Reference. Mature spinach, one of the most commonly consumed leaf vegetable in the United States, has about 0.28 mg/g of total ascorbic acid. The VCC of our micro-greens broccoli samples is in the range of 0.33–0.56 mg/g. Worth to note is that the VCC measured in this study is the content of free ascorbic acid so the total ascorbic acid content would be higher.Therefore,micro-greens broccoli, especially the soil-grown ones from the local farm, can be regarded as good sources of vitamin C. Comparing VCC in vegetables grown in soil and hydroponically has created mixed results. For instance, a study by Treftz et al. found significantly higher ascorbic acid content in soil grown raspberries yet lower in soil grown strawberries, compared with their hydroponically grown counterparts. Buchanan et al. reported significantly higher total ascorbic acid content in three of the four hydroponically grown lettuce varieties than their soil grown versions, while the fourth one showed higher but non-significantly total ascorbic acid content. Although not fully understood, vitamin C is synthesized in plants as a response to oxidative stresses and through the l-galactose pathway using mannose or galactose. Therefore, the growth conditions, including nutrition in soil and hydroponic growing media, and environmental stresses may all impact vitamin C biosynthesis in micro-greens and result in the VCC difference in this study.Similar to the results of TPC, no significant difference was observed in the TAC of micro-greens samples from different sources and grown conditions . It has been well established that the consumption of vegetables is inversely associated with morbidity and mortality from chronic diseases and antioxidants play a significant role in the beneficial effects.

The lettuce cultured using the 2-fold ozonetreated sludge dilution solution showed significantly higher contents of chlorophyll, soluble sugar and Vc. The 2-fold ozone-treated sludge dilution solution improved the quality of hydroponic lettuce compared to the modified Hoagland nutrient solution used traditionally. It was also observed that unsuitable concentrations of ozone-treated domestic sludge dilution solution could inhibit lettuce growth. High sludge concentrations inhibited lettuce growth and at low sludge concentrations the nutrition provided was insufficient to satisfy the lettuce growth demand . The leaf number, plant height, fresh weight and dry weight in the lettuce cultured by the modified Hoagland nutrient solution were significantly higher than those cultured by any of the ozone-treated sludge dilution solutions. While the nitrate content in the lettuces cultured using the 1-, 2-, 3-, and 4-fold ozone-treated sludge dilution solutions were 34.3, 53.93, 55.34, and 68.60% lower than that in the modified Hoagland nutrient solution treatment, respectively, although the chlorophyll, soluble sugar and Vc contents increased. The various physiological indexes of lettuce were not independent and there was a certain relationship between them . As shown in Table 3, nitrate content had a significant or extremely significant correlation with leaf number, plant height and dry weight, and the correlation coefficient were 0.99, 0.86, 0.87, respectively. The result indicated that the nitrate content would increase with the leaf number, plant height and dry weight increasing. Thus, the leaf number, plant height and dry weight could be appraisal indexes of nitrate content. So, the nitrate content could be controlled within the scope of the security by the leaf number, plant height and dry weight.

And dry weight had a significant or extremely significant correlation with leaf number, plant height and root length, with the correlation coefficient of 0.89, 1.00, and 0.90, respectively. This was consistent with others reports . But the correlation for chlorophyll or soluble sugar content with other physiological indexes was not significant. Additinally, Vc had a significant correlation with leaf number. Some researches showed that the significant correlation among physiological indexes and quantitative trait loci  of corresponding physiological indexes may exist chain effect . Therefore, vertical farming racks the correlation among physiological indexes could be understood further by the correlation analysis among physiological indexes. The results could provide basic theory for genetic analysis of lettuce stress resistance in the future. The ozone-treated domestic sludge dilution solutions investigated in this study were thought to be an efficient liquid fertilizer substitute. Here, improvements in terms of physiological, biochemical and nutritional characteristics and safety in the lettuce growth process were observed. Before the sludge was treated by ozone, the number of fecal E. coli bacteria was measured. The results showed that the number of the feces in the sludge before ozone treating ismore than 1×105 L–1, which was measured with the most probable number  method, while the number of the feces in the modified Hoagland nutrient solution is zero. And the fecal E. coli was not detected in the 1-, 2-, 3-, and 4-fold ozone-treated sludge dilution solutions after ozone treated for 120 min, so ozone treatment showed strong bactericidal ability, which was beneficial to the effective control of pathogenic microorganisms. But heavy metal distribution in the sludge solution and lettuce cultivated with sludge must be studied in subsequent research. And further studies will also be performed on food safety.

