In greenhouses, CO2 gains are proportional to the losses in classrooms, though with lower concentrations because of the larger greenhouse area. Fig. 9 displays the hourly effects on CO2 levels and air temperature due to airflow integration, and Table 8 depicts the maximum and average CO2 concentrations. Average CO2 levels decrease only 133.35 ppm in classrooms and increase 74.89 ppm in greenhouses. Air exhaustion lowers peak levels in classrooms by 1943.6 ppm and 817.3 ppm for schools A and B, respectively. The maximum gains in greenhouses are 1001 ppm in archetype A and 366 ppm gains in archetype B. This difference between schools is due to classrooms and greenhouse sizes.Fig. 10 shows the theoretical thermal load and EUI for both archetype schools and assessed RFs. Heating is provided by centralized gas boilers with a coefficient of performance  of 0.92 and cooling by electrical splits with a 2.6 COP. For comparison purposes, conventional insulated roofs were included. Only considering the thermal load, RTGs are the most efficient solution achieving an average 48.2% reduction, followed by iRTGs  and lastly, eGRs. Authors do not expect optimized eGRs could surpass RTGs performance because soil thickness only affects energy load by 0.02% for depths between 5 cm and 60 cm, and water content only 13.2% for saturations between 20 and 100%. A similar trend was shown for eGRs and soil RTGs in Italy, where the latter outperform other roofing systems for non-cooling scenarios. However, that study showed that insulated roofs have a better annual performance because, even thoug RTGs and eGRs reduce energy demand by 50% in summer,nft growing system in winter their performance is lacking.

However, in Quito, vegetated roofs outperform insulation due to their active heat sink effect through which plants process energy according to their immediate needs. Similar results were obtained for moderate climates in North America, comparable to Quito’s climate.For Quito schools, RTGs raise the operative temperature in classrooms by 0.8 ◦C for 112 schools and by 1.25 ◦C for 102 schools. This temperature increment represents an annual thermal load decrement of 45% or 7872.67 MWh. At the city scale, iRTGs show only 5% lower thermal performance than RTGs but improve air quality in classrooms by decreasing in half the number of occupancy hours when the concentration of CO2 exceeds recommended levels. iRTGs operational electricity demand is 1813.85 MWh for the hydroponic cultivation system and 399.81 MWh for mechanical ventilation. This demand represents an increment of 15.6% and 3.5% from the current educational stock electricity demand. While the thermal load improvement far surpasses the electricity demand for RTGs and iRTGs, their operation is still a liability due to increased economic expenditure. The literature suggests that RTGs have better environmental performance than conventional greenhouses because negative impacts are amortized between the host building and the farm. However, for retrofitting free-running buildings, iRTGs environmental impacts need to be considered independently. A previous study on Quito’s schools showed that eGRs were preferable due to their social acceptance, water storage and runoff reduction capacity, and compliance with local urban and construction codes. Additionally, eGRs installation and maintenance costs are lower than RTGs compliant with building construction codes. In RTGs, the LDPE envelope requires replacement every 3–4 years and the hydroponic system every ten years, thus increasing its life-cycle costs. With a focus on harvesting, eGRs require tight control for irrigation, fertilization, waste management, pest control, wind exposure and weeding to keep a productive state. However, the operation of hydroponic farms has a higher environmental impact because of the chemical fertilizers and artificial substrates. Consequently, protected GRs or soil cultivation in RTGs may provide the best compromise between thermal comfort improvement and electricity demand.The proposed co-simulation is a step forward in the energy modelling for BIA as it inputs crops’ energy balance into host buildings’ energy simulation. This strategy takes advantage of the capabilities of two software tools: complete building energy modelling in E+ and complex mathematical functions in MATLAB for crop’s modelling.

Table 9 summarizes the pros and cons of the proposed co-simulation. Its main advantages are: 1) using indoor climate as the boundary condition for the crop’s sub-models which enhances model performance by up to 20%; 2) including radiant and advective terms for plant’s energy balance which is of particular importance for naturally ventilated greenhouses; and, 3) implementing a dynamic LAI to model transpiration which allows a more accurate simulation of crop’s heat balance throughout its growth stages. The E+ EcoRoof module used for eGRs requires fixing crop and soil parameters. The workaround proposed in this article allowed researchers to include a dynamic LAI and plant height – through a concatenation of multiple simulations runs – but soil’s thermal characteristics could not be altered. Moisture content can vary substrate’s specific heat by 1.5 times and double its thermal conductivity from dry to saturated conditions. Although EcoRoof includes water balance models, substrate properties are assumed constant and can produce errors of 2 ◦C in surface temperature. The conduction transfer algorithm in the E+ EcoRoof model has numerical limits for some plant parameters and, therefore, extreme values cannot be set, e.g. LAI between 0.001 and 5.0. Such is the case for pre-harvest lettuce that surpasses LAI’s upper limit, therefore under calculating the crop’s energy balance in pre-harvest. However, as extreme high LAIs are only proper for enclosed crops, other plants should not be limited by this. The EcoRoof model is based on the Army Corps of Engineers’ FASST atmospheric vegetation model but adapted to be used with a thin soil layer. The Stanghellini model is an adaptation of PM model for use in protected environment farms. Both models require crop-specific parameters and resistances that are difficult to quantify, which is why many studies on greenhouse climate management use simplified PM models that rely on weighting coefficients. A key point of this co-simulation is the capability to simulate different evapotranspiration models according to data availability and environmental conditions. On this matter, Penman-Monteith and a simplified PM model were modelled in MATLAB alongside the Stanghellini model described previously. Fig. G.1 in Appendix G depicts the crops’ heat fluxes and greenhouse indoor air temperature using these three transpiration models. For LAIs below 4, the PM model predicts higher latent heat fluxes. As LAI increases, PM and Stanghellini latent heat flux estimations become similar. The simplified PM model always underestimates heat flux but to a lesser degree during the first growth stages. The higher temperature estimates using both PM models suggest more substantial heat gains for RTG and iRTG scenarios, beneficial for indoor comfort in classrooms but disadvantageous for lettuce crops due to overheating risk – 24 ◦C maximum temperature. The Stanghellini model consistently predicts a higher sensible heat loss from the air to the crop, generating a temperature drop during sunlight hours. These higher values are due to the direct use of LAI in the Stanghellini model, whereas PM uses effective LAI. Vegetation cover fraction accounts for the effective agriculture area and should be further considered in transpiration models. This parameter is usually proportional to LAI, but for closed crops such as lettuce, both parameters are non-linear. For lettuce, the cover fraction increases rapidly during the first growth stages and then remains constant despite leaves densify.

Therefore, LAI should be corrected to reflect better the transpiring surface.Arsenic  is a potentially hazardous element that can pose serious risks to living organisms and the environment. It is a non-essential element for plants and often produces strong phytotoxicity,which can be caused both directly, through phosphate replacement by arsenate in some biochemical reactions and arsenite binding to sulfhydryl groups in proteins,and indirectly, through the formation of free radicals and other reactive oxygen species  during As detoxification. As a consequence, plants subjected to As stress due to exposure to As-rich waters or contaminated soils undergo oxidative damage. Plant cell disruption due to As toxicity may arise through membrane integrity alteration, inhibition of photosynthesis and respiration, and metabolic disturbance. The remediation of soils and waters contaminated with As has become a major environmental issue. A suitable option for environmental restoration, in many cases, is phytoremediation, a cost-effective technique based on the use of plants for the treatment of a wide range of pollutants in the environment. In particular, the potential for phytoremediation of wetland plants has attracted increasing attention, as some of them can grow naturally in contaminated areas and in constructed wetlands. Common reed  Trin. ex Steud, a macrophyte species with a very wide geographical distribution, possesses several characteristics of interest regarding the accumulation and sequestration of pollutants. High biomass production, a well-developed roots and rhizomes system, and high tolerance to potentially toxic elements  confer on common reed its suitability for the remediation of aquatic and soil environments contaminated by PTEs. However, high concentrations of PTEs in this species may still lead to oxidative stress, including DNA damage and misfolding and loss of functionality of key enzymes and proteins. In this sense, further understanding of how common reed responds at the physiological level to PTEs toxicity may be crucial regarding its possible use in future phytoremediation actions. Although the efficiency of common reed with regard to PTEs phytoremediation is widely reported in the literature, its detoxification of As has received less attention and needs to be studied further. The use of common reed rhizofiltration represents an interesting choice for As removal from contaminated groundwater. In addition, new approaches like the formation of an iron plaque in the roots to increase As retention  or the use of common reed biomass for the generation of bioenergy  make this species of particular interest regarding phytostabilization. However, nft hydroponic system the roles of the different As species in the tolerance and/or accumulation of this element by common reed remain largely unknown. The effects of As in plants have been studied previously, as it is known to be the dominant species in waterlogged and organic matter rich soils. Contrastingly, very little information can be found regarding.

As uptake and toxicity in common reed plants, even though As has been found to be the only species present in pore water  extracted from flooded and unflooded strongly oxidized contaminated mine soils  and in water extracts of compost and biochar amended mine soils. We hypothesized that the response of common reed plants to As may differ along a gradient of toxicity, and that this would be reflected in their physiological and oxidative status and in the transformation of As and its compartmentalization as different As species. Therefore, the aim of this study was to elucidate novel aspects of common reed As tolerance by exposing plants to distinct As concentrations in an original hydroponic experiment. The effects of As accumulation on the plants were evaluated through nutritional and oxidative stress parameters, which were finally related to the concentrations of the major As species in the different plant compartments.The presence of As in the nutrient solution did not cause any evident damage in the common reed plants, as no significant differences were found for the growth and yield parameters between the control and the different As treatments. Nevertheless, the roots showed a certain sensitivity to the As mass concentration in the nutrient solution, with the lowest increase in length occurring at the highest treatment dose. The photosynthetic pigments analyzed  did not show significant differences among the different treatments  and external symptoms of toxicity in the plants were not observed. The Chl-a mass fractions determined were within the range found for this species in previous studies. The ratio Chl a/b, used as an indicator of contaminant-induced plant stress,ranged from 2.7 to 3.1 in our plants  and was very close to the ratios previously reported for common reed plants exposed to Cu and Cd contamination. Detoxifying mechanisms might have efficiently mitigated the adverse effects of As exposure, since the plant growth and photosynthetic rate, two of the most common indicators of PTEs toxicity in plants, were barely affected. Plant nutrition can be altered by PTEs toxicity, in turn affecting transport processes of the cellular membrane and transpiration. The fact that phosphate and arsenate ions have the same charge and chemical structure is responsible for most of the toxicity due to As, based on its capacity to disrupt cell functions. In the present experiment, the levels of P in the roots slightly increased when common reed plants were exposed to 0.5 mg As L− 1,but then decreased significantly as the As mass concentration in the nutrient solution increased. These results point to the competition of arsenate for phosphate transporters in plant roots. Contrastingly, the levels of P in the aerial part of the plants were very similar in all the treatments, indicating that As did not compete with P for carriers involved in translocation.

The results indicated that Ag+ has rapid and highly toxic effects on cowpea roots at concentrations similar to those which are toxic to freshwater biota. Similarly, high phytotoxicity effects of Ni2+  and Cd2+  have also been reported by Kopittke et al.  and Li et al.,respectively. The highly toxic effects of these ions may be due to their accumulation in the outer cortex and the meristem of roots. They usually compete for biologically active sites with toxic ions, thereby reducing the toxicity of the latter. However, they can also inhibit plant growth at high concentrations. Kopittke et al.  studied the toxic effects of 26 metals on cowpea root and reported a 50% inhibition in root elongation at approximately 14,000 μM for Mg2+, indicating that the toxicity of Mg was far lower than that of toxic ions. Based on the QICAR method, separation of ions on the basis of their classification according to HSAB theory improved the linear regression predictive effect for root toxicity. log {EC50} of soft ions was significantly correlated with eight physicochemical properties. Z, which had the greatest correlation coefficient with log {EC50} for soft ions, was also the most effective variable for establishing predictive equation. Z is one of the auxiliary criteria by which soft and hard ions were distinguished. Generally, the toxicity of soft ions is attributed to soft–soft interactions,and soft receptors are usually characterized by low charge or large radius. Thus, the ion charge is of overriding importance for the toxicity of soft ions. Hard ions exhibited significant correlations between log {EC50} and seven physicochemical properties. Similar to the results from soft ions, the single-variable linear relationship based on Z of hard ions showed the beat prediction of toxicity effects.

