Month: July 2022

One explanation could be that the higher likelihood for influence outweighed the effect of status, since relatively lower status farmers could still influence an entrepreneurial attitude towards organic. The status mechanism in the high threshold scenario can, therefore, explain why, e.g., entrepreneurs enter the organic market, and/or why homophily in farming styles in markets develop. An interesting result of the model, is that new entrants are important for the diffusion of organic farming. The influence of new entrants in this research went via two factors: their farming styles and their peer influence. A higher chance that the successor had the same farming style as his/her predecessor led to fewer organic farmers and organic pigs, irrespective of the parametrisation of the social influence mechanism in the model. This result was confirmed by the experts. Moreover, it is in line with previous research findings that associated organic farmers more often with an urban background , assuming that farmers with an urban background have a different farming style through different peers. It is also in line with the social identity theory , assuming that successors have a higher chance for different farming styles than their predecessors through different reference groups. More research in the farming styles of successors, including the diversity of farming styles and development of new rationales, would be interesting for gaining insight in diffusion of alternative farming practices. It should be noted that this result also means that when farmers have a smaller chance to have a successor, the number of organic farmers would decrease and the diversity of organic farmers’ farming styles as well. The importance of successors in conversion to organic is, however, in contrast with studies that found no correlation between age and early or late adopters of organic farming ,vertical farming racks or between age and farmers with a conservation identity.

The role of successors in adoption of alternative farming practices might, therefore, be different per context. Finally, in this research we used a predefined network for farmers, predefined characteristics for farmers and markets, including the distribution of markets and farming styles, and the instantiation of attitudes among farmers with a specific farming style. Different instantiations could have an effect on model results. For example, a distribution of age that better resembles reality , could affect more changes in farming styles in the model and, therefore, more organic farmers, or more quitters. Future empirical research on farmers’ characteristics including their network and/or sensitivity analysis on the instantiation of market parameters, attitude distribution, and pig farmer characteristics including their network can give more insight in the effects of different initial situations on diffusion of added-value markets. In addition, it would be interesting to gain more insight in social influence mechanisms among consumers that affect demand of organic meat, given a static price, through e.g. agent-based modelling. Currently it is challenging to find out how empirical and sociological knowledge can be brought to bear upon policy advice. In this research we used the social identity theory to gain better insight in the mechanisms behind social influence. As discussed above, this gives a good starting point for operationalizing social influence and in some parametrization scenarios similarities can be found in model outputs and trends in the pig farmer population. In others, contradictions still exist . Agentbased modelling serves as a good method to further explore how social identity theory affects decision-making and macro patterns, by identifying reference groups, status symbols per reference groups, and changes in reference groups. Specific model operationalisations should, however, still be explored further, such as the value for thresholds, and the effect of only positive social influence versus positive and negative social influence. If policy makers aim to promote alternative farming strategies, they have several policy instruments at their disposition.

There are legal instruments for labelling and certification. There are financial instruments, such as payments for conversion and continued organic production, investment grants, and biodiversity offsets to penalise intensive farming practices through taxes. Finally, there are communicative instruments that focus on changing social norms in society . Since supply is in reality is largely regulated by an organic slaughterhouse, and consumer prices are kept stable by supermarkets, the level of demand for organic meat cannot be addressed via consumer price, and the price elasticity of demand cannot be exploited. This is a constraint on diffusion of organic farming, since our results suggest that price elasticity of demand is an important mechanism to increase the trend in demand to stimulate diffusion of organic farming. Given the importance of the trend in demand for organic pork meat, two alternative financial policy instruments can be suggested. Both need further research to support the policy instrument that best fits organic farming diffusion. First, structural payments to organic pig farmers can decrease the cost price for organic pigs. A decrease in production costs should lead to a decrease in farm gate price, which in turn might lead to a decrease in consumer price. Given the high price elasticity of demand for organic pork meat , a lower consumer price for organic pork should lead to a high increase in demand for organic pork. Increase in demand then leads to increase in supply.If we assume that conventional pork consumers can be triggered by price changes to start consuming organic pork,biodiversity offsets to penalise conventional pork can equalize prices between conventional pork and organic pork. This does require more empirical research into cross-price elasticities of organic meat in comparison to conventional meat. Also, two communicative policy instruments can be interesting given the currently regulated market. First, communicative policy instruments can be developed that target consumer demand by, e.g., explaining the benefits of organic farming to consumers as to try and change social norms among consumers in favour of organic pork instead of conventional pork . The second direction for policy is to focus on new entrants, as this might contribute to diversity in farming styles in the farmer population and therewith diffusion of alternatives.