Using ozone treated sludge dilution solution as fertilizer in hydroponic systems has the potential to outperform traditional fertilizers and to cut costs for growers, however, further research is necessary.It is evident from this study that increased accumulation of bio-active compounds, antioxidant properties and mineral composition can be achieved without compromising the yield by selecting less N sensitive varieties that can use N effectively. N application generally improved leaf area and attributed increased chlorophyll content due to the effective use of photosynthetically active radiation . However, the morphological response of the green lettuce varieties to different N supply rates differed from the reports of Mahlangu et al.  where, Lollo Bionda Lobi leafy lettuce grown in non-circulating hydroponic system  failed to show any increase in fresh weight although the form of N source was NH4 NO3 . In this study, fresh weight in the green varieties showed strong correlations with increasing N supply . However, discrepancies in results could be due to the type of growing system used or it can also be due to the genotype. In red lettuce varieties, photosynthetically produced carbon molecules are used more for the production of phenolic compounds than for the use of plant growth and fresh weight . This relates to the C/N shift theory that shows a shift to carbon based metabolites under N deficiency for the biosynthesis of phenolic acids and flavonoids . The reports of Becker et al.  further confirmed that the activity of phenylalanine ammonia-lyase that is responsible for the biosynthesis of polyphenol propanoids are higher at lower N concentrations, which further explains the observed increased phenolic acids  and flavonoids  and lower fresh leaf weight in var. Multired 4 in this study. Slight divergence in the trends were observed with the biosynthesis of phenolic acids in green varieties  with respect to N application rates in this study and the reports of Becker et al. . The lettuce genotypic differences could have attributed to these observed differences.

Increasing N application rates were shown to reduce phenolic acids and flavonoids in butter head lettuce cv. Egery . However in this study the response to increasing N supply with regards to the accumulation of different phenolic acids and flavonoids composition differed . N supply affects leaf chlorophyll content  and in this study N application rates between 90 to 120 mg L–1 were sufficient to maintain the chlorophyll content to carry out photosynthesis effectively in both green cultivars . This observation coincides with the findings of Mahlangu et al.  on the green lettuce cultivar Lollo Bionda Lobi grown in a non-circulating hydroponics system where 100 to 120 mg N L–1 was reported to be sufficient to maintain photosynthesis. However in cultivar Lollo Bionda Lobi, the relationship model between the applied N rates and the chlorophyll content fitted linearly  whereas in Mulitigreen 1 and Multigreen 3, they showed a polynomial model . The observed discrepancies in chlorophyll content could be due to the lettuce genotypic differences. Increased rates of N applications have been reported to reduce the ascorbic acid content in different fruit and vegetable crops . On the contrary, Chiesa et al.  reported that high N  increased ascorbic acid content in lettuce. Variation in accumulation of ascorbic acid content with respect to different rates of N applications varied with the findings of Mahlangu et al.  where the ascorbic acid concentration was higher at 100 to 120 mg N L–1. In this study, the highest ascorbic acid content was obtained at 60 mg L–1 in both green and red varieties  and as shown by Mahlangu et al.  it declined with increasing rates of N application. The observed divergences with regards to the N application rates and ascorbic acid content in lettuce can be explained due to the type of N source and application methods. Type of fertilizers such as ammonium nitrate  or urea  was shown to increase the ascorbic acid content compared with ammonium sulphate . Abd El-Rehem and Abd El-Baky  demonstrated that the use of ammonium nitrate reduced the ascorbic acid content with higher application rates. IM and FNB  recommended daily intake  for an adult male and female is 90 and 75 mg of ascorbic acid. It is evident in this study that 100 g of Multired 4 provides 17 mg of ascorbic acid at 60 mg N L–1 and similarly 100 g of Multigreen 1 and Multgreen 3 contained 14 and 15.5 mg of ascorbic acid, respectively.