In general, the establishment of a good toxicity predictive model requires a large volume of data, grow table which are lacking for many metals. Modeling based on the soft–hard ions classification required more toxicity data for metal elements, which caused difficulties in achieving modeling. In addition, the types and number of metal elements decreased after soft–hard ion grouping. Although R2 was improved, this effect may be the result of spurious correlation due to a reduction in sample number. As a consequence, the classification of soft–hard ions, using QICAR, needs to be verified over a larger number of elements. Alternatively, we explored the relationship between metal toxicity and the physicochemical parameters σCon, HLScale and log K, based on the QICAR method. The parameters showed different predictive effects for metal phytotoxicity. The correlation between toxicity and σCon values was not significant when considering all metal ions,but was improved after excluding La3+ and Mg2+. This indicated that σCon may not explain the toxicity of all ions. Kinraide  investigated the relationship between the σCon values and toxicity of low-valence ions  and found that the combination of charge and σCon could predict toxicity well. However, this relationship was limited to only low-valence metal elements. In the current study, we found that the toxicity predictive effect based on the HLScale method was even worse  than that based on σCon. On the other hand, Kopittke et al.  investigated the relationship between HLScale and rhizotoxicity of 26 metal ions to cowpea, and found that the closeness of fit was good. There are several possible reasons for these disparate results between the two studies. Firstly, roots from different plant species may contain different ligands. The cowpea  used by Kopittke et al.  may contain more of the same ligands that Kinraide  used to calculate HLScale. Thus, the HLScale can predict the toxicity of metals to cowpea roots better than to the roots of the wheat  we used. Secondly, there are limited data on metal species and toxicity involved in the HLScale method.

The HLScale method we used only involves eight metal elements, including two subsequently excluded outliers. This small number of elements has a strong influence on the regression relationship between HLScale and metal toxicity. Thirdly, there are different phytotoxicity mechanisms for different metal elements. Previous studies have pointed out that binding of metal ions to hard ligands is an important, non-specific mechanism that causes toxicity directly by inhibiting the controlled relaxation of the cell wall required for cell elongation. However, this may not apply to all ions. In other words, toxicity may not be associated with the binding strength to the hard ligands for all ions. For example, some soft ions, such as Ag+, may strongly bind to the R-S-functional group  in metallothionein, and in this process, the metal complex is bound to glutathione and sent to the vacuole, thereby exerting toxicity effect. Analogously, Kopittke et al.  investigated the toxicity mechanism of As3+ in cowpea roots and found that the phytotoxicity of As3+ is due to its reaction with dithiol groups on proteins and the inhibition of enzyme reactions that require free sulfhydryl groups. The theory of different phytotoxicity mechanisms for different metal elements is also supported in the current study by the SEM images,whereby the soft metal ions, Cd2+ ) and Ni2+ ), seriously damaged the roots and were more toxic than Cr6+ or Mg2+, indicating that they bound strongly to the soft ligands. On the contrary, Cr6+ ) and Mg2+ ) were less toxic and bound only weakly to the hard ligands, indicating that the binding strength of these metal ions to different ligands varied. Our results confirmed that the K value, based on BLM theory, can predict the phytotoxicity caused by metal elements well. The BLM considers all ligands together, calculating parameters based on toxicity. This parameter  is obtained for a specific plant species tested, so it has more advantages in predicting phytotoxicity. However, the available K values are limited as the BLM involves only a few metal elements. In addition, although some elements, especially those which are essential for plant growth, such as Mg and Ca, have K values, these need to be excluded in the establishment of QICAR due to their low toxicity or non-toxic nature. Further study on the toxicity of different elements toward different organisms is required. A common ligand parameter suitable for all organisms by averaging and normalizing their corresponding K values may further improve the application of this parameter in QICAR.School buildings require high indoor comfort levels because children are sensitive to their environment.

A study in primary schools in the UK found that classroom environment affected academic performance by 25%. Similarly, Chatzidiakou et al. determined that, for temperatures between 20 and 25 ◦C, a reduction of 1 ◦C resulted in a 2–4% increment in learning performance. The majority of the European and South American school buildings stocks were built between the 1960s and 1980s; therefore, it is unsurprising that non-refurbished buildings’ indoor environmental quality  does not comply with current comfort standards and indoor air pollution levels. The educational stock’s high energy use intensity  relates to infrastructure deficiency, only worsened by age, intensive use and lack of maintenance. Many energy efficiency retrofit projects exist for educational stocks, especially in Europe, where combinations of envelope improvements and efficient active equipment achieved near-zero energy goals. However, extrapolating these strategies to free-running stocks is unsuitable as passive strategies should be prioritised. The urgent need for replicable envelope retrofit strategies is known. However, there are scarce studies on passive retrofits for school buildings. Recently, schools began incorporating orchards and farms because of the benefits in children’s development fostered by nature. Rooftop farms  diminish the roof’s heat exchange with the outdoor environment whether because of the increment in thermal mass – soil in green roofs – or the creation of a new external envelope, e.g. in rooftop greenhouses  the building’s roof becomes the greenhouse’s floor. As such, RFs are suitable strategies to improve the energy performance of uninsulated schools. Additionally, schools’ RFs have pedagogical, nutritional and health-related benefits,can foster a circular economy and social cohesion, reduce the environmental impact of food chains, and increase food security. Since the 2000s, the advancement in agricultural technologies sprouted diverse RFs systems as low-tech soil-beds, high-tech controlled greenhouses, and, more recently, building-integrated agriculture. BIA refers to the bi-directional integration of exhaust flows between buildings and farms. RFs are constrained to the roof’s load capacity and require technical solutions for water, energy and waste management not relevant to ground agriculture. On this subject, there is a growing body of literature comparing building-based agriculture to its ground counterpart regarding environmental performance, cost-efficiency, and triple-bottom sustainability. To date, limited studies assess RFs systems from a building energy perspective. Initial studies quantify the performance of a specific farm system, their potential improvement, and the benefits of one-directional integration.

Difficulty in modelling transient heat and mass transfer between plants and their surrounding environment led researchers to neglect these in energy quantifications, to use static loads to represent plants, ebb flow table or to use offline independent models. Furthermore, only a few studies compare the energy and thermal benefits of building-based agriculture. Though these studies provide the first quantification of vegetation effects in buildings, there is a need for a more accurate energy modelling of RFs through the online closed-loop calculation of building and crop energy balances. Crops mathematical models are easily implemented in numeric computing platforms. Therefore, energy co-simulation is the most practical option. In addition, considering the deficiencies in school building stocks and the educational and social benefits of green envelopes, there is the need to evaluate the feasibility and replicability of RFs as passive retrofit strategies. To understand better the energy and thermal benefits of buildingbased agriculture, this study quantifies the improvement in building performance by thermodynamic coupling of different RFs. For this purpose, this study assesses two Ecuadorian archetype free-running schools and three RFs systems: edible green roofs,hydroponic rooftop greenhouses,and thermally integrated rooftop greenhouses. To move forward the simulation of building-based agriculture, this study couples the heat and mass balance of crops with building simulation engines through co-simulation. This cosimulation incorporates crops growth as a time function, includes airflow exchange between farm and building environments, includes the electricity requirements for farms operation, and relies on validated archetype buildings to extrapolate results to educational stocks. The following sections are: 1) theoretical background, 2) methodology description, 3) results and discussion on the three RFs studied, and 4) conclusions.Two real-reference building schools  were used as host buildings for RFs, one for typology A and the other for typology B. Historically listed buildings were not considered because of their specific requirements. These archetypes were selected using an engineering bottom-up urban energy model; a full description of their selection and validation is in Ref.

Fig. 3 depicts the archetypes, and Table 1 describes their envelopes. Archetype A is a building complex with two blocks, one cross-shaped and the other rectangular. Both blocks are single storey, have lightweight construction systems, pitched roofs, and a window-to-wall ratio  of 30%. The pitched roofing in archetype A is incompatible with any RF system. As such, for RFs scenarios evaluation, this was replaced with a flat 10 cm composite deck-slab with a 5 cm cement screed and indoor ceiling. Archetype B is a two-storey L-shaped concrete-frame building. Its classrooms are single-sided, accessible from an exterior hallway, and have a WWR of 21.3%. Schools standard working schedule is from 7 a.m. to 1 p.m. for weekdays and unoccupied during weekends. Summer vacations are from mid-July to the first week of September. Buildings are occupied for a total of 1477 hours annually. The average number of students per classroom is 35, and the children’s median metabolic rate is 74 W/ person. Both archetypes are uninsulated, naturally ventilated, and free-running, i.e. there is no heating, cooling, mechanical ventilation, or domestic hot water. Both schools have LED lighting fixtures on classrooms and teachers’ areas with an average power density of 7.5 W/m2 ; and compact fluorescent lighting  on halls, toilets and storage areas with a power density of 10 W/m2. Appendix A presents a detailed description of internal gains. At normal pressure, archetype A infiltration rate is 0.5 ACH, and Archetype B is 0.4 ACH. These values were taken from a detailed air network simulation of both buildings.For evaluating RFs’ energy performance, three theoretical RFs scenarios on these two real-reference school buildings were assessed using dynamic simulation: edible green roofs,hydroponic rooftop greenhouses,and thermally integrated rooftop greenhouses. Fig. 4 shows these RF scenarios in both case-study schools.

The standardization of STS aimed at facilitating the combination, but there was a large gap between scores. However, as the STS was ordinal data recommended by Egdane et al,it had a greater priority than the growth character for tolerance selection. Therefore, the standardization was considered still relevant to determine genotype tolerance. SaTI results showed a normal distribution pattern in the population; and thus, it was comprehensive and effective. Moreover, the SaTI had an additive pattern. Formation of normal distribution or standardization was also carried out by Peternelli et al  through discriminant analysis in the formation of their selection index. The effectiveness of SaTI  was also shown in a more selective selection than that only based on STS values. Anshori,who used the same genetic materials and methods, found 45 good tolerance genotypes to salinity stress using only STS. The genotype adaptability index for tolerance to salinity stress based on a combination of GAI and SaTI was an indirect selection approach for a targeted environment. The indirect approach aimed at optimizing the potential of two different characters. Simultaneous selection with two or more characters would be tighter if they were not correlated. Our results indicated that GAI and SaTI were not correlated, and therefore the IASI model became important to obtain appropriate genotypes in saline areas.

Based on the correlation of the Sukra yield, IASI had the highest significance correlation to the yield. That is, the combination of GAI and SaTI was more selective and effective in predicting the actual yield in saline environments than the selection solely based on tolerance. Besides,mobile vertical farm the number of selected DH lines  was considered quite appropriate for preliminary selections. This indicated that IASI was a good method for preliminary selections of DH lines under salinity or other stresses. The validation in Sukra showed that the combination of both experiments was more effective than a focus on the tolerance selection in hydroponic screening because the yield is not correlated to the tolerance trait in early vegetative stages. Therefore, the selection of sustainable genotype under stress should combine agronomic and tolerant characters. IASI was an appropriate indirect approach in selecting the adaptive genotype under a saline environment. Its correlation value to the Sukra yield reached 0.69. The low repeatability showed that the environmental factors affected the yield variance and influenced the bias in correlation and regression analyses. Although the regression had a low variance value in predicting the Sukra yield,the genotype distribution was still within prediction interval,except for one genotype. In addition, based on direct adaptability index  in Truntum as a saline prone area  in the dry season,IASI also has a significant correlation to DAI with the value of 0.59. This indicated that IASI is still relevant in predicting the response of adaptive genotypes in saline areas. DH lines have high homozygous levels; however, they do not ensure stability in stressful areas. Stressful areas can be strongly influenced by the environment and G × E interaction. Hidayatullah et al  showed that DH rice lines have significant G × E interactions in all agronomic characters. In addition, the heritability of DH wheat lines can decrease for all characters in a saline environment. 

IASI may increase the effectiveness and efficiency in DH line selection, especially in the initial stage of breeding during the development of DH lines adaptive to salinity stress. In conclusion, multivariate analysis in the selection process can improve the effectiveness of DH rice line selection in preliminary selection stages. We found that GAI formula was 0.465 yield + 0.433 NPT + 0.31 NFG. In all, 24 DH rice lines were considered to have good agronomic characters. SaTI was developed by an average of SSI based on discriminant analysis and standardized salinity tolerance score. There were 34 DH rice lines with good tolerance to salinity stress in hydroponic culture. SaTI, in selecting tolerant genotypes, was more effective than that based solely on STS. IASI was effective based on the correlation in Sukra validation and DAI, and 28 DH rice lines were selected as adaptive to salinity stress.The bio-fortification of staple foods has been accepted as a practical and cost-efficient way to alleviate malnutrition by increasing the concentrations of essential nutrients in crops and ultimately in humans. Paddy rice,one of the most widely planted staple crops and the source of 80% of the daily caloric and micro-nutrients for over half of the world population, is the most suitable candidate for crop bio fortification strategies. Compared with medical supplementation and dietary diversification, it is easier to directly benefit the malnourished conditions of people living in poor rural regions via agronomic or genetically bio-fortified rice. Rice is adapted to diverse local edaphic and climatic conditions, resulting in the development of thousands of varieties and genotypes by selective breeding,however, yield and environmental adaptation are likely the most important factors for varietal breeding to enrich micro-nutrients content. For example, superfluous soil iron  is one of the most significant conditions decreasing rice production in Southeast Asia and South America. 