Communicative policy instruments can focus on improving the image of farming within society for the younger generation. Financial policy instruments can focus on easing the entrance of young farmers into the farmer population. Both are interesting directions to further explore. The European Green Deal includes an action plan aimed at promoting the efficient use of resources by moving to a clean and circular economy and aimed at restoring biodiversity and reducing pollution. The European Commission has developed guidelines to support the achievement of these objectives by “investing in environmentally friendly technologies” and by “supporting industry in innovation” . Precision farming has the potential to meet the challenges posed by the public ambition to produce more while consuming fewer resources . PF has been defined as “a concept of agricultural management based on observation, measurement and response to inter- and intra-field variability in crops or livestock aspects” . Not only are European institutions supporting it as a fundamental practice for the development of agricultural sustainability in the future, but they are also focusing on the political need to act to improve farming practices, for which it is necessary to “boost investments and uptake of new technologies and digital-based opportunities such as precision agriculture” . Studies have addressed the crucial role that data management will play in making farmers’ work more efficient through the use of combined technologies , remote sensors, smart tractors and operational focuses . In addition to these strictly technical factors, socio-ethical and environmental variables linked to the application of technological innovation , in terms of good or negative impacts on agriculture in the future, should be considered . Today, new responsibilities have been assigned to agri-food systems along with the challenge posed to innovation processes to produce a desired scenario, especially in environmental and socio-ethical terms for the agriculture of the future . In this sense, the need to boost a more responsible adoption of innovation paths is emphasised within the field of responsible research and innovation . Only a few studies have examined the extent to which RRI principles have been applied to PF . In particular, the development of a framework of responsible innovation in agriculture requires the study of 4 dimensions : anticipation, inclusion, reflexivity, and responsiveness. Anticipation is related to the ability of research and innovation policy to anticipate an unfavourable scenario in terms of potential environmental and socioethical risks, while reflexivity has been defined as the promotion of new reflection processes around innovation processes by including all prospective actors to reduce negative impacts while enhancing positive outcomes.

Furthermore, the inclusion of stakeholders and bottom-up governance strategies have been proposed as principles for promoting engagement in innovation processes and innovation trust. The last principle, responsiveness, includes the ability to support a change of course within innovation processes once new knowledge, emerging challenges or needs have been discovered. The RRI literature has risen to prominence in relation to numerous technologies that have high potential but considerable uncertainty. However, applications to precision farming technologies are limited . Analysing the desirability of PF through the RRI lens may be very useful since scholars have highlight the notable difficulty in the transfer and adoption of TI. As a demonstration, many authors have described several barriers to adoption, from the costs incurred to the difficulty of use , which can be fully captured by the concept that Vecchio et al. defined as the “complexity” perceived by farmers. Starting from these considerations, the aim of this study is to understand farmers’ perspective on the theme. Specifically, “In my opinion, precision farming is…” is the relevant issue from which this research starts. To answer this question, it is necessary to emphasise that the optimisation of production processes involves many dimensions of farms, such as the technological, economic, institutional, and behavioural dimensions . These aspects can be interpreted as pieces of a conceptual puzzle, vertical rack system of which some have been widely investigated, while others have not. In the field of research on agricultural innovation, the aspects involved in the sphere of the self of the farmer, that is, “a set of behavioural aspects such as motivation, emotion, relationships, perception and cognition” , have been less explored. Only a few authors have explored this dimension, mainly addressing the theme of perception . These studies have described perception as a functional variable in the adoption process, defining the main links with other pieces of the puzzle. In the complex adoption framework, our aim is precisely to colour the picture of farmers’ sphere of self and, more specifically, farmers’ perceptions. This study, however, intends to attribute to perception a character of exceptionality due to its subjective nature as an element belonging to the cognitive sphere. For this reason, this work proposes to study the adoption process by proposing a theoretical framework in which perception is isolated and analysed on a different level than that of the other pieces of the puzzle. Such analysis enables us to capture the views of farmers by overcoming the inevitable bias caused by the design of surveys aimed at establishing functional links . To that end, our survey focuses exclusively on farmers’ perceived meaning of the term “precision farming”. Furthermore, we accept the relevance of subjectivity in filling the content of the sphere of the self. Going into the cognitive sphere in the context of the self means carrying out an in-depth investigation. As with any survey and methodology chosen, it involves having biases but at the same time being able to collect very detailed information that is difficult to capture with quantitative techniques and tools. Although the results obtained cannot be generalised, they represent important insights and enrich the knowledge of the subject under analysis. Consistent with these aims, we use the Q methodology , theorised by Stephenson in 1935 and defined by Brown as a “bridge”. According to Durning and Ellis et al. , the QM is useful for analysing the transition between positivism and post-positivism aimed at the study of subjectivity. It mixes qualitative approaches, which are necessary to capture the multiple facets of subjectivity that escape numerical reduction, with quantitative techniques that help to measure results.