Phenolic acids, vertical rack system flavonoids and ascorbic acid are known as antioxidants and play a major role in human nutrition. Stefanelli et al.  reported that the changes in total phenol content and the antioxidant power  revealed similar trend with increased rates of N applications and that the highest FRAP activity and total phenolic content was attained at 400 mg N L–1. Heimler et al.  established a positive correlation between total phenolic content and antioxidant capacity in 5 out of 10 lettuce cultivars. The trend in antioxidant activity in this study differed from the findings of Mahlangu et al. , which showed higher activities at 120 mg L–1. In this study in all three cultivars the antioxidant power  and capacity  were higher at lower N concentrations  and decreased with higher rates of N application.Differences in nitrate accumulation between the green lettuce and red lettuce varieties were noted in this study and a similar observation was reported by Becker et al. . There is discrepancy between the findings of this study and the reports of Becker et al.  with regard to increasing nitrate content in the leaves with increasing rate of N application. Becker et al.  stated that the nitrate residue did not increase steadily with increasing N concentration in the nutrient solution. Permitted nitrate content for greenhouse lettuce is set at 4 500 mg kg−1 of fresh weight when grown from 1 October to 31 March and at 3 000 mg kg−1 from 1 April to 30 September by the European Community . Although the nitrate levels in the leaves were calculated on a dry weight basis in this study, the contents were much lower than the levels set by the European Community. It can be recommended to use less N sensitive varieties to promote the production of phytochemicals and to reduce accumulation of nitrates in a controlled production system.

Reports of Stefanelli et al.  stated that Ca increased while B, Mg, Mn, and Zn significantly decreased with increasing N application rates. In this study N application rates helped to improve the mineral composition  in the leaves and different accumulation trends were noted with respect to the different cultivars with the red cultivar responding well. Petropoulos et al.  showed the influence of N application on Mn and Zn in lettuce leaves . However, in this study, N application influenced the accumulation of Mn in Multigreen 3 . An increase in minerals especially Fe, and Mn that are an important source of micro elements in lettuce will add value by enhancing the nutritional value in the food chain. Rice is the foremost staple food in Asia, feeds more than 60% of the population in China and constitutes up to 40% of the total national grain production . At present, there is an urgent need to shift rice production toward mechanized farming . Mechanical transplantation is an emerging rice planting method in China and represents an important opportunity for scaled, simplified, commercialized, and modernized development . Mechanical transplantation has several advantages, such as high and stable yields and high efficiency , and has developed rapidly in China in recent years; the use of mechanical transplantation will guide the future direction of national agricultural production to solve the problems concerning severe shortages in the rural labor force and to achieve full mechanization of rice cultivation. However, traditional rice seedling cultivation methods for mechanical transplantation are time consuming and laborious, and collecting topsoil for the boxes can disrupt soil structure and subsequently reduce crop yields. All of these factors limit the development of mechanical transplantation of rice in China. The use of hydroponically grown long-mat rice seedlings  represents a novel seedling cultivation method that reduces labor costs and improves efficiency . This method presents two main features: first, non-woven cloth is used as the medium instead of soil, and the seedlings are cultivated hydroponically; second, nursery seedling boxes that are 3 to 6 m in length are used rather than traditional small rectangular boxes . The grain yield and economic benefit of HLMS were equivalent or superior to those of traditional nutritive soil methods because of the robust seedling quality, better transplantation quality and higher dry matter accumulation during late growth of the former ; in addition, the root entwining force of HLMS is enhanced, resulting in significantly increased flexibility of seedling age and enabling high-yielding and -efficiency rice production .

For blended DAP and KNO3 , both reverse fluxes of potassium and nitrate were similar regardless of the FS tested, but smaller than with KNO3 alone. Therefore, it is hypothesized that the Js of K+ and NO3− are more dependent on DS than FS composition. However, it is worth mentioning that when using blended salts, nitrate reverse fluxes were even lower with Na+ in FS than when using only DI water  due to lower ion exchange. Thus, the presence more than the type of salt in the FS influenced the Js of nitrate ions. The obtained results show the complexity of ion interactions because the tested DS ions  behaved differently and were influenced, to different degrees, by both the presence of salts in FS and the DS composition. Forward fluxes  were minimal for all ions except for Na+ in tests with DAP and blended salts in DS, with Jsf ranging from 2.1 to 2.9 mmol m− 2 .h− 1 . As indicated by Hancock and Cath , feed solutes with larger hydrated radii , had better FO membrane rejection than monovalent ions . High sodium fluxes  are commonly reported , and in this study its presence influenced ammonium reverse fluxes. This is because Na + has higher diffusivity than Mg2+ and therefore its transport to the DS facilitates the ammonium transport to FS and vice versa . The percentage of mass of ion passage from FS to DS  showed higher Na+ passages with the pair of monovalent ions , because of the retarded sodium diffusion due to the divalent SO42− presence in the case of Na2SO4 in the FS . Similarly, lower anion  passages were experienced in presence of the divalent Mg2+ . These results point out the importance of FS composition for the performance of the FO process. Concerning draw solutes, divalent ions from FS showed lower forward fluxes, and thus the final FS and DS were less contaminated with ions from the opposite solution when divalent ions were present on both sides of the membrane. Fig. 6 indicates the distribution of solutes in FS and DS at the end of the tests.