Farmers likely prefer rice with a lower Fe uptake to ensure optimal yield. Therefore, it is important to screen for the elemental concentration status of rice varieties. Moreover, plants take up and translocate nonessential elements through sharing the same pathways as essential elements due to similar chemical properties. For instance, zinc  and cadmium  share the same zinc-iron transport protein  influx transporter into plant roots ; and plant roots can absorb arsenic  and selenium  via the phosphate transporter.These nonessential elements can be enriched through the food chain from crops to humans, and the consumption of contaminated crops can pose a significant health risk. Rice is easy to accumulate toxic metal under flooded conditions due to high affinity. Many previous studies have reported the public concern and health risks of Cd and As,and the radioactive contaminant elements cesium  and strontium  in crop grains harvested from contaminated areas, such as Fukushima in Japan and Chernobyl in Ukraine. Hence, increasing the essential nutrient uptake and reducing nonessential elemental contamination in rice is a pressing issue for human health. Elements with similar chemical properties sharing the same transportation pathways can result in competitive uptake between them. Ionomics, the study of the elemental profiles of all essential and nonessential elements in organisms, tissues and cells, provides a rapid screening strategy for elemental interactions and varietal identification of plant species using high-throughput element analytic methods, such as inductively coupled plasma-mass spectrometry  and ICP-atomic emission spectrometry. Numerous ionomic studies for the rapid identification of elemental interactions among plant varieties have been conducted. Chen et al  performed ICP-MS to quickly characterize Lotus japonicus mutants with altered ionomic profiles. Chu et al  screened extreme Cs accumulation among Amaranthus species to find the ionomic basis for low Cs varietal breeding.

Watanabe et al  used ionomic methods to screen vegetable varieties to determine the characteristics of mineral accumulation and nutritional values. Therefore, it is feasible to screen rice varieties for bio-fortification and safety by ionomic-based research methods. Consequently, we cultivated 120 rice varieties at the early seedling stage in identical hydroponic systems to the external environment. The concentrations of nutrients, trace elements and anions of the rice species were analyzed by ICP-MS and capillary electrophoresis  to screen for rice varieties with high concentrations of nutrients or low concentrations of toxic minerals or both. The ionomic interactions among genotypes and the ionomic variations corresponding to phylogenetic relationships were also characterized.Meanwhile, a principal component analysis  using correlation coefficients conducted to compare the correlations among subspecies, indicated a 34.0% of the variance was partitioned in the first PCA axis, and root and shoot subgroup samples were separated on that axis. Root samples also showed a greater degree of separation on the 2nd PCA axis, indicating that the correlations among subspecies showed a differentiation between the shoots and roots. In addition, the PCA correlation scores in the roots among the subspecies were located in the same first quadrant, while the location in the shoots, showing a highly significant difference among subspecies, was in a different quadrant. Consistent to PCA, the elemental correlation patterns of roots were similar among subspecies, while that of shoots were largely different which the strongest correlation pattern was in japonica, and the less was in aus. K and Mg correlated with only a few elements in aus and indica, and P and Al only correlated with more elements in the shoots of japonica and roots of aus, respectively. In contrast, Ca, Cu, Zn and Sr were detected significantly related with many elements in all plots. Mg in the roots of japonica correlated significantly negatively with As, but positive relations showed in the shoots of japonica and indica. The same situations were also detected in Mn and Co, Mn and Cs, Zn and Cs. Although different rice subspecies and organs showed different elemental interactions, many still remained the same.

Obviously, correlations between S and SO4 were always significantly positive. Ca interacted significantly and positively with Ba and Sr, and Ba also significantly and positively correlated to Sr in all rice organs.To identify the ionomic differences between organs of the subspecies and to observe the geographic factors, we compared the element concentrations in all samples,shoots  and roots  using PCA, and displayed the loading plots. As shown in Fig. 4-A, there was a significant separation between shoots and roots in all the rice genotypes. Meanwhile, all the macro-nutrients along with Mn and B loaded on the negative x-axis to explain the shoot ionomes, vertical farming racks while the root ionomes were mainly determined by the micro-elements and anions. The PCA results in Fig. 4-B and -C showed that the clusters were largely determined by the different origins of the varieties, whereas the rice subspecies from the same origins were not significantly separated in shoots or roots. For example, the japonica shoots from Japan were separated from japonica shoots from South Asia and Southeast Asia, but not from the indica shoots from Japan in the PCA results. According to the loading plots, the differences in shoots and roots from Japan were mainly explained by most nonessential and toxic elements such as As and Cd, whereas K, Na, Li and anions mainly contributed to differences in rice varieties from South Asia.There were significant differences in the concentrations of all elements except Ba among japonica, indica and aus, but the magnitudes of element concentrations generally displayed similar, indicating that the ionomic variations were within the pre-framework of the phylogenetic factors of the genus Oryza, although the genomic differences among subspecies have been. As shown in Figs. 1 and 2, all rice subspecies follow the rule that trace elements and anions were concentrated higher in the roots than in the shoots, and it was confirmed by the separation between shoots and roots in all the rice varieties, as well as the loading of micro-elements on roots in PCA. Rice root is the main barrier to limit translocations of heavy metal and toxic element to shoots by chelation and compartmentalization. However, micro-nutrients for the goal of bio-fortification, such as Fe and Zn, are also indiscriminately fixed in roots. Interestingly, opposite to S, the SO4 2- concentration in the roots was higher than that in the shoots, primarily due to most of the inorganic S being fixed in root vacuoles or converted to organic S  in leaf for protein synthesis. Thus, it would be a meaningful challenge to identify absorption and translocation mechanisms for specific micro-elements in roots. As a response to the element concentrations, dry biomass weights among subspecies were showed in boxplot. The concentrations of essential elements in the shoots or roots of japonica were markedly the lowest, while that of harmful elements in japonica showed the highest. Correspondingly, the biomass weights of both shoots and roots in japonica were the lowest among subspecies. The results showed that improving nutrients and reducing toxic elements also showed a crucial correlation on improvement of the biomass and yield of rice. Significant differences among diverse rice genotypes detected in most elements in both shoots and roots showed that the phylogenetically-changed elements were more marked in the shoots than in the roots, and further indicated that the variations in elements among the rice varieties were mainly attributed to their translocation from roots to shoots, consistent with previous studies. 

Prior to the beginning of the experiment, 75% of the total volume of the aquaculture subsystem was filled with artesian well water. The remaining 25% was filled with water from 60-day mature inoculums from RAS and BFT systems previously performed. These inoculums were used to ensure that the microbial communities of the MBBR and bioflocs were already mature. For the maintenance of the biofloc microorganisms and the C:N ratio of the water at 15:1, liquid molasses was added to the BFT and DFP fish tanks three times a week as a complementary carbon source. The amount of molasses was calculated based on the input of N by fish feed. Calcium hydroxide was added to the fish tanks when the alkalinity was under 80 mg L− 1.Nile tilapia  juveniles were purchased from a commercial hatchery  and acclimatized for 7 days after arriving at Caunesp. In all treatments, 114 masculinized juveniles  were stocked, totaling an initial biomass of 0.43 kg m− 3 and density of 300 fish m− 3 in each fish tank. After 4 weeks of culture, the number of fish per tank was managed in order to readjust the densities. Thus, 70 fish per tank were kept, resulting in a density of 184 fish m− 3 and a calculated biomass of 3.6 kg m− 3. During the 56-day trial, the fish were hand-fed with the test diets four times a day at 08:30, 11:00, 14:30 and 18:00 h.

The amount of feed was calculated based on the percentage of body weight recommended by a commercial feed industry,ranging from 12 to 5% according to the average fish weight. A sample of at least 20% of the total number of fish in each tank was weighed weekly to adjust the amount of feed in all treatments. At the end of the experiment,vertical grow all tilapia juveniles were counted and weighed and 15 fish from each tank were individually measured. Final individual body weight,total fish length,weight gain,yield,specific growth rate,feed conversion ratio  and survival  were assessed.For this, three different samples were taken:  30 individuals from the initial fish population;  10 from each repetition per treatment at the end of the experiment; and  15 g of each diet. The fish were anesthetized and euthanized. Subsequently, the whole bodies were weighed, packed and frozen at − 20 ◦C for later analysis. The frozen fish were ground, homogenized in a meat grinder  and lyophilized. The lyophilized matter was used to determine the percentage of dry matter and crude protein,according to the methodology of A.O.A.C., Association of Official Analytical Chemists. The same analyses were applied to determine the composition of the diets. All analyses of the proximal composition of the tilapia tissue were made in duplicate.Two trials of butter lettuce  production in different phases were carried out. In Cycle 1, the seedling phase was evaluated in a 14-day trial. For this, hydroponic seedlings at 7 days after sowing  and 0.59 ± 0.08 g were grown until 21 d.a.s. In Cycle 2, the final production phase was performed, in which new hydroponic seedlings at 21 d.a.s. and 2.04 ± 0.57 g were planted and cultivated for 21 days until harvest. In both cycles, 8 plants were distributed in each hydroponics subsystem with a density of 19 plants m− 2.These four lettuces per tank were selected randomly at the beginning of the trials and the same were weighed weekly throughout each trial.

At the end of Cycle 1, all plants were weighed and the following growth parameters were evaluated: leaf and root height,total wet weight,total dry weight,number of leaves per plant,productivity  and specific growth rate. At the end of Cycle 2, the following growth parameters were evaluated for seven lettuces from each plant tank: leaf and root heights,wet leaf and root weights,dry leaf weight,number of leaves per plant,and productivity. Also, in both cycles, a visual analysis was applied to identify the non-marketable plants. Plants that contained up to 33% of abnormalities on the leaf surface, i.e., with a yellowish color, burns or wrinkles, were considered non-marketable. For the control of plant pests, twelve traps  were distributed through the greenhouse. A visual scan of the presence of plant pests or diseases was performed daily on all plants and no sign of them was seen during the trials.Prior to the beginning of both trials, 50% of the total volume of each plant tank was filled with water from the aquaculture subsystem and, for the remaining 50%, artesian well water was used. The tanks of HP treatment were only filled with artesian well water. In the DAPS plant tanks, the water was collected from the upper-middle portion of the MBBR of the DAPS aquaculture subsystem. In the DFPs treatment, the water of each DFP fish tank underwent a decantation and filtration process before being directed to the plant tanks. This means that the water was pumped into the RFS and remained there for 20 min until the biofloc particles were decanted. After that, the RFS supernatant was directed to a bag filter  and then to the PTs. The initial volume of water taken from each aquaculture subsystem, which was used to supply two PTs,was replaced with artesian well water. Between the two cycles of plant production, all the PTs were emptied, cleaned and all the aforementioned procedures for filling the PTs were repeated.

When the EC in all PTs was stable, the seedlings were planted. For plant nutrition during the experiment, water from each aquaculture subsystem or well water  was added manually to the PTs, at the proportion of 2% of the initial volume of the PT since it was the estimated volume of water evaporation in the PTs. In the DFP systems, the biofloc decantation and filtration procedures were always carried out and the decanted bioflocs returned to the fish tanks, except for samples collected in the beginning, middle and end of the experiment. In both plant cycles, the commercial fertilizer solution was added according to the equation above and only if the registered EC values were below the expected ranges. In Cycle 1, the EC was maintained between 1.1 and 1.3 mS cm− 1 and a unidirectional water flow between fish and plant tanks occurred once a day on alternate days. In Cycle 2, the EC was between 1.6 and 1.8 mS cm− 1 and a unidirectional water flow occurred once a day, six days per week. Aiming to maintain the pH in the PTs at between 5.5 and 6.5, diluted phosphoric acid  was added when the pH exceeded 6.5.FLOCponics systems were run in a decoupled layout with the aim of enabling proper management of each subsystem; thus, taking advantage of the nutritional benefits of the biofloc-based culture to produce tilapia juveniles and lettuce. The findings of this study suggest that some critical points usually associated with FLOCponics systems were addressed by individualizing the aquaculture and hydroponic subsystems. For instance, the difficulty of maintaining a low concentration of solids in the hydroponics subsystems and, at the same time, providing a sufficient amount of bioflocs in the fish tanks,has been reported as an issue of coupled FP systems. Another challenge of coupled FP is regulating the water pH within the appropriate range for fish, bioflocs and plant growth. The trade-offs related to solids concentration and water pH were tackled in the present study through the use of decoupled FLOCponics. The physical-chemical parameters of the water were monitored in each subsystem of all treatments to interpret the production results.