The models of the use case ‘Added value weeding data’ are described in more details as an illustrative example. Table 4 provides an overview of the applied control models. Point of departure in all use cases is a particular farm crop or animal, i.e. potato, tomato, lettuce, cow or pig. These objects are nested in high-level objects, such as fields, greenhouses and stables. The plant use cases all also predict the expected output of the farming process, i.e. potatoes, tomatoes or lettuce to be harvested. Two use cases also virtualize equipment used, i.e. a weeding machine or truck. All use cases analysed combine multiple types of Digital Twins, starting with monitoring the actual state of objects and then predicting future states e.g. expected yields or animal health. Most of the use cases also include intelligence to advice interventions. The crop farming use cases also process these advices into prescriptive Digital Twins, e.g. by defining task maps. Most use cases focus on the usage phase of a lifecycle and do not include imaginary or recollection Digital Twins. Only the use case ‘Added value weeding data’ applies a recollection Digital Twin for optimisation of machine settings based on historical data about machine behaviour. None of the use cases have implemented yet autonomous Digital Twins. Table 5 lists the main technical components that are used to implement the layers of the Digital Twin technical architecture. It shows that in the Device Layer all use cases apply domain-specific sensors and three use cases also use specific actuators. Most technologies for technical communication are based on standardized protocols of both conventional technology such as wired networks and recent wireless IoT networks such as LoraNet. The use case ‘Happy cow’ has chosen to apply a custom-built network consisting of distributed access points that enable communication up to several kilometers. Also in the IoT Service layer a combination of technologies is used.

Process-based orchestration of services is not yet addressed. Only the first use case ‘Within-field management zoning’ includes some Modelling Services. In the Digital Twin Management Layer all use cases provide services that combine and store data from diverse data sources and represent harmonized virtual entities. These services also include intelligence for simulation or decision support dependent on the supported control functions . In the application layer,hydroponic bucket all use cases provide dedicated dashboards for the interaction with users and three of the use cases also integrate with existing farm management systems . Finally, all use cases comprise some generic technical functions for the service organisation, security and management. When growing organic vegetables, weeding is one of the most important and frequent activities to control both the quality of the field and its produce . In recent years, automated intra-row weeding machines have entered the market, enhancing the weeding process significantly. The most advanced weeding machines use machine vision applications to distinguish crops from weeds. These camera data can not only be used for automated control of the weeding task, but also as a valuable information source for farm management. This use case uses these location-specific camera data of a weeding machine as a main data source to provide actual insights into the number of lettuce heads growing on the field, the plants’ growth status, weed prevalence and best harvesting moment. As such it creates Digital Twins of a field, plants and weeds to monitor crop growth and to predict the crop weight and size of lettuce. The applied control model of the use case ‘Added value weeding data’ is shown in Fig. 10. The main farming processes are sourcing and planting young lettuce plants, producing lettuce in the field, harvesting lettuce which is ready for consumption and delivering it to the market. The main physical objects involved are planting machines and young plants, fields containing weeds and growing lettuce, weeding machines, harvesting machines and harvested lettuces. The Digital Twins of this use case are used for monitoring weed pressure and crop growth, controlling the weeds to be removed and predicting the optimal moment of harvesting. To do so, the sensor function uses processed camera images to calculate crop parameters such as size.

Furthermore, crop growth sensing adds weather data and field properties, including temperature, relative humidity, wind speed and direction, solar radiation and soil moisture . The data acquisition function also includes external weather data. These data are then transformed into Digital Twins. The virtualisation in this use focusses on the field, which implies that the main Digital Twin is a high-precision and actual heat map of a field. A field map comprises weed density and the number and size of crops , and the expected final weight and crop size of the lettuce . Planting seedlings are excluded. The Digital Twins of the individual lettuce crops and weeds are used by farmers during the weeding activity and afterwards the calculated parameters of every plant in the field are also available remotely. Furthermore, Digital Twins of the weeding machine is used to optimize machine settings afterwards . The discriminator function uses the Digital Twins of weeds and growing lettuces to monitor weed pressure and crop growth, i.e. crop size and crop distance. The decision maker function translates the weed pressure into a planning of the weeding activities.The user sets a target value for crop weight and then the optimal harvest moment is determined. Based on this information the optimal moment of harvesting is determined and the harvesting is planned. For lettuce, growers get paid by lettuce head in the right weight class. Finally, the effector function executes the planned weeding task. The weeds are automatically removed, controlled by the actuators in the weeding machine that apply machine instructions based on Digital Twin of the weeds. Because of the high-precision weed density maps, fields can be weeded partially, only where needed. Also the planned harvesting activities are executed by harvesting machines but they do not use customised machine instructions. The control cycle partly takes place on-site within the weeding machine. Camera data are directly processed into local Digital Twins that distinguish crops and weeds. These Digital Twins are then instantly translated to actuator instructions and the weeds are removed without human involvement. However, all other control activities are done remotely by farmers who interact with the Digital Twins via cloud-based systems.