A high percentage of draw solutes passed to the feed side, resulting in almost 100% fertilizer losses to FS in some cases . Such observations are of utmost importance as they jeopardize the interest of the FDFO concept. This reinforces the fact that KNO3 cannot be used alone as DS for fertigation. Comparatively,hydroponic bucket using DAP or blended fertilizers reduced the losses, which however were still very high for ammonium, nitrate and potassium in most cases as soon as salts were present in the feed solution. Ideally, osmotic equilibrium should be achieved by an equal EC between original FS and DS solutes without nutrient losses, and not because of large reverse flux of DS solutes to FS, limiting the DS dilution. However, the obtained Js and nutrient losses were very high, osmotic equilibrium was reached, and water fluxes decreased as a consequence of the salinity buildup in FS caused by the reverse fluxes of DS ions. Existing studies are controversial since some of them point out that the solute fluxes from DS to FS are not influenced by the presence of salts in FS , while others indicate the opposite . In this study, while the presence of salts in FS did not influence PO43− behavior, it did clearly influence both Js and losses of NH4+. Although the influence of salts in FS was not clear in terms of reverse fluxes of K+ and NO3− , Fig. 6 shows that it strongly influenced the passage of K+ and NO3− ions to the FS. Adequate nitrogen and potassium dilution for direct hydroponics application was achieved in some tests with magnesium ions in FS . For the rest of the cases, due to the high reverse fluxes, nitrogen and potassium concentrations were below the target ranges. In contrast, phosphate reverse fluxes, as well as mass dilution were minimal, and phosphate concentrations in the final DS were well above desired concentrations and toxicity levels. Therefore, the presence of salts in FS plays a fundamental role in the final concentrations of NPK in DS. Additionally, one of the main problems of sodium diffusion in FDFO is its final concentration in the DS, since the DS is intended to be used as a nutrient solution for direct application in hydroponics. Sodium concentrations over 50 mg.L− 1 are toxic for the plants .

Average sodium concentrations in the final DS of tests with NaCl and Na2SO4 in FS were above the level of toxicity . These results indicate that Na+ forward fluxes may compromise the quality of the final DS and the general efficiency of the FDFO process. Nanotechnology is now considered one of the most promising applied disciplines and involves the manipulations of materials at the atomic level and the manufacture and assembly of metal, semimetal, and non-metal nanoparticles and their various industrial and agricultural applications . However, all NPs manufactured are ultimately released into the environment and eventually reach cultivable soils. Accordingly, NPs are considered an emerging class of contaminants . ZnO-NPs and CuO-NPs are among the most studied nano metal-oxides in terms of their environmental impacts, but their effects on cereal crops like maize have not been well researched. For example, Cu-based pesticides account for a sizeable proportion of the global demand  , and research interest had shifted towards the use of CuO-NPs rather than Cu bulk or salt-based pesticides , due to their nano-specific properties such as higher S/V ratios, surface energies, and bioreactivities. CuO-NPs are also used as catalysts and in sensors, batteries, solar energy converters, textiles, optics, and microelectronics . Similarly, ZnO-NPs have potential applications in the manufacture of solar cells, sensors, piezoelectric devices, cosmetics, and medical devices . Given the potential usage of CuO-NPs, it is inevitable that as a result of discharge, soil and water become major nanoparticle sinks. Therefore, NPs pose potential issues concerning the metabolic activities, structures, and functions of plants and for human health due to the consumption of edible plants and the contamination of water . Plants have large surface areas and are potential ecological receptors for NPs, and thus, NPs pose the risk of adversely affecting crop production .