Except for the maximum values of settleable solids,the other results for water quality in the aquaculture subsystems were within the acceptable range for tilapia  and also for BFT and RAS microorganisms. The mean results of settleable solids were within the recommended range of 5 to 50 mL L− 1 for tilapia ; however, in some measurements the values exceeded 100 mL L− 1. No issues regarding a high amount of solids in the aquaculture subsystem have been reported so far in the research on FLOCponics. As already mentioned, low concentrations of bioflocs have usually been indicated as a drawback of coupled FLOCponics systems. Hargreaves  stated that the values of settleable solids above the recommended value of 50 mL L− 1 do not favor fish growth or nutrition, but might result in oxygen depletion and a higher electricity demand in biofloc-based cultures. The high amount of settleable solids in the fish tanks, associated with the unexpected increase in environmental temperature, was probably the main factor that caused the sudden drop in DO and the unviability of the DFP-36 treatment. For the other treatments, the DO values were always higher than 3 mg L− 1 despite the recorded values of settleable solids. The accumulation of solids was probably a result of the methodology adopted for regulating the C:N ratio, in which the addition of the carbon source was performed periodically based on the amount of N inputted by the feed. Given the need for minimizing the risks associated with high solids concentrations, it is recommended to test the methodology based on the concentration of ammonia in the water to regulate the C:N ratio  or to remove and reuse the solids  in FP systems. The differences found for the mean values of DO, pH, EC and TDS in the aquaculture subsystem were a result of the different input of N and carbon source  in each treatment. The input of the carbon source seems to be the main factor in these results, indoor growers since in the DAPS the mean values of pH, EC and TDS were distinct from those recorded in the BFT and DPF-32, even though all of them received the diet with 32% crude protein.

For the nitrogenous compounds and orthophosphate results,it is hard to conclude whether or how the dietary protein or integration with plant production affected the variation of these nutrients during the experiment. Further studies with a focus on the nutrient flows between the BFT and hydroponic subsystems and the carrying capacity of DFP systems are still required to understand the efficiency of recovering nutrients from the BFT effluents by plants. In the hydroponic subsystems, except for the pH values in Cycle 1, the other parameters of water quality remained within the expected ranges in both cycles. The pH values should remain between 5.5 and 6.5 to enable higher bioavailability of nutrients, whether they come from the aquaculture subsystems or from the extra commercial fertilizer. In Cycle 1, the pH was above the recommended range in all treatments and the highest values were recorded in the DFP treatments. Phosphoric acid was added in the plant tanks to regulate the pH when it exceeded 6.5. Another factor that interfered with the pH was the buffering in biofloc-based cultures. Despite these issues with pH, they exerted no negative effects on the growth of lettuce seedlings. As expected, the results for tilapia growth demonstrated that the well-known benefits of BFT for juvenile nutrition are also found in the DFP systems. Tilapia juveniles fed with 32% CP and grown on both biofloc-based treatments  grew 22.7% more than those in DAPS also fed with 32% CP. Luo et al.,Long et al.,and Hisano et al.  showed the same tendency of improved zootechnical performance for tilapia grown in BFT compared to RAS, although both were fed with the same amount of CP. They indicated the uptake of the microbial bioflocs as a complementary feed by tilapia as the main reason for these results. Not finding differences in FCR among the treatments is somewhat surprising, since better feed conversion is usually related to biofloc-based culture compared to RAS. Nevertheless, the results of PER,PPV,and CPwg  show the highest efficiency in using the dietary protein in the fish produced in the biofloc-based system  compared to DAPS  with 2.93, 40.11% and 13.70%, respectively. These results suggest that even in an integrated system the in situ food present in biofloc-based systems is used by tilapia juveniles to complement their dietary protein needs. The similar results for tilapia growth in DAPS and in DFP fed with 8% lower CP reinforce this statement. The zootechnical results of tilapia fed with lower CP suggest positive economic and environmental implications of DFP. Since protein is usually the most expensive component in the diets,the use of lower CP levels will result in lower feed costs.

Data about total soluble solids of the Beefsteak and Cherry tomato cultivars were significantly different among all genotypes. Data presented in Table 2 illustrated the similar trend of TSS among Cherry cultivar, but it was significantly higher than the group of Beefsteak tomatoes. In comparison between Beefsteak and the Cherry tomato comparatively higher total soluble solid was found in cv. Aria  while the minimum in cv. Grandy. Mavromatis et al.  reported that the values of the total soluble solid of different varieties fall in the range 2–4%, but the concentrations of total soluble solid  vary with the variety and ripening stages due to the breakdown of polysaccharides. Titratable acidity was statically analyzed  and it showed significant variation among the nine cultivars of tomato and its values ranged from 2.45 and 3.97 g/L. These results are consistent with the data reported by Romero-Rodriguez et al.,within the range of 2.9–4.4 g/L. Significantly more concentration of TA was calculated in Cherry cv. Cheramy  which was similar to cv. Nactar  and Beefsteak cv. Grandy. Ascorbic acid plays an important role in the human diet because it cures the chronic disease, stress and scurvy. Ascorbic Acid of Beefsteak and Cherry tomatoes showed significant differences at P ≤ 0.05 with a range of 15.48–23.24 mg/100 g. These values were supported to the outcome of Mavromatis et al.  and Chattopadhyay et al.. Data presented in Table 2 illustrated that the maximum AA concentration was found in the Cherry cv. Claree  followed by cv. Aria,Nactar,Cheramy  and the minimum in Beefsteak cv. Dirk. The overall comparison between Cherry and Beefsteak, maximum AA concentration was recorded in cv. Claree  and the minimum in cv. Dirk. 

A possible reason for the differences obtained in AA content for the same variety can be explained for the incidence of light in tomato at the end of harvest, temperature conditions during pre-harvest, stackable planters harvest time and post-harvest. The level of AA is higher at the time of maturity and then went down. The biochemical parameter of the Beefsteak and the Cherry tomatoes revealed significant differences among all cultivars. ‘Aria’ of the Cherry group showed the significantly higher amount of total sugar,reducing  and non-reducing sugar. The cultivars including Beefsteak group were shown the statistically lower concentration of total sugar, reducing and non-reducing sugar than that of the Cherry group. The indexes are used to evaluate the quality of the fruits and it showed relatively significant variation among the cultivar. The color indexes  were statistically similar in the Beefsteak cvs. Dirk, Grandy, Naram, Vernal and Sahel but lower than the Cherry group. Significantly more darkness and yellowness were seen in cv. Aria while redness observed in cv. Claree.For years the human population of our planet has tended to be concentrated in urban centres, which increases the demand for food in cities. This, together with the growing concern regarding environmental sustainability and food security, is leading to new and complementary forms of local and sustainable food production that are practised for domestic use or self-consumption in backyards, rooftops and communal vegetable gardens. This is especially interesting in more deprived areas for people with low resources. Although the volume of food produced with more sustainable farming methods, at the domestic level or by small peri-urban producers, has a very low contribution to the overall demand for food in large cities, there is a growing demand due to the quality of the products obtained and the concerns arising about sustainability in urban areas. In addition, maintaining food security and water scarcity have emerged as a critical challenge. Sustainable food production by saving and recycling water and nutrients is considered one of the possible solutions. This is, precisely, the basis of aquaponics, a biointegrated system that links aquaculture with hydroponics and is considered a model of sustainable food production,by taking advantage of wastewater from aquaculture and using it as nutrients for plant growth.

Domestic aquaponic systems are an option when it comes to producing food for self-consumption,contributing in improving food security and self-sufficiency, which in cities such as Sydney  could reach 15 %. They have the ability to adapt to being a form of peri-urban cultivation to produce various vegetables, such as leafy greens, which are considered part of a more balanced and healthy diet. This system also has a great potential for indoor food production regardless of climatic conditions. Hence, aquaponic production systems can be located on non-arable land close to markets, thus shortening the supply chain and reducing the carbon footprint associated with rural farms and transporting produce to city markets. It could also become an integrated agricultural system that plays a crucial role in future so-called “Smart cities”, especially from an environmental, socioeconomic and sustainable perspective, as it employs innovative systems to provide, through short supply chains, fresh food. Although there are no official censuses by countries of aquaponics facilities for domestic use or self-consumption,it seems to be an extended practice. One of the characteristics that make domestic aquaponic systems difficult to evaluate is that they are very heterogeneous, with a variety of designs adapted to the different socioeconomic and climatic conditions, also using different species of fish and polycultures of different vegetables. It is very difficult to find quantitative data on fish and vegetable polyculture production in these systems, or if one does, they do not appear to have been evaluated with a scientific procedure. Aquaponic systems with a tank size of 1000 L and a growth space of about 3 m2 can be considered micro-scale,and are suitable for domestic production for a family home, with the purpose of producing several vegetables and herbs at the same time for their subsistence. In the majority of cases, especially in commercial systems, only one crop and one fish species are produced at the same time. In micro-scale systems, producing a variety of crops  is most common, particularly when they are intended for selfconsumption. There are very few research works related to polyculture aquaponic systems and the existing ones include few species cultivated in them. However, aquaponic systems aimed to provide a healthy and complete diet to their users require the production of a high and varied number of vegetal species.

With this in mind, the main objective of this study was to assess an aquaponic production of more than 20 vegetables, fruits and herbs, together with a fish species, with a self-consumption purpose, in a marginal neighbourhood of the city of Seville,taking into account the climatic limitations during summer and winter. In this regard, operating an aquaponic system in locations subjected to low temperatures during some periods constitutes a challenge if fish species not adapted to these conditions are used. This sometimes leads to excessive energy consumption, which often represents one of the main costs of aquaponic production. Hence, it is important to define strategies to avoid having high energy costs and to achieve economic sustainability of this type of production. Therefore, as a specific objective, two methods were tested to avoid excessively low water temperatures in the fish tanks, in order to optimise the operation of the aquaponic systems in winter.The study was conducted in a greenhouse located at IES Joaquín Romero Murube secondary school in the Polígono Sur neighbourhood,which has one of the highest rates of social exclusion and economic poverty in Spain. The greenhouse, 9 m long, 5 m wide and 3.5 m high,had a clear plastic polyethylene film covering. The most extreme temperatures were reached in December and June. From June 21st to October 15th, the plastic cover of the roof of the greenhouse was replaced by a shading mesh  to avoid extreme high temperatures. Therefore, temperatures during these months did not exceed 40 ◦C. Two identical self-constructed aquaponic facilities were built based on FAO designs for micro-scale aquaponic systems,with some changes made to improve the performance and handling of the facility. Cheap and easy-to-obtain materials were employed. The total investment costs for the construction of the facilities were estimated to be around 1300 € for each of the MAS.

Each of the MAS consisted of a tank for the fish production and a conjunction of three different hydroponic sub-systems:Nutrient Film Technique,Grow Bed  and Deep Water Culture. In each MAS,stackable flower pots the total cropping area was 4.56 m2,of which 3 m2 correspond to NFT, 1.2 m2 to GB and 0.36 m2 to DWC. The total volume of recirculating water was 1.8 m3. The fish tank consisted of an IBC  type tank  with a 0.7 m by 0.7 m opening trimmed on top to allow the operations with the fish. Two meshes were placed on the opening, one to avoid the fish jumping out of the tank and another to shade. A correct level of dissolved oxygen in the water inside the tanks was ensured by means of a 5 w air pump with a flow of 0.48m3 h− 1. The water in the tank was maintained with a constant level in order to always ensure the same volume. The fish tank was connected through a 50 mm PVC pipe to another 0.45 m3 tank acting as a clarifier, in order to avoid anaerobic conditions due to an excessive accumulation of solids. This tank had a cylindricalconical shape with a drain valve at its bottom. The water from the fish tank entered the clarifier through an elbow, causing a circular motion which facilitated the sedimentation of solid waste, while the clean water flowed upwards. Water came out of the clarifier through a 75 mm PVC tube attached to a bulkhead connector located 9 cm below the top edge and passed to the top of the grow bed tank. This was made with an IBC tank cut in half with a capacity of 0.45 m3 filled with 0.4 m3 of prewashed expanded clay. This tank acted at the same time as a bacterial bio-filter and as a GB hydroponic sub-system. From there, the water passed through a bell syphon to the sump tank,containing 0.54m3 of water. A 0.04 m thick extruded polystyrene foam sheet  covered 1/3 of its surface, and three holes  allowed placing plants inside it, separated 0.25 m from each other. From the sump, 80 % of the water was sent back to the fish tank thanks to a submersible SunSun JTP 4800Lh− 1 and 32 w pump. The remaining 20 % was recirculated by an NFT hydroponic sub-system, and back to the sump again. For the NFT, five PVC pipes,placed on concrete blocks levelled to obtain a slope of 1%, were used. Each of the pipes had 12 holes with a diameter of 0.05 m at 0.25 m from each other.A total of 22 crops were grown during a year in both MAS. A polyculture of fruits, vegetables and herbs was selected  with the objective of providing a family with a diversity of products that enabled achieving a nutritionally healthy diet.