The next section elaborates on how this is technically implemented. Digital Twins can be seen as a new phase in smart farming. Using Digital Twins as central means for farm management enables the decoupling of physical flows from its planning and control. As a consequence, farmers can manage operations remotely based on real-time digital information instead of having to rely on direct observation and manual tasks on-site. This allows them to act immediately in case of deviations and to simulate the effect of interventions based on real-life data. The main contribution of the paper is that it has proposed a conceptual framework for designing and implementing Digital Twins for smart farming. The framework builds on an analysis of literature and a clarification of the concept of Digital Twins, which is still developing. An important novelty of the framework is that it adds a typology of Digital Twins based on the life cycle phases of the objects being virtualised. Depending on the perspective, the emphasis is currently often on monitoring or predictive Digital Twins. However, Digital Twins can already be created in the design phase of a life cycle and support the creation of its physical, real-life sibling. During operational usage, Digital Twins can not only be used to monitor and simulate the effects of interventions, but also to remotely control an object by using actuators. Finally, Digital Twins are also very valuable after disposal of a physical object e.g. for traceability, compliance and learning. So far, these distinct Digital Twin types are not explicitly addressed in the literature, which results in conceptual confusion. This paper has contributed to avoid this by introducing a typology and by defining the distinct control capabilities of each type in a control model. The case studies show that there are already applications in the agricultural domain that are not framed as Digital Twins. This is not surprising, since Digital Twins are building upon existing technologies especially for precision farming, internet of things and simulation. However, especially more advanced applications, including e.g. predictive and prescriptive capabilities across the lifecycle, are still in an early stage of development. The designed framework was useful to explicitly describe and analyze how Digital Twins are used in practice. As such, it has provided a new perspective on the cases that originally focused on the innovative application of Internet of Things technologies to farming. It also showed the value of not yet applied Digital Twin types, which inspired the use cases about potential redesign scenarios.

For this reason, we expect that applying the Digital Twin concept, as described in our framework, can accelerate the development and adoption of Digital Twin solutions for smart farming. However, future research is needed to provide evidence for this hypothesis. Furthermore, the implementation model of our framework only deals with implementing the enabling information technology. We did not take into account organisational and behavioural issues, such as the impact on supply chain collaboration, data ownership and governance, stackable planters the potential emergence of disruptive business models based on Digital Twins, ethical considerations, and so forth. We would like to encourage researchers in these disciplines to also study Digital Twins, since these non-technical issues might be decisive for the success of Digital Twins. Our intended follow-up work is related to the further development of the framework. In particular, we plan to elaborate the conceptual framework into an information architecture framework, which will comprise a consistent set of architectural viewpoints for modelling Digital Twin-based software systems . This architectural framework will be the basis for developing Digital Twin applications that cover the entire life cycle. Many farming systems in Europe are struggling with substantial challenges resulting from fundamental changes in their economic, technological, demographic, ecological and social environment . The resilience of farming systems, i.e. their ability to cope with and respond to shocks and stresses, has therefore become a major concern . The Covid-19 pandemic and the measures for its containment – e.g. lockdowns, travel restrictions and border closures – were expected to add another shock to farming systems. Using 11 indepth case studies, this paper investigates the extent to which different farming systems across Europe were affected by the crisis, which resilience strategies they adopted, and which characteristics enabled or constrained their resilience abilities. This paper contributes to a fast-growing literature on impacts of the Covid-19 pandemic on different parts of agricultural and food systems, e.g. food value chains, marketing channels, trade patterns and food security . Impacts on different farming sectors, e.g. due to production and demand distortions, have also been discussed . Others have reflected on the resilience of food systems at large in the light of Covid-19 . However, a systematic assessment how characteristics of farming systems have enabled or constrained their responses to the Covid-19 crisis is missing. By using an elaborate framework to assess and compare the resilience of farming systems before and during the pandemic, this paper aims to enhance our understanding how different farming systems were exposed to the crisis, which resilience capacities were revealed and how resilience was enabled or constrained by the farming systems’ social and institutional environment. Section 2 explains the SURE-Farm framework to assess the resilience of farming systems and the special data collection on Covid-19. Results are presented in Section 3, followed by discussion and conclusions in Section 4. Following the social-ecological tradition of resilience thinking , we define the resilience of a farming system as its ability to ensure the provision of its desired functions in the face of often complex and accumulating economic, social, environmental and institutional shocks and stresses, through anticipating, coping and responsive capacities . The resilience of a farming system is affected by specific system characteristics, and by the enabling or constraining environment, in particular institutional arrangements and resource availability .