Recent studies have shown that CuO and ZnO NPs are accumulated by some terrestrial plants at high levels and cause phytotoxic effects, which may be due to the uptakes of Cu2+ or CuO-NPs. Furthermore, the addition of maize root exudates during the co-cultivation of maize seedlings in the presence of 25 mg L− 1 of CuO NPs for 7 days increased CuO-NP accumulation and hampered seedling growth . On the other hand, ZnO-NPs at 100 mg L− 1 alleviated Zn deficiency in maize seedlings after adsorption on seed endosperm and internalization . The majority of studies on maize/ZnO-NP interactions have been performed on agar media up to the seedling stage, and in one such study, ZnO-NPs were found to reduce seedling root growth by 51% at 1000 mg L− 1 . Nonetheless, we considered that further comparative studies were required to investigate in more detail the response of maize plants to different concentrations of ZnO-NPs and CuO-NPs in different growth media. Metal-oxide based NPs penetrate plant cells by endocytosis or other passive/facilitated transport routes and are then either accumulated at locations near entry points or are transported to other plant organs, and their accumulations and interactions with cellular components damage plant anatomies and physiologies . Furthermore, it has been reported the uptakes, translocations, and bioaccumulations of NPs in plants depend on physicochemical features, genotypes, and plant anatomies . On the other hand, free metal ions such as Cu2+ and Zn2+ are known to adversely impact crop plants, and due to their solubilities in water and deposition in soils to pose an increasing environmental challenge.Maize was selected for the present study due to its economic importance. Maize is an edible flowering plant of the family Poaceae and is the third most-produced cereal crop after rice and wheat .

It is considered a human staple food and animal feed in most developing countries and is rich in proteins, carbohydrates, essential minerals, fat, iron, and vitamins. Furthermore, maize has also been reported to interact with some metal-based NPs such as TiO2-  and Fe3O4-NPs . Little is known about the comparative impacts of nano and bulk ZnO or CuO or Zn2+ and Cu2+ ions on maize cultivation in different media, in which the effects of nano, bulk, and ionic species may be differentially influenced by their different mobilities and interactions with maize plants. Although the use of soil-less media is desirable for the toxicity assessment of NPs on crop plants, stackable planters soil-based approaches are more relevant because soil effects can alter material activities. Thus, the uncontrolled disposal and improper recycling of CuO-NPs and ZnO-NPs in the environment is likely to increase plant exposure, which in turn is likely to affect their accumulation kinetics and other ecosystem trophic levels. Therefore, the objective of the study was to assess the phytotoxic impacts of nano and bulk ZnO and CuO and Zn2+ and Cu2+ on maize using plate assay, hydroponic conditions and soil environment. The depths of ZnO- and CuO-NP penetrations were detected and visualized by SEM, EDX, and elemental mapping analyses, and NPs aggregates and ultrastructures in maize root and leaf cells were examined by TEM. Accumulations of Zn and Cu from NPs, bulk materials, and ions in maize roots and metal translocations to upper ground parts were investigated by ICP-MS. The presence of chemical functional groups and possible alterations were observed by FT-IR of lipids, proteins, and carbohydrates in roots and shoots. Root cells were also analyzed by flow cytometric analysis to detect the apoptotic growth phase . In addition, we assessed the impacts of NPs, bulk materials, and ions on phosphorus accumulation, soluble protein and chlorophyll production, and on the inductions of proline, oxidative radicals, and antioxidant enzymes. Few reports have been issued on the adverse impacts of metal-oxide NPs on the growths and biochemical attributes of maize plants , but few are available on the toxic effects of ZnO-NPs and CuO-NPs on physiological and morphological parameters of maize grown in different media or compared these effects with those of bulk ZnO or CuO or Zn2+ or Cu2+. Similar to the current study, ZnO-NPs at 400 mg l− 1 reduced maize seed germination maximally by 53%, but Zn2+ had no negative impact .

In an earlier study, CuO-NPs at 100 mg L− 1 significantly inhibited root elongation of maize seedlings but CuO-bulk did not . Because of their increased uptakes and consequent accumulations, soil adsorbed ZnO- and CuO-NPs interact with many cellular components, and thus, interfere with the metabolic activities of maize and cause cellular damage that might reduce crop production. To check the adsorption of NPs to soil constituents, SEM-EDX scanning of soil before and after adding CuO-NPs  showed the presence of a prominent Cu signal in the EDX spectrum that was not detected in non-treated control soil . Reduced water flow and limited root hydraulic conductivity caused by the physical adsorption of NPs of ZnO or CuO into the nano-sized pores of maize roots restrict growth , and this reduction in water availability inhibits maize leaf growth and transpiration by physically blocking apoplastic flow. Frequency of occurrence of smaller sized particle aggregates after addition of ZnO and CuO NPs  in soil was higher than that of non-treated soil  which suggests the availability of added NPs and their role in physical blockage of maize root pores . Furthermore, resulting reductions in the supplies of water/nutrients and the higher intracellular concentrations of Zn and Cu could disturb root and shoot transcriptomes and suppress the expressions of genes that participate in primary, cellular, and secondary metabolic processes . At lower concentrations, Zn and Cu species improved the biological parameters . This could be due the fact that Zn when present at below threshold levels in plants, acts as a co-factor for many essential enzymes of plants.