The species used were: Raf tomato,Roma tomato,Water melon,Eggplant,Cucumber,Italian frying, Lamuyo and Goat horn pepper,Basil,Onion,Stevia, Pumpkin,Melon,Chard,Broccoli,Cauliflower,Cabbage,Chinese cabbage,Strawberry,Potato,Zucchini,Lettuce. The different species were planted according to their optimal growing season and the seedlings were obtained from commercial plant nurseries. These seedlings were selected with at least four true leaves and approximately one month after germination. For planting, the root system of the seedlings was carefully washed and inserted in the corresponding hydroponic subsystem. For each species, the different maintenance tasks usually used in their hydroponic production  were performed.Before starting the aquaponic production, water was recirculated through the MAS during 40 days without fishes or plants, so the nitrifying bacteria could proliferate. For the initial loading of both MAS, a mixture of rainwater and drinking water from the public network was employed to achieve a pH between 7 and 7.4. After that, only dechlorinated water coming from the public network was used to replenish water losses by direct evaporation and evapotranspiration. The MAS were monitored throughout a complete year from April 24th, 2018 to April 24th, 2019. Management and maintenance actions were carried out in the same way as a family would do for a self-managed aquaponic production. An electricity meter was installed in each of the systems to record consumption.

The controller has a 32-bit ARM processor that can be interfaced and simultaneously communicate with 8.0 driver boards via Ethernet. The control signals can be generated by the crop models algorithms coded into the processor or cloud-based streaming systems. At the same time, environmental sensors can collect measurements, store data on an SD card, and transmit data directly to a web server or via wireless communication to a gateway using LoRa modulation. A detailed description of this framework for greenhouse tomatoes is explained in Rezvani et al.. Some of the specific features of the IoT automation system shown in Fig. 13 include simultaneous switching control of up to 33 actuators from any location, multiple and independent voltage lines for DC and AC actuators, open-source programming, and waterproof IP66 enclosure, and external charging circuits. The wireless sensor nodes and the controller boards are shown in Fig. 13 were used with a conventional PID temperature controller to maintain an experimental hydroponic greenhouse. The air temperature responses were collected at ten samples per minute using LoRa sensors with different sensor probes and plotted on the IoT dashboard, as shown in Fig. 14. ach data point was assigned a unique ID that represented the collection time and location and stored on a private cloud database with a secured API key address to be accessed and used by the IoT controller as the feedback of the control algorithm. Several data collection samples were carried out, and all samples were analyzed to ensure the reliability of IoT control. Results showed that no single data point was lost during the tests as long as the WiFi network was available.

The system’s reliability can be significantly improved by increasing the number of WiFi access points so that the controller can switch between the networks. The response of the controller demonstrates the robust performance of the IoT-based automation with a high spatiotemporal resolution and excellent stability in data transfer at 10.0 readings per minute within 1.0 km distance from the wireless controller, hydroponic net pots which can be considered a reliable approach for adjusting growth parameters for CEFP. It can be concluded that the integration of wireless communication, distributed data analysis, and a web-based data monitoring dashboard provides flexible automation and an online assessment tool to investigate the effects of structure design, covering materials, cooling and heating techniques, and growing seasons on the optimality and comfortability of growth parameters for CEFP. The limitation in wireless communication due to the high-density plants and other disturbances can be overcome by increasing the number of the sensors and repeater nodes which reduces the loss of connection and data interruptions even in mesh applications or using antennas with cable for higher positions, higher mesh density, multiple gateway nodes, and higher output power. Hydroponic fodder production is likely to play an increasingly important role in livestock production with the current dwindling supply of grazing land, water, and increased food demand. As fodder could be grown hydroponically in CE, most grains are still not feasible to produce indoors. So, the effective use of agricultural land for grain production would be beneficial in fighting against world malnutrition problems by transforming the fodder and pasturing fields into crop production fields. Also, controlled environment hydroponic fodder farmings could contribute to climate mitigation objectives if complemented with effective energy management and land use policies.

A study showed that incorporating hydroponic barley production in CE can reduce GHG emissions with increased seed-to-fodder output, efficient energy management, and the integration of renewable energy for operation. Fig. 15 shows the sensitivity in GHG emissions with the seed-to-fodder output ratio in a hydroponic fodder production system powered by renewable energy in Canada. The results indicate hydroponic fodder production outperforms conventional open-field production only when the seed-to-fodder ratio is more than 5.5.In general, the feasibility of fodder production in CE is being challenged by some criticism regarding nutritional benefits for livestock, high capital and operational costs, and social difficulties for using grains as animal feed. In the coming decades, food demand is expected to grow rapidly, while agricultural land is being degraded as a consequence of climate change and unsustainable agricultural practices . Greenhouses are used as one of the strategies to mitigate these problems and their area has been growing rapidly. Greenhouses can have high food productivity in limited space, while being less dependent on environmental conditions. Greenhouses with soilless forms of agriculture, also known as hydroponics, have significantly higher efficiencies for water and nutrients use than conventional agriculture. However, soilless forms of agriculture generally rely heavily on synthetic fertilizers, as organic fertilizers still result in significantly lower yields . For the creation of nitrogen fertilizers fossil fuels are used, while phosphate fertilizers are manufactured from non-renewable sources. Their discharge into the environment causes severe damage to aquatic ecosystems through eutrophication . Consequently, the production of synthetic fertilizers has significant negative effects on the environment.

The introduction of the aquaponics concept, the combined cultivation of fish and crops, is an attempt to reduce the use of synthetic fertilizer and to further improve nutrient and water use efficiencies of hydroponics . Nowadays, an industrial aquaponics system consists of a recirculating aquaculture system and hydroponic system , with water flowing between the two to exchange nutrients . In the RAS, fish are reared in tanks and a screen filter and nitrification reactor are used to remove solids and to convert ammonia to nitrates, respectively. In permanently coupled aquaponics , the nutrient-rich RAS water is used as a hydroponic solution to grow plants in the HPS and the water from the HPS is returned to the RAS after the plants have taken up a fraction of the nutrients . However, this single-loop system inherently creates sub-optimal conditions, as the plants and fish have different needs for nutrient levels and pH . To eliminate this problem, an on-demand coupled aquaponics system has been developed , where the water flow from the HPS to the RAS becomes optional, allowing more control over both systems . To increase the nutrient use efficiency in on-demand coupled aquaponics systems a remineralization loop was added, in which an anaerobic digester converts the filtered-out solids from the RAS into plant-available nutrients for the HPS, leading to a multi-loop system . In addition to the remineralization loop, a desalination loop, in which nutrients in the discharged RAS water are concentrated and clean water returned to the RAS through reverse osmosis filtration, was also proposed . The concentrated nutrient solution is subsequently transferred to the hydroponic solution. Besides concerns regarding fertilization, highly productive greenhouses are also known for their high energy consumption. In northern latitudes, the majority of greenhouse energy demand is for heating, resulting in a heavy reliance on fossil fuel . For a standard greenhouse in the Netherlands, Kempkes et al. reported that heating can account for up to 90% of the energy use and 25% of the production costs. Reducing the energy consumption for heating is crucial for reducing greenhouse gas emissions and reducing the dependency on fossil fuels.

In the past decades, there has been plenty of innovation and research on saving energy in greenhouses, driven by environmental regulation and cost reduction . To achieve this, a mix of technologies and operational strategies can be used. For example, dehumidification and heat recovery can be used to control the indoor climate, which decreases the need for ventilation . In terms of operational strategies, changes in the temperature and humidity ranges can decrease the need for heating in a dehumidification process . With increasing latitude, seasonal differences generally increase, while average temperatures decrease. In addition to an increase in heating demand, Goddek & Körner showed in a modeling study that this increased seasonal variation makes an aquaponics system more difficult to manage and less efficient. This is because the main water transfer in an on-demand coupled aquaponics system is driven by transpiration, which in turn is mainly related to global radiation. Thus, greenhouses at higher latitudes have much potential for energy use reduction. Several studies already cover the effects that different energy-saving strategies have on crop production . However, its effect on nutrient use efficiencies in aquaponics systems is largely unknown and complex due to dynamics and interactions in such coupled systems. This study aims to investigate and explore what effects various operational strategies and design parameters have on the optimal design and operation of an on-demand multi-loop aquaponics system in a cold and varied climate. For this, a model was used to simulate a multi-loop aquaponics system, growing lettuce and Nile tilapia , using weather data from the Netherlands. Subsequently, several design and operation parameters were varied to find their influence on water and nutrient use efficiency,energy use and the growing environment. Special interest was paid to the variation in crop transpiration, which was used for the comparison of different scenarios.

To better support the theory proposed in this article, the production of tomatoes was explored, as well. Additionally, two new buffering strategies were investigated, intending to further increase the performance of the aquaponics system in varied climates.The aquaponics system in this study mostly follows the design of Dijkgraaf et al. and consists of three compartments: the recirculating aquaculture system , anaerobic digester and hydroponic system.These elements are shown in Figure 1 and explained in the following sub-sections.The main component of a RAS is a set of fish tanks, blueberry grow pot where fish is produced in staggered cycles at high densities. Nutrients are added to the system in the form of fish feed, which is either consumed or wasted by the fish. From the consumed fraction, nutrients partly contribute to fish growth and leave the system with the harvest of fish, whereas the other part is excreted as feces and urea. The urea dissolves in the water, increasing the nitrogen concentration, while the solids remain in suspension. In addition to the fish tanks a RAS has a treatment train with a drum filter to remove these solids and a moving bed bioreactor to prevent the build-up of ammonia, as shown by Goddek & Keesman . The discharge of RAS water is mainly directed to the HPS, while a small fraction of the water remains with the separated solids.The solids form a sludge that is transported to the AD. To maintain a constant water volume in the RAS, fresh water from tap, rain or groundwater is added to compensate for the discharge. The dilution of RAS water takes place when the ammonia or nitrate concentration surpasses a prespecified limit.To prevent, to a large extent, the loss of nutrients in the fish sludge,it is treated in an anaerobic digester system.This system consists of two sequential UASB reactors with different pH levels for optimal performance, as in Goddek & Körner . The chemical oxygen demand present in the fish sludge is digested in the first reactor, resulting in the production of biogas which can be utilized for heating the reactor, fish tanks or greenhouse. In the second reactor, as a result of the remineralization process, a large fraction of the nutrients in the fish sludge becomes soluble . Combined with the direct RAS discharge, this highly concentrated effluent flow makes up the nutrient source from fish to crops. The whole AD system is explained in great detail by Delaide et al..The hydroponic system is similar to conventional soilless, closed-loop cultivation systems.Several irrigation techniques can be used, such as Deep Water Culture,Nutrient Film Technique or drip irrigation, each with its own range of suitable crops.The advantages and disadvantages of many soilless systems are described by Maucieri et al. . For this study, lettuce is grown, either in DWC or NFT. The main difference concerning this study is the overall water volume of the nutrient solution,with DWC having a far greater volume than NFT.For both technologies the nutrients not taken up by the plant is returned to the sump for recirculation, forming closed loop systems. In this study, the preparation of the nutrient solution is different from standard hydroponics, as it is largely based on a mix of RAS water and digestor effluent, topped up with synthetic fertilizers.

The development of novel biopolymers based on chemical modification of naturally occurring polymers can be a highly cost-effective alternative with diverse opportunities for engineering novel delivery systems. Lignin is a byproduct of the pulping industry; 50–60 million tons of lignin are produced every year . Given its origin and properties, LN can be sustainably used for agricultural purposes given that its degradation products are humic acids that will enhance soil fertility. Lignin is a highly complex and heterogeneous polyphenolic polyether with a wide range of molecular weights. The complexity and abundance of the functional hydroxyl groups of lignin provides significant potential for chemical modification. For example, we previously successfully grafted LN with poly acid to form a novel LN-g-PLGA amphiphilic polymer which was further assembled into core-shell nanoparticles. The hypothesis of this study was that LN-g-PLGA nanoparticles would facilitate targeting and controlled release of a nonsystemic agrochemical. Methoxyfenozide , a commonly used non-systemic pesticide, was used as a model to determine release and translocation from soybean roots to shoots. MFZ is a diacylhydrazines insecticide used for the control of lepidopteran larvae; the pesticide is most effective when ingested by the larvae. MFZ has a low solubility in water and given its non-systemic nature, quantifying its presence in plants would be an ideal indicator for the effectiveness of this platform. To test this hypothesis, 28 days old soybean plants were treated with MFZ-loaded LNPs at three concentrations in a hydroponic system. After 6, 12, and 24 h, treated plants were harvested and MFZ content in the roots, stems and leaves was determined by liquid chromatography-mass spectrometry.

If the hypothesis is proven correct, this will allow for creating a bio-inspired platform to enable targeted and precise in planta delivery of non-systemic analytes. The development of a bio-inspired platform to enhance the efficient of non-systemic pesticides can be a novel and powerful approach in sustainable nano-enabled agriculture. The plants were exposed to free MFZ at three concentrations 0.27, 2.7, and 27.03 μg/ml for the low, fodder system for sale medium and high doses; and to the equivalent concentration of nanodeliverd MFZ, corresponding to 0.01, 0.1, and 1 mg/ml LNPs. Statistical analysis demonstrated that the time and the LNPs had a significant influence on the translocation of MFZ in all tissues .The concentration of MFZ in the roots increased over time when applied with a nanodelivery system, whereas the MFZ concentration in tissues of plants treated with free MFZ was consistent over the three time points tested. Similarly, MFZ concentration in the stems was higher for nanodelivered MFZ, especially at the longer times, 12 and 24 h, in the low and medium concentrations . The effect of time was most significant in the leaves, however.This is to be expected given that it will take time for the LNPs entering the roots to translocate the agrochemical, either still associated with the LNP or as free released analyte, from the roots to the leaves.