The physiological response of the common reed plants to As toxicity and its possible relationships with the growth parameters and nutrient composition of the plants were evaluated through a PCA. The analysis resulted in a total of eight components, of which the first four explained 75.5% of the variance. The first component  related positively the photosynthetic pigments levels in the leaves with plant yield,height, and P mass fraction,as well as with oxidative stress parameters in the roots  and in the aerial part of the plants. This component therefore shows that the growth and photosynthetic system of the common reed plants were not affected by the increasing presence of As in the nutrient solution, likely as a consequence of the formation of those compounds that acted as an efficient defense mechanism against oxidative stress. The second component  related the different As species determined in the plants positively with each other, and negatively with Cu and Zn in the aerial part of the plants and with P and K in the roots. This confirms that As accumulation did not affect the plant’s photosynthetic and oxidative status, but may have limited macro and micro-nutrients uptake in the roots and their transport to the aerial part of the plants. The third component  simply related the values of Cu, Fe, Mn, and Zn in the roots,while the fourth component  related the levels of Mn and Zn in the aerial part of the plants negatively with those of MDA and proline in the aerial part and with that of N in the roots. The latter component seems to relate the deficiency of certain nutrients, likely a consequence of As uptake and accumulation in the plants,with the formation of MDA and proline in the plants, which is also indicative of As toxicity. 

When the regression factors generated in the PCA were plotted,the highest As dose  was clearly differentiated from the rest of the treatments, linked to a high accumulation of As in the roots. The control replicates grouped together opposite those of treatment T10, associated with the levels of nutrients in the plants,hydroponic nft system while the rest of the treatments showed an elevated variability and no clear or consistent relationships with any of the parameters determined in the plants.Malachite Green,a triphenylmethane dye, is a multipleuse compound that is mainly used in textile industries and partly used in aquaculture in fungicides and ectoparasiticides. While the effects of MG on aquatic invertebrates and algae have been scarcely elucidated,Hidayah et al.  reported that MG in wastewater from either industry or aquaculture has been widely reported to be toxic to many kinds of fish with lethal effects at a concentration of less than 1 mg/L, with the dye and its derivatives being accumulated in aquaculture products such as fish, prawn and crab. It also possesses carcinogenic and genotoxic properties which pose a potential risk to humans and therefore, this dye has been banned in Europe, the USA and several countries. However, MG is still being used in some parts of the world because it is highly effective and easily available at low cost. It is also used domestically as a treatment for diseases of tropical fish and can be readily obtained by the public ; hence, concern about its illegal use exists. In Asian countries such as Bangladesh, MG has been reported to be used for the eradication of external parasites and fungal diseases in fish farming. However, removal of MG from aquaculture wastewater has received little or no attention compared to other pollutants. Consequently, contamination of MG in aquaculture waste could be expected with harmful consequences to the surrounding environment. Effluents from aquaculture usually contain high amounts of nutrients such as nitrogen ,phosphorus and organic compounds that either potentially cause algal bloom in receiving water  or, if high enough, can support vegetable production. 

To reduce water pollution problems, fishery industries in many countries including Thailand have been forced to treat their effluent in proper ways such as by the rational use of water and by the recovery of substances from wastewater. Hence, effluents from aquaculture have been used for garden applications or the production of hydroponic plants as a secondary treatment in the waste management procedure. In some management practices, such as the study by Somboonchai and Chaibu,four vegetables were grown in a hydroponic system integrated with catfish culture. However, the effluents from aquaculture such as shrimp farming contain not only nutrients but also other chemical substances such as antibiotics, herbicides and fungicides that potentially impact on the environment. A review by Carvalho et al.  indicated that pharmaceutical products, including antibiotics, hormones, analgesics and anti-inflammatory drugs, chemical compounds used for disinfection and cleaning, and endocrinedisrupting compounds can be assimilated by the plants. Therefore, while the potential for biomass production and nutrients recovery from wastewater are primary concerns in wastewater management systems,bioaccumulation of toxic substances is another aspect of concern. Additionally, increasing water scarcity in either dry regions of the world or in developing countries makes the reuse of wastewater in agriculture more important. Nevertheless, it is of interest to identify whether or not a practice is productive and safe for both the environment and human health. Several plant species can tolerate toxic substances by accumulating them in non-toxic forms or transforming them to either nontoxic or less toxic products. Most studies showed that textile dyes can be either adsorbed and accumulated or transformed to less or non-toxic substances by detoxifying enzymes, predominantly peroxidase, in plant cells. The dye MG was found to be transformed to 4-dimethylamino-cyclohexa2,4 dienone in Blumea malcolmii Hook. using enzyme laccase and the products had less toxicity toward Phaseolous mungo and Triticum aestivum when tested. Rai et al.  found that biodegradation of MG by Aloe barbadensis resulted in nontoxic metabolites, suggesting the possibility of using treated, dye wastewater for irrigation. Torbati  reported that activity of antioxidative enzymes, namely SOD, POD and CAT, in Spirodela polyrhiza L. was increased with increased MG in the bathing medium.

The activity of these enzymes allowed the species to tolerate MG at concentrations of 10 mg/L and 20 mg/L. However, knowledge on the degradation of synthetic dyes by vegetable plants is scarce since phytotransformation has been studied mainly in non-edible plants. Nowadays, the trend toward eco-friendly and sustainable production of any kind of product strongly influences consumers. The current study investigated the application of wastewater containing MG from aquaculture for the production of pak choy,a vegetable that is produced commercially in many Asian countries. It was hypothesized that being a member of the genus Brassica whose species usually have high antioxidant enzyme activity upon exposure to toxic substances,B. chinensis may have ability to degrade MG dye and, hence, tolerate the dye at the low concentration used in aquaculture. If so, the reuse of water contaminated with MG could be applied. However, some Brassica species such as cabbage  and Wisconsin fast plants  could take up and accumulate some toxic substances in their tissue, especially in the roots. Therefore, on the other hand, the MG dye in water may be accumulated in plant tissue and inhibit growth of the plant. Thus, the aims of this study were: 1) to study the effects of MG on the growth of B. chinensis and 2) to evaluate the accumulation of toxic substances in the edible parts of B. chinensis. The findings from this study will be useful for consideration in a wastewater management strategy, particularly for the reuse of aquaculture wastewater in crop irrigation.When the seedlings were age 7 d, the nutrient mixed solution was applied replacing water, and the seedlings were allowed to grow under ambient conditions to age 14 d before being transferred to the growth medium used in the growth experiment. Fourteen-day-old seedlings with 3e5 leaves and an average height of 8 cm were selected for the growth experiment. The seedlings were grown in nutrient mixed solution for 1 wk to allow for acclimation to the hydroponic growth conditions. The nutrient mixed solution was prepared from tap water and 1 ml/L of commercial A and B nutrient solution for hydroponic planting. The pH and electrical conductivity  of the nutrient mixed solution were monitored and maintained at 6.0e6.5 and 1.5e2.5 ms/cm, respectively.After 1 wk acclimation, 48 seedlings were distributed to four levels of MG concentration treatments: 0 mg/L,1 mg/L, 2 mg/L and 4 mg/L. The basal part of each seedling was fitted in a small plastic basket to hold the plant in an upright position and the baskets were fixed on the lids of 5 L plastic tub containers. One container with 12 seedlings was used for each treatment. The chemical formula of the MG used was C23H25N2Cl. The concentration of each treatment was obtained by adding the appropriate volume to make up 500 mg/L of MG stock solution to the nutrient mixed solution which hereafter is called the growth solution.