The MFZ concentrations in the leaves at the low, medium, and high LNP exposures increased over time; they measured 0.05, 0.15, and 0.22 μg/g at 6 h; 0.06, 0.48, and 0.44 μg/g at 12 h, and 0.08, 0.96, and 1.72 μg/g at 24 h of exposure and they were in most cases significantly different from the controls . To assess the ability of the LNPs to shuttle MFZ from the roots to the leaves, translocation efficiency was calculated as the percentage of MFZ translocated to the plant upper section as a function of the total amount taken up by a plant. TE has been used by others to demonstrate the efficiency of a delivery system to translocate a molecule from the roots to the shoots of plants. The TE achieved with LNPs was 0.065 at 24 h at a concentration of 0.01 mg/ml LNPs while at the higher concentration tested the TE decreased to 0.006 after 24 h, which is similar to results obtained by others.The decrease in the TE with increasing LNPs concentration can be attributed to the higher accumulation of MFZ in the root tissue at these high concentrations. The same reasoning explains the higher TE of the controls relative to the treatment. Even though the TE was higher for the controls, the total amounts translocated were significantly lower than the treatment and did not change significantly over time . The total amount of nanodelivered MFZ to roots, stems and leaves indicated that LNPs delivered ~7-fold more MFZ than the free form, and as high as ~17-fold with the low LNP concentration treatment after 24 h . Even though the size of the LNPs suggested lower to no translocation to the upper section of the plant, LNPs still showed a noticeable TE compared to other delivery systems with similar sizes. This can be attributed to the highly negative charge of the particles , which may have facilitated the translocation of the LNPs within the vascular system.

The specific mechanisms for NP translocation in plants are still not well understood and different species of plants posses distinct interactions with NPs as NPs must traverse different plant systems in order to be translocated to the leaves and shoots of the plant. There are two well known pathways for the internalization of analytes from the root epidermis to the xylem located in the endodermis; nanoparticle properties will determine which of these uptake pathways is followed. Apoplastic transport makes use of the capillary network located in the epidermis and cortex and requires osmotic pressure and a concentration gradient to transport certain minerals, whereas other compounds are more selectively accumulated. This mechanism is known as passive transport because it does not require energy from the plant. Symplastic or transcellular is an energy requiring active-transport mechanism for the uptake of nutrients; molecules are bound to carrier proteins to be introduced in the cell via endocytosis. Particle size, hydrophobicity, surface charge, and aggregation are all known to alter NP- cell wall/membrane interactions. Particle size in particular is a determining factor in the uptake of NPs; smaller particles have a greater tendency for long distance transport whereas larger particles, either from original size or through aggregation, more often accumulate in tissues. Cell walls are semipermeable and limit the passage of different molecules, but because pores in plant cell walls have an average size of 3–8 nm, nanoparticles exceeding this size are most likely to be transported by the symplastic pathway. Interaction of the cell wall with particles through endocytosis can lead to formation of pore cavities that facilitate the access of even more particles increasing the uptake of NPs to the plant. A number of studies have demonstrated that nanoparticles of specific chemistries and exceeding a certain size do not translocate at all. For example, most AgNPs with a size up to 40 nm remained adhered to the root caps of seedlings treated for four weeks and did not translocate. Given the ~100 nm size of the LNPs tested herein, we expect translocation to have occurred through the symplastic transport mechanism, with improved translocation due to the negative charge of the particles.

In general, a positive charge is associated with higher NP adsorption on root surfaces and greater NP content in roots, whereas negatively charged NPs are associated with higher internalization rates and translocation efficiency. As noted above, the goal of this study was to improve translocation of a non-systemic agrochemical and as such, the negative charge of the LNPs was desired as a contributing factor to reach this goal. Nanoparticle delivery systems have the potential to be used as tools to improve the effectiveness of agrochemical delivery by facilitating translocation from roots to leaves or vice versa as shown here in a hydroponic system. The interaction of the LNPs with the soil is expected to be different from what was seen in a hydroponic solution due to possible aggregation, degradation and interaction of NPs with soil constituents. These interactions are affected by their particle size, shape, and surface charge, as well as the presence of organic matter, pH, and microorganisms which can affect how particles behave. Because the LNPs have a highly negative charge and no tendency to aggregate under a wide pH range, they are expected to remain in suspension unless they encounter positively charged soil constituents to which they will electrostatically be attracted to. Further studies are required to completely understand their interactions with soil and support the hypothesis that LNPs can shuttle agrochemicals to plants grown in soil. Cadmium is a class-I carcinogen and extremely toxic metal to human being; exposure to Cd primarily damage kidneys and can cause other very serious health problems for the cardiovascular, pulmonary and musculoskeletal systems . Bioaccumulation of Cd in rice grain is a serious concern, particularly in Asian countries where people ingest rice two to three times each day. It is a critical issue for rice because nearly half of the population worldwide is dependent on rice as a dominant staple food . The world noticed its first evidence of Cd poisoning in the 1950s in Japan through the occurrence of the “Itai-Itai disease” caused due to consumption of Cd contaminated rice .

Rice plant has the tendency to amass Cd more efficiently as compared to other cereals and also constitutes the major source of dietary intake of Cd in some countries like China and countries located in the sub-continent including Sri Lanka, India and Bangladesh . Cd is a trace metal without any notable function in the growth and development of animals and plants . Agricultural soils have been contaminated by Cd as a result of anthropogenic activities, for instance metal smelting, fossil fuel incineration, sewage sludge and phosphatic fertilizer application . Cd is readily acquired through roots of crop plants and transferred to the upper parts of the plant, including the grain, and this procedure relies on how much Cd is available in soils as well as the inherent capacity of the plants . Crops that are grown in Cd-polluted soils may have ahigh amount of Cd in their edible portions, and this could be a precursor to consumers risking chronic toxicity if the intake is large enough . Aquatic plants such as the rice plant ,fodder growing system common cattail and common reed release oxygen from root aerenchyma by radial oxygen loss that oxidizes Fe2+ in submerged soils to Fe3+ oxides that precipitate on the surface of the roots in the form of Fe plaque . The presence of Fe plaques on surfaces of wetland plant roots may offer a means of exclusion and attenuation of metals . A study showed that rice varieties with greater radial oxygen loss capacities tended to form higher rates of Fe plaque on the surfaces of roots and hence limiting the transfer of Cd from root to above-ground parts including grain . Dong et al. discovered that Fe plaque formation led to the deposition of heavy metals onto the external surfaces of roots, subsequently restricting Cd in rice tissues being taken up and transferred. A study showed rice tissues Cd content as well as the citrate-bicarbonate-dithionite -Cd of roots were greatly influenced by Fe and Cd supplementation into growth solution . Exogenous Cd addition in the culture solution remarkedly enhanced the concentration of Cd in rice tissue and CBD extracts, and these concentrations were undetectable in the absence of exogenous Cd additions. It was observed that Fe application displayed a significant role in the Cd accumulation and transportation in rice plants and root cells, rather than in the Fe plaque existing around the roots . Another research study revealed that Fe plaque deposited around the root decreased Cd absorption and that Fe addition reduced the harmfulness of Cd to the rice seedlings grown hydroponically . The study confirmed that application of 0.2 mM Fe to the solution of 10 µM Cd did not suppress the root growth of the rice plant and the root and shoot Cd content and translocation of Cd from the nutrient solution to the shoots with a combined treatment of Fe and Cd, were diminished by 34.1%, 36.0% and 20.1%, respectively, compared to only Cd addition. Understanding the root plaque formation and its impact on Cd accumulation for various rice varieties is crucial as this will generate key information on Cd accumulation by rice genotypes to assist in minimizing the human health risks associated with rice consumption.

To examine whether genetic adaptation to the plant root had taken place during the EE, single evolved isolates from the evolved populations were saved as frozen stocks and subjected to phenotypic and genotypic characterization. To represent different populations and time points during the EE, three isolates were randomly picked from each of population 3, 4, 6, and 7 at transfer 12, 18, and 30 . To detect possible changes in colony morphology, ON cultures of the ancestor and evolved isolates were spotted on LB agar and colonies inspected after 48-h incubation. On LB agar, the ancestor formed a round colony with a wrinkled periphery, whereas different colony morphologies were observed among the evolved isolates . At T12, some isolates displayed a colony morphology resembling the ancestor, e.g. isolate 3 from population 3 and isolate 2 from population 7 , referred to as the ‘‘Wrinkled’’-type. Several other isolates formed a colony with a white sharp edge along the wrinkled periphery , hereafter referred to as the ‘‘Sharp-Wrinkled’’-type. Additionally, isolate 7.1 formed a hyper-wrinkled, white colony, referred to as the ‘‘Snow’’-type. These distinct colony morphologies were also observed at later time points . Interestingly, the Snow-type was only observed in population 7. Furthermore, the three isolates from population 6 at T30 formed slightly less wrinkled colonies compared with the ancestor. We note that three isolates do not represent the entire population, and isolates with other colony morphologies could be present in the populations. Nonetheless, the appearance of isolates with altered colony morphologies in the four populations indicates the presence of genetic changes. Furthermore, the occurrence of isolates with altered colony morphologies already at T12, and especially the presence of three different types in population 7, at this early time point, suggests rapid diversification of B. subtilis during EE on A. thaliana roots. Such diversification into distinct morphotypes was also observed in our previous study on EE of B. subtilis on plant roots and has additionally been observed in EE of B. subtilis pellicle biofilms,dutch buckets indicating successful adaptation to the selective environment.

The design of the EE employed in this study should enable selection for bacteria that efficiently colonize the root. We, therefore, speculated whether the altered colony morphology of some of the evolved isolates was associated with improved productivity on the root . To test this, the ancestor and evolved isolates from the final time point were tested for individual colonization of A. thaliana seedlings under the same conditions applied during the EE. CFU quantification revealed that most evolved isolates tended to show increased root colonization, with five isolates from three different populations displaying significantly increased productivity on the root, with an up to circa 1.3-fold change relative to the ancestor . To track down when such improved root colonizers emerged during the EE, the randomly selected evolved isolates from T12 and T18 were similarly tested. Three and five evolved isolates at T12 and T18, respectively, displayed significantly increased productivity relative to the ancestor. In addition, a single isolate from T12 was significantly reduced in root colonization. These results confirm that indeed the genetic adaptation of B. subtilis to the plant root took place during the EE. Furthermore, the observation of improved root colonizers already at T12 indicates that B. subtilis rapidly adapted to plant root colonization during the EE.While multiple evolved isolates displayed increased individual root colonization relative to the ancestor , we next wanted to test whether the evolved isolates had a noticeable fitness advantage over the ancestor during competition on the root. For this purpose, two selected evolved isolates from independent populations from T30 were competed against the ancestor on the plant root. Following 48 h of root colonization, CFU quantification revealed that both evolved isolates had out competed the ancestor on the root, and statistical analysis confirmed that the evolved isolates had a significantly higher fitness relative to the ancestor . This result was further supported by CLSM imaging: regardless of the fluorescence labeling combination, the two evolved isolates formed biofilms on the roots, as evidenced by aggregates along the root, whereas the ancestor was scarcely present .

Noticeably, the fluorescent images revealed that Ev6.1 formed fewer and smaller aggregates along the root compared with Ev7.3 , consistent with the individual root colonization of the two isolates . To test whether the fitness advantage of Ev6.1 and Ev7.3 over the ancestor was specific to the plant root environment, the two evolved isolates competed against the ancestor in a non-selective environment, i.e. in LB supplemented with xylan, a plant polysaccharide found among others in the secondary cell walls of A. thaliana , and under well-shaking conditions. In this non-selective environment, neither of the evolved isolates out competed the ancestor but instead seemed to suffer a fitness disadvantage compared with the ancestor, although the difference was statistically not significant . Similar results were obtained for the permuted fluorescent combination . These results demonstrated that the evolved isolates had a fitness advantage over the ancestor specifically in the plant root environment. Furthermore, the loss of fitness in a different, non-selective environment suggests an evolutionary cost of adaptation to the plant roots .To identify the genetic changes contributing to the increased root colonization and fitness advantage over the ancestor during root colonization , the genomes of selected evolved isolates were re-sequenced. To represent independent populations, the isolates from populations 6 and 7 at T30 were included. Furthermore, to track molecular evolution over time, the three isolates from population 7 at T12 and T18 were also re-sequenced. Finally, one isolate from population 1 at T30 was included for re-sequencing owing to its ‘‘Smooth’’ colony morphology and reduced root colonization . In the 13 re-sequenced isolates, we observed in total 51 unique mutations of which 37 were non-synonymous . Isolate Ev1.1 harbored several mutations in gtaB encoding a UTP-glucose-1-phosphateuridylyltransferase that synthesizes a nucleotide sugar precursor essential for the biosynthesis of exopolysaccharides and for the synthesis of wall teichoic acids and lipoteichoic acid .