The pH and EC of the growth solution were monitored and maintained as mentioned above and the growth solutions were renewed weekly. The experiment was maintained under ambient conditions with the air temperature 24e29 C,nft channel relative humidity 41e60% and natural sunlight. Decolorization of MG in the growth solution at day 7 was detected spectrophotometrically using an ultravioletevisible spectrophotometer. The solution from each treatment  was sampled and measured for absorbance at 400e800 nm compared with the absorbance of freshly prepared solution at the same concentration. After 4 wk of growing, growth parameters  were measured. Then, all plants were harvested and each plant was separated into root and shoot  parts, and the shoots were stored at 70 C in a freezer for further tissue analysis. The roots were abandoned since it is normally a non-used part of this vegetable and it was impossible to separate the plant roots from the supporting sponge. The weight of the shoot was measured after drying in a hot-air oven at 60 C for 48 h and the final dry mass was determined. The experiment was conducted between February and March.The effects of MG on plant species have been mostly tested using seed germination and the plant seedling stage, with germination and seedling development being generally inhibited. The current study found that MG contamination in water also caused negative effects to B. chinensis, particularly at concentrations greater than 1 mg/L. From the results, the negative effects of MG were strongly evidenced on root growth which was reduced by 50% upon exposure to MG of 2 mg/L and 4 mg/L compared to the control or 1 mg/L MG treatments. Similar effects on root growth were found in Arabidopsis thaliana grown on medium supplied with 4 mg/L of Crystal Violet and MG. The stunted roots may contribute to the overall reduction in plant growth since the uptake of water and nutrients could occur only via root transport under the hydroponic growth conditions used in this study. Nevertheless, the overall growth of B. chinensis in the current study indicated that the plant tolerates MG at a concentration of 1 mg/L. MG has been suggested to be toxic to plants as it could be strongly absorbed on the surface of cellulose  and taken up through the roots and accumulated in plant tissues. Saranya et al.  found that the chlorophyll contents in Hydrilla verticillata decreased with increasing Basic Violet 14 dye concentrations from 5 mg/L to 25 mg/ L, although the difference was not significant at 5 mg/L and 10 mg/L, and this result supported the inhibition of the dye on chlorophyll biosynthesis. In the current study, there was no evidence of chlorosis in the B. chinensis leaves at the concentrations applied. However, the effect of MG on chlorophyll biosynthesis in this plant species should be better explained by pigment analysis. Dye contamination in either the water or soil usually causes a reduction in the total content of macromolecules such as proteins and carbohydrates whilst it usually induces the activity of several enzymes used for dye degradation in exposed organisms. Triphenylmethane dyes such as Crystal Violet and Methyl Violet could cause lower protein synthesis which consequently inhibited cell growth in Bacillus subtilis. Jayanthy et al.  found that soil contaminated with dyes from dyeing industries caused decreasing protein, total free amino acid and carbohydrate contents in Vigna radiata, whilst in the same plant, there were increases in the proline, glutathione and methyl glyoxal contents in either leaf or root tissue which indicated a response to abiotic stress. Moreover, the activities of lignin peroxidase, veratryl alcohol oxidase, laccase, tyrosinase and DCIP reductase were induced in Aster amellus Linn. and Glandularia pulchella  Tronc. upon exposure to the dye Remazol Orange 3R. 

In the marine environment, they have been reported in coastal waters, surface waters, the water column, deep-sea sediments, estuaries and fjords , including in northern waters . MPs can have a wide range of negative effects on biota, from reduced feeding to immune system alterations . Depending on size and shape, MPs can cross body barriers, e.g. be translocated from the digestive tract to other tissues and organs, and be taken up into cells . MPs have been detected in liver and muscle tissue of both farmed and wild salmonids . Plastic microfibers have also been detected in several organs from blue mussels exposed to concentrations of 2000 particles/L, including mantle and foot . Accumulation of MPs in edible tissues might in turn constitute a health risk for human consumers . Negative effects of MPs on organisms may derive from plastic additives as well as the plastic polymer particles themselves . Additionally, persistent organic pollutants can sorb to MPs due the hydrophobic nature these chemicals, and act as vectors for POPs in the marine environment . For example, several studies have documented sorption of PCBs, PAHs, pesticides and other POPs, on MPs collected from marine environments . POPs sorbed to MPs may be transferred to the organisms after ingestion, increasing and complicating the impact of such particles and pollutants . The role of MPs as vectors of pollutants to organisms has been suggested to be irrelevant compared to dietary or environmental exposure. In addition, MPs could pass through the gastrointestinal tract without leaching chemicals . Nevertheless, translocation of small polluted MPs into cells, tissues and organs of organisms only exposed to low levels of POPs could have a significant impact in such individuals. In such cases, MPs might be a pathway for uptake and bioaccumulation of POPs in marine organisms . Dioxins are chemical substances formed as byproduct during combustion reactions in the presence of chlorine .