Two of the three isolates from population six at T30 harbored a nonsynonymous point mutation in the fliM gene, encoding a flagellar motor switch protein, part of the basal body C-ring controlling the direction of flagella rotation . All three isolates in population 7 at T30 harbored a mutation in the intergenic region upstream from the sinR gene encoding a transcriptional repressor of the genes responsible for matrix production . Interestingly, this mutation was also present in the three isolates in population 7 at T18 and in one of the isolates in this population at T12, suggesting that this mutation arose rather early in the EE and rose to a high frequency in population 7. Indeed, sequencing of the seven endpoint populations revealed that this mutation upstream from sinR was fixed in population 7 at the final time point, i.e. the mutation had reached a frequency of 1 in this population at T30 . Furthermore, anon-synonymous mutation within the sinR gene was detected at high frequencies in populations 2 and 3. In addition, non-synonymous mutations in genes related to flagellar motility were besides population 6 also observed in populations 3, 4, and 5 . Finally, mutations in genes related to cell wall metabolism were identified across all seven populations . The detection of mutations within genes related to biofilm formation, motility, and cell wall metabolism across independent populations supports the role of these mutations in the adaptation of B. subtilis to A. thaliana roots.Next, we wanted to elucidate which bacterial traits were altered during such adaptation to the plant root. For this purpose, evolved isolates from the final transfer were subjected to further phenotypic characterization. Given the detected mutations and that both biofilm formation and motility are important for successful root colonization by B. subtilis , we hypothesized that these two bacterial traits would be under selection during the adaptation to the plant roots. To this end, plant polysaccharides including xylan have been shown to induce biofilm formation in B. subtilis in a non-biofilm inducing medium .

One way of adapting to the plant root could thereby be through enhanced biofilm formation in response to such PPs. To test whether the improved productivity on the root by the evolved isolates was associated with more robust biofilm formation in response to PPs, the ancestor and evolved isolates were tested for pellicle biofilm formation, a biofilm formed at the medium-air interface , in LB supplemented with xylan . Importantly, a rich medium rather than the minimal medium was used in this assay to provide the bacteria with plenty of nutrients, allowing us to assess only the ability of the evolved isolates to form biofilm in response to xylan, and not the ability to utilize xylan for growth. We observed that a few isolates from T30 developed a pellicle biofilm similar to the ancestor, i.e. Ev4.1, Ev4.2, and Ev6.1 . In contrast, the remaining isolates developed more robust pellicles with highly structured wrinkles indicative of enhanced matrix production. Especially the three isolates from population 7 developed hyper-robust, white pellicles, consistent with the Snowtype colony morphology observed for these isolates . The biofilms developed in response to xylan by the evolved isolates generally correlated with their productivity on the root. For example, isolates Ev4.1, Ev4.2, and Ev6.1 developing similar pellicles as the ancestor and isolates Ev7.1, Ev7.2, and Ev7.3 forming hyper-wrinkled, robust pellicles in response to xylan were among the ones showing the smallest and largest increase in individual root colonization , respectively. This is in accordance with Chen et al. demonstrating that the ability of B. subtilis mutants to form robust biofilms in vitro correlated with that on the root. These results suggest that improved productivity on the root was associated with robust biofilm formation in response to xylan. To test whether this enhanced biofilm formation was specific to the presence of PPs, grow bucket the ancestor and evolved isolates were tested for the ability to form pellicles in LB in the absence of xylan. In this medium, the pellicles developed by both the ancestor and evolved isolates were less robust . For most isolates, the improved biofilm formation was specific to the presence of PPs, whereas the isolates from population 7 displayed robust biofilms also in the absence of plant compounds suggesting a general improvement in biofilm formation in these isolates.To test whether the evolved isolates were affected in motility, the ancestor and evolved isolates from population 6 and 7 were tested for two types of motility: swimming motility, a single cell movement in aqueous environments powered by flagella rotation and swarming motility which is associated with a rapid multicellular movement of hyper-flagellated cells across a surface facilitated by self-produced surfactin.

Interestingly, most isolates were significantly impaired in both swimming and swarming motility . Swimming motility was observed for the ancestor and evolved isolates after 4 h . However, after 6 h only the ancestor and Ev6.2 had reached the edge of the Petri dish, whereas the remaining isolates reached at the most half of the swimming distance of the ancestor. Swarming was observed for the ancestor after 4 h which continued until the expanding colony almost reached the edge of the Petri dish after 8 h . In contrast, the evolved isolates showed reduced or a complete lack of swarming throughout the experiment. The evolution of motility-impaired isolates in independent populations could indicate that motility is not important for root colonization in the selective environment. Notably, during the EE the 48-well plates were continuously shaking at 90 rpm. We speculated, that these mildly shaking conditions could allow the bacteria to get into contact with the root by chance and thereby reducing the impact of motility on root colonization in the selective environment. To test whether motility is important during root colonization under shaking conditions, the ancestor was competed against a Dhag mutant, deficient in the production of the flagellin protein, for three successive rounds of root colonization under static or shaking conditions.Under static conditions, the Dhag mutant was significantly out competed by the WT.In contrast, under shaking conditions, the Dhag mutant was able to co-colonize the root to similar levels as the WT. These results demonstrate that motility is important for competition on the root under static conditions but is not required under shaking conditions. Thereby, impaired motility of several of the evolved isolates is not expected to negatively influence the fitness of these isolates in the selective environment.

The control treatment with the 100% hydroponic fertilizer had significantly the highest concentrations of phosphorus and nitrogen . The subsequent substitution of the biogas slurry with the hydroponic fertilizer from 20% to 60% resulted in a direct increase in the concentration of P and N. Relative to the control treatment, the 20% substituted treatment showed a 5.6 and 4.8 times less P and N, respectively. Unlike for P and N, the concentrations of the cations Na, Ca, Mg and K were significantly higher in the treatments with biogas slurry, with the control having the lowest concentrations. The concentration of Ca and K in the 20% substituted treatment was 107.7% and13.9% more relative to the control treatment. However, for Mg the highest concentration was observed in the 60% substituted treatment, whilst for Na, the highest concentration was observed in the 40% substituted treatment. Several trace metals which include As, Cd, Cr, Ni, Zn and Pb were measured in this study. However, it was noteworthy that the concentrations of most of these metals were below detectable limits except for Pb and Zn only . Of these two metals, there were no significant differences observed between the treatments on concentrations of Zn, whilst significant differences were observed for Pb. The concentration of Pb ranged from 0 to 0.0015 mg/L whilst that of Zn ranged from 0.08 to 0.15 mg/L. The pH in the different treatments was not significantly different, with all values being neutral ranging from 7.69 to 7.91. However, pH was slightly higher in the treatments with biogas slurry, resulting in an average 2.4% difference relative to the control. The electrical conductivity was also significantly higher in the treatments with biogas slurry whilst it was lowest in the control treatment. On average, EC within the treatments with biogas slurry was 23.8% higher compared to the EC in the control treatment. Across all the treatments, EC ranged from 275.7 to 356.7 μS/cm.

The chlorophyll content of a plant is a general indicator of nutrient status and nutrient response of the plant. In our study, chlorophyll content was expressed as an index ranging from 1 to 45, with 1 being the lowest. There were significant differences observed between treatments on chlorophyll content index , with the control having the highest values whilst the 20% substituted treatment had the lowest index . Relative to the control treatment, the 20%, 40% and 60% substituted treatments resulted in 39.4%; 22.8% and 8.7% significantly lower CCI. The marketable fresh fruit yield was significantly different among all treatments, with a clear trend where the higher the fertilizer substitution, the higher the tomato yield. Relative to the control, the 20%; 40% and 60% treatments resulted in 14.4; 4.6 and 0.8 times less fresh fruit yield, respectively. It was important to note that substitution of the biogas slurry even with 60% of the hydroponic nft channel fertilizer did not result in fruit yields comparable to those in the control treatment. On another note, the sugar content which was measured in Brix units, increased with a decrease in nutrient substitution of the different treatments . There were statistically significant differences in sugar content with the 20% substituted treatment having the highest sugar content whilst the control had the lowest content. Relative to the control treatment, the 20%, 40% and 60% resulted in 80.3%; 46.5% and 30.6% more sugar content, respectively. The last parameters measured to determine yield were the number of fruits and flowers between the treatments. Similar to fresh yield results, there were significant differences observed among treatments, with the control having the highest number of fruits and flowers, whilst the 20% substituted treatment had the lowest numbers . Compared to the control treatment, the 20%; 40% and 60% treatments resulted in 7.4; 2.6 and 1.1 times less number of fruits, respectively. In a similar trend, relative to the control, the 20%; 40% and 60% treatments resulted in 4.8; 1.1 and 0.9 times less number of flowers, respectively .For the cations Ca, Mg, K and Na in the tomato plant biomass, the 20%substituted treatment resulted in significantly the highest uptake of Ca and Mg . For Ca uptake, the 20% substituted treatment resulted in 102.7% more Ca, whilst the 40% and 60% substituted treatments resulted in an average 2.9% more Ca only, relative to the control. For K concentration, as the concentration of the hydroponic fertilizer substitution increased, the uptake of K by the plant decreased.

The K in the tomato plant biomass under the 20% substituted treatment was 20.4% more than that of the control treatment. The lowest concentration of Mg in the plant biomass was observed in the control treatment, which was 122% less than that observed in the 20% substituted treatment. However, there were no significant differences observed between the 40% and 60% substituted treatments for Mg concentration. It was interesting to note that Na concentration was significantly the highest in the control treatment, whilst the biogas digestate based treatments did not record significantly different concentrations . On average, the Na concentration in the control treatment was 116.3% more relative to all the three biogas digestate based treatments. The concentration of most of the measured trace metals in the tomato plant biomass was below detectable limits except for Pb and Zn only . The concentration of Pb in the plant biomass showed an unusual trend across the treatments, with the 60% substituted treatment having the highest concentration whilst the 20% substituted treatment had the lowest concentration. The concentration of Pb in the 40% and 60% substituted treatments was 22.7% and 96% higher, respectively, compared to the control treatment, whilst the 20% substituted treatment recorded a 78.6% less Pb, compared to the control. On the contrary, the concentration of Zn in the plant biomass significantly decreased with an increase in the level of substitution of the hydroponic fertilizer . The 60% substituted treatment recorded plants with the lowest concentration of Zn followed by the control. The concentration of Zn in plants within the 20% substituted treatment was 57.8% higher, relative to the control.Biogas digestates have been reported to have high levels of biological oxygen demand, suspended solids and low dissolved oxygen, whilst being rich in nutrients such as N and P . However, studies that have evaluated the fertilizer potential of these digestates in hydroponics crop production are scarce . In our study, the raw biogas digestate diluted at 20% and 40% resulted in crop phytotoxicity as indicated by the low germination indices. Germination index is a very sensitive parameter used for evaluating the phytotoxicity of organic materials and GI indices below 50% have been reported to indicate materials unsuitable for use in crop production . These lower GI have been attributed to heavy metals, ammonia and low molecular weight compounds that reduce seed germination and root elongation . Though heavy metals were significantly low in the raw biogas digestates , it is possible that the presence of higher levels of ammonia versus nitrates and other organic compounds in the digestates used in our study could have resulted in elevated phytotoxicity at higher concentrations of biogas digestates.

Moreover, the observed phytotoxicity cannot be explained by the electrical conductivity or medium salt content, as these were well within the recommended values. It was also noted that different crops showed varying phytotoxicity responses to the biogas digestates, indicating the difference in their tolerance levels. Several researchers have indicated that biogas digestates are very high in phosphorus and nitrogen, as these are not utilized during the anaerobic digestion process . However, the concentration of these nutrients is highly dependent on the source of the manure. In our study, the manure was collected from animals that had been feeding on natural shrubs from the Namib Desert, whose macro nutrient content is very low. This could explain the low macro nutrient contents observed in the biogas slurry, which were not in the same range as reported by Juan et al. . The low macro nutrient concentrations therefore informed the need for substitution of the biogas slurry with the different levels of inorganic hydroponic fertilizer. Even under substitution with hydroponic fertilizer, the macro nutrient levels in the biogas digestate based treatments remained low in our study. This clearly indicates the lack of positive benefit from the cow manure based biogas digestate especially on the concentrations of macro nutrients in hydroponic nutrient solutions. However, the concentration of the cations was increased under the biogas based treatments, which is in agreement with results of Wang et al. even when only 2.6% biogas digestate were used with mineral fertilizers. The results of our study therefore indicate that the diluted biogas digestate can be an important source of micro nutrients such as Ca and Mg, though supplementation with other sources of macro nutrients is required. It was interesting to note that the concentration of heavy metals in the biogas digestate were lower than the recommended limit for irrigation water . These higher concentrations of elements like Ca and Mg as well as the lower concentration of heavy metals observed in the tomato plant biomass can be explained by the nutritional composition of the nutrient solutions used in this study.

The yield parameters which include chlorophyll content, fresh yield and number of fruits all showed a direct response to the concentration of N and P in the different solutions. These reductions in yield properties of the tomatoes could be attributed to the variability and imbalance in nutrient composition of biogas digestate based hydroponic solutions . According to Moller and Muller ,nft growing system in crops like tomatoes, conversion of ammonia to nitrates and nitrites together with supplementation of micro-nutrients may be required before biogas digestates are used in hydroponic culture. Based on the results of our study, the high nutrient demand for tomatoes suggest that other less demanding crops such as lettuce may be more suitable for cultivation using cow based biogas digestates. However, the sugar concentration was inversely related to the nutritional composition of the hydroponic growth media in our study. Similar results were reported by Wang et al. and this has been attributed to plant physiological response to high salt content under the biogas digestate based treatments.High demand has bolstered trade in medicinal plants and subsequently, creating the need for commercial cultivation of these plants. However, successful commercial cultivation of medicinal plants relies on achieving consistently high quality and quantity of medicinal materials at a reasonable cost of production. Consequently, efficient technologies and techniques are being developed. For example, greenhouse technologies circumvent the setbacks which are normally associated with open air conditions, such as variations in biotic and abiotic factors, and limited arable land . A plethora of studies have examined the effects of varying levels of specific abiotic factors on the production of secondary metabolites . On the contrary, much fewer studies have investigated the effects of biotic factors on nutraceutical and medicinal plants . Some entomopathogenic fungi are endophytic; some are easily mass-produced in vitro; some are rhizospheric; they are quite ubiquitous . An endophytic fungus forms a mutually beneficial symbiotic relationship with a plant; it lives inside a plant’s tissues without causing disease to the plant, meanwhile, boosting plant defenses and in return the plant acts as the host.

The increase in quantity and diversity of secondary metabolites in endophyte-containing plants are probably responsible for the reduction of insect herbivory on plants. The endophytic fungi-plant relationship can be explored for cultivation of targeted high value medicinal plant species with the view to optimizing medicinal properties by increasing quantity and quality of secondary metabolites in these plants. Beauveria bassiana, an entomopathogenic fungus which occurs naturally and ubiquitously in the soil , is endophytic and an interesting candidate for the evaluation of endophytic fungal colonization on the medicinal properties of plants. Plants belonging to the genus Allium including garlic, onion, and Chinese chive are known for their medicinal properties; they have proven pharmacological and nutraceutical activities. They contain organosulfur compounds such as allicin and phenolic compounds like gallic acid, quercetin, coumaric acid, and ferulic acid . Phenolic compounds are bio-active substances that show antioxidant, anticancer, anti-inflammatory, and antimicrobial activity, and prevent chronic disease .

The two Synechococcus strains were distinct in that UHCC0524 reached stationary phase on the third day, while UHCC0527 did not display any growth during the entire experiment. micro-algal strains belonging to the genera Selenastrum, Scenedesmus or Tetradesmus have been extensively used in municipal and industrial wastewater research. Compared to other waste streams such as municipal or poultry wastewater, the hydroponic effluent presents a significantly lower COD and BOD content but higher amounts of nitrogen and phosphorous. Lower COD and BOD content generally allows better light penetration which, combined with rich nutrient concentrations, demonstrates the suitability of hydroponic effluent as a growth medium. The genus Apatococcus represents a highly versatile group of mixotrophic photobionts often establishing symbiotic relationships with lichens and having the ability to survive in terrestrial habitats as free-living cells or in biofilm colonies. To the best of our knowledge, this was the first time an Apatococcus strain was applied for agricultural wastewater remediation, although the presence of this genus in wastewater streams has been previously reported. The Chlorococcum genus has received significant attention due to its growth performance in wastewater streams and ability to synthesize high lipid content potentially suitable for bio-diesel.Likewise, the genus Monoraphidium has been known for the ability to proliferate in industrial waste waters and accumulate saturated fatty acids appropriate for bio-diesel.

The Microcystis genus is commonly identified as a dominant species in harmful algal blooms; however, it is worth noting that not all strains produce microcystins, and in this study a non-toxic strain was used. Its moderate growth in hydroponic effluent suggests that this isolate may not be as robust as previously reported strains. The Nostoc genus represents a group of N2-fixing heterocystous filamentous cyanobacteria that have demonstrated ability to grow in sewage and synthetic waste waters. The unicellular Synechococcus strains are used in wastewater remediation either as immobilized or free-living organisms. In this study, the inoculum of both Synechococcus strains were maintained as free-living cells in low-light intensity, grow table hydroponic and therefore light stress might have inhibited the growth of the UHCC0527 strain. The results of the screening experiment confirmed that the hydroponic effluent is a promising alternative source of nutrients for photosynthetic microorganisms. Several micro-algal and cyanobacterial strains can effectively proliferate in the effluent without any adjustment . The results also showed the potential of the NordAqua Nordic Culture Collection by uncovering several isolates suitable for bioremediation of hydroponic effluents. Considering our extensive knowledge of the performance of UHCC0027 in synthetic and municipal waste waters and previous scale up experience we continued to work with this Finnish isolate of the Scenedesmaceae family in the following up scaling experiments. Besides, the micro-nutrient concentrations of the hydroponic effluent  were within growth promoting ranges for the Scenedesmus genus .

The application of waste streams for the large scale cultivation of micro-algae presents different challenges regarding the elimination of competitors, nutrient composition, and the content of organic matter. Employing suitable pre-treatment methodologies to the source and characteristics of any given effluent can aid the efficient proliferation of the seeded culture. To this end, three different pre-treatments were compared for their potential impacts on the growth of UHCC0027: Coarse filtration , micro-filtration , and bleach . The selection criteria for the applied methods were the presence of native microorganisms in the hydroponic effluent and the feasibility of integration into a pilot scale PBR. A control group was grown in autoclaved BG11 medium. Non-seeded flasks containing only pre-treated effluent were also included to evaluate the impacts of each method on the indigenous microbial community. The results demonstrated the ability of UHCC0027 to proliferate without significant differences between the tested pre-treatments . Additionally, there were no significant differences in the nutrient removal efficiencies with UHCC0027, demonstrating an ability to remove 36–52% and 99% for N-NO3− and P-PO43− , respectively . Interestingly, the non-seeded CF-treated medium demonstrated an increase in OD750 after 7 days of incubation . At day 12, the CF non-seeded medium displayed a significant increase in turbidity  followed by an increase of the total chlorophyll content, suggesting the presence of an indigenous photosynthetic microbial community . Assuming that indigenous microbes were also present in the CF-treated flasks seeded with UHCC0027 but its growth did not significantly differ from the control , it can be concluded that the chosen microalga was able to outcompete the indigenous microbial community.

A negligible increase in OD750 in non-seeded MF and BST flasks by the end of the experiment indicates that these methods are effective in reducing indigenous microbial activity. The effluent used for pre-treatment experiments had an average concentration of 186.17 ± 2.94 mg L− 1 for N-NO3− and 11.77 ± 0.02 mg L− 1 for P-PO43− . The N:P ratio of 35 indicates an unbalanced nutrient ratio that can negatively affect the growth of Scenedesmus species with implications in lipid and protein content . At 22 ◦C, over 90% of the P-PO43− was consumed in the beginning of our experiment, similar to what has been observed previously, although cellular adsorption has been reported to partially contribute to this process. This may have enabled the growth of UHCC0027 for 2 weeks with only residual amounts of P-PO43− in the medium. Thus, uptake of N-NO3− continued until all P-PO43− was consumed. The constant supply of 3% CO2 also favored nutrient uptake by improving the C:N:P ratios during the experimental period until the nutrients were depleted from the medium. At the end of the experiment, microscopic observations of the CF blank samples showed the complex food webs of the indigenous microbial community which were characterized by the presence of several taxonomic entities. In contrast, the microscopic observations of the seeded CF samples demonstrated the dominance of the microalga UHCC0027 . Therefore, since the different pretreatments did not significantly affect the growth or the nutrient removal efficiencies of UHCC0027, and, the culture was able to overcome the presence of indigenous microorganisms in lab scale experiments, a decision was made to combine the BST method with the two step filtration system  for the PBR inoculation. However, the BST method was not applied during the turbidostat mode to ensure the feasibility of daily operation and to facilitate handling large volumes of effluent.

Nonetheless, when compared with the previous setup, the outcome of Setup 2 demonstrated no significant impact in terms of nutrient uptake when the DW of the culture was reduced to one half. Additionally, this result shows that Setup 1 was operated without limitation, presenting an opportunity for increased DW in future experiments. This could improve the removal efficiencies of the system. The following setups, 3 and 4, were planned to evaluate the performance of the culture under varying pH. The transition period between Setups 2 and 3 was the longest undertaken, due to changes in both biomass concentration and pH . A two-step approach was used, whereby the culture was grown to a DW of 1 g L− 1 before the pH was changed. In Setup 3, the pH was lowered to 6.8 and the hydroponic effluent had the highest N:P ratio, oscillating between 32 and 37 . This variability did not compromise N uptake which remained stable at 86.68 ± 6.5 mg g− 1 , but led to a significant reduction of P uptake to 11.84 ± 0.42 mg g− 1 . The removal efficiencies were 31.5 ± 0.9% for N-NO3− and 68.9 ± 5.9% for P-PO43− .By this time, the P-PO43− in the hydroponic effluent was at its lowest concentration which explains the increase in the removal efficiency . The stress induced by different setups, particularly from this transition period onwards, was observed as a gradual bio-fouling effect. In Setup 4, pH was increased from 6.8 to 9 and the N:P ratio varied between 26 and 27. These factors,grow table in combination with several days of high solar radiation  led to a significant increase of P uptake to its maximum value of 21.30 ± 0.96 mg per 1 g DW . During this period, the system met the requirements of the EU directive  for wastewater maximum nutrient discharge for P-PO43− . However, the growth rate was lower than in the previous setups, which contributed to a longer HRT . The contribution of bio-fouling to this outcome should also be considered, since it continued to accumulate throughout Setup 4. Shifting the pH from 6.8 to 9 led to changes in dissolved inorganic carbon species from CO2 to HCO3. The supply of CO2 on demand through set point injection may have created a scenario where different inorganic carbon species were present simultaneously. This detail may have caused extra stress to the culture if the cellular metabolism was activated to favor the absorption of one inorganic carbon source to the detriment of the other.

Nonetheless, loss of growth rate due to a pH as high as 9 has previously been reported for Scenedesmus strains. The N:P ratio of the hydroponic effluent varied significantly between each setup of the PBR . During continuous cultivation, the N:P ratio of the hydroponic effluent varied between 16 and 37, which did not affect the biomass productivity to the extent observed for the chlorophyte C. vulgaris grown in municipal wastewater. Our data suggest no strong correlation between the N:P ratio and the volumetric productivity throughout the course of the experiment. This outcome may result from the high concentrations and constant availability of N-NO3− and P-PO43− in the media as well as the cellular physiological saturation of nutrient uptake. The scenario of nutrient repletion throughout the course of continuous operation is a likely factor in the moderate removal efficiencies observed for N-NO3− and P-PO43− . This, combined with cultivation in a PBR and the steady abiotic conditions provided by the greenhouse infrastructure may also explain the higher biomass productivity observed compared to other studies on Scenedesmus species. Additionally, due to the nutrient replete conditions, it can be hypothesized that the rate of assimilation did not follow a linear relationship with the rate of supply. This trend is described by the theoretical kinetics model of Michaelis-Menten and has been observed by n batch experiments. Despite this, luxury uptake of nutrients may have occurred during the last setup of the PBR run, since the growth rate was at its lowest, but the nutrient uptake was not substantially suppressed . In a continuous cultivation system, the HRT is negatively correlated with the growth rate of the species. If the growth rate decreases, the inflow of media is reduced, which increases the retention time. This trend was verified throughout the course of the experiment, indicating that the transition periods were crucial to stabilize the culture between each setup . Longer retention times yielded higher amounts of biomass to a total maximum of 90.2 ± 0.02 g of DW obtained at the 5th day of the batch operation. During the turbidostat operation the maximum value of total biomass was 67.32 ± 1.85 g of DW, observed in the last period of the PBR run . In our system, the coefficient of correlation between the turbidity sensor and the DW was obtained with a suspended algal culture. Therefore, the gradual accumulation of biofilm presented a threat to the sensor light path and turbidostat operation. For that reason, the PBR was stopped after 36 days of continuous operation and over 1 m3 of hydroponic effluent circulated.