In vertebrates, dioxins are carcinogenic and can disrupt the immune, nervous and endocrine systems, and affect reproduction and development . Considering the increasing numbers of wildfires as result of climate change ,hydroponic grow table and that dioxins tend to accumulate in polar regions due to slower degradation in cold areas and the grasshopper effect , more attention should be paid to the impact of dioxins in the environment together with MPs. To our knowledge, there is no record of levels of dioxins adhered to MPs in the environment except for the levels found in charred MPs . Therefore, interaction of MPs with certain POPs such as dioxins should be studied more in depth. Atlantic salmon is one of the most consumed fish species in the world and the commercially most important farmed marine fish species . The levels of pollutants associated with Atlantic salmon farming have long been a concern. Salmon feed contains about 10% of fish oil , which is the main source of POPs to farmed salmon. Analysis of 20 salmon feed samples randomly collected from salmon farms in Norway in 2017 showed concentrations of polychlorinated biphenyls of 3 μg/kg, sum of dioxins and dioxin-like PCBs of 0.6 ng TEQ/kg, and concentrations of polybrominated diphenyl ethers of 0.38 μg/kg . In addition, fish feed also contained traces of organochlorine pesticides such as DDT , toxaphene and endosulfan, and organophosphorus pesticides such as chlorpyrifosmethyl and pirimiphos-methyl. In recent years, replacement of fish oils with plant oils has led to reduced levels of POPs in farmed Atlantic salmon in Norway . However, the use of plant ingredients in aqua feeds has increased the levels of other organic pollutants in fish feed, such as PAHs and pesticides . In Norway, about 1.3 million metric tons of Atlantic salmon is produced annually . For each kg of salmon produced, about 0.5 kg of feces and unconsumed feed pellets are generated . This waste slips through the open-cage net pens and spreads in the environment, depending on local physical, chemical and biological factors. Open fish farms thus represent local point-sources of pollution. Yet little is known about how MPs might contribute to the spreading of POPs near fish farms. Fish farms benefit from the lightweight, strong and flexible plastic in permanent installations. Net pens, ropes, floats and pontoons are some of the framework structure made of plastic polymers such as HDPE, PP, PET or PVC . Abrasion and loss of plastic items inevitably lead to the release of MPs into the environment. A recent study showed the presence of MP polymers such as PP, PE, PVC and PET in sediment, seawater and suspended matter around Atlantic salmon farms of Norway .

In Norway, about 1000 commercial salmonid farm facilities are placed in the marine environment . These are estimated to use a total of 191,799 tons of plastic materials, of which 35,571 tons are nets , 17,201 tons are mooring ropes , 4440 tons are feeding pipes, and 108,405 tons are cage floaters, handrails and walkways . Based on the degradation rates of PE , nylon and PP ropes in the marine environment calculated by Welden and Cowie , the total amount of nets and ropes deployed by fish farms in Norway are estimated to release 3137 tons of MPs/year and 805 tons of MPs/year into the environment, respectively. Feeding pipes used in salmon farming have recently been shown to release an average of 0.25 g/m/day MPs along with the fish feed . Based on this study, feeding pipes used in Norwegian aquaculture are estimated to release 225 tons of MPs/year in the water. In addition, cage floaters are estimated to release 5.4 tons of MPs/year based on the assumption that plastic degrades at an annual rate of 0.5% in the Norwegian marine environment . Based on all the above, fish farming in Norway might realease more than 4172.4 tons of MPs annually into the marine environment. Thus, understanding the potential role of MPs as vectors of pollutants from fish farming is important for countries such as Norway. The objective of this study was to evaluate whether MPs can sorb POPs released from fish feed significantly, and consequently act as potential vectors for these chemicals to the surrounding environment. As a step towards better understanding the impact of different MPs on the environment, we assessed the capacity of HDPE, PP, PET and PVC to sorb POPs. For this purpose, MPs of four different types of polymers commonly found in the environment and detected in the vicinity of salmon farming facilities were placed close to two salmon farms for three months. For comparison, MPs were additionally deployed in two sites not influenced by salmon farming; one low-polluted and one polluted location. Furthermore, a positive control for fish feed pollutants was set up under laboratory conditions. POPs sorbed to MPs were then qualitatively and quantitatively analysed. Blue mussels were collected from a reference site and placed next to the MPs at all stations, with the objective of documenting the pattern of POPs in the environment. This filter-feeder is often used in water monitoring studies as a bioindicator of water pollution . Recently, mussels have also been suggested as bioindicator for marine MP pollution, since they are widely distributed and have been shown to indicate differences between sites with large differences in MP concentrations . Blue mussels are currently used in coastal environmental monitoring programs in Norway as bioindicators for heavy metals, organic pollutants and MPs .

Two replicates of each of the PP, HDPE, PET and uPVC polymers incubated at each site were used for chemical analysis. Frozen samples were treated gently to remove the biofilm on the MPs through an enzymatically and strong alkali driven cleaning process. Each sample was sequentially incubated at room temperature with a mixture 1:20 of Celluclast and 1 mL Viscozyme enzymes in 0.1 M PBS at pH 6.0 for 24 h and with 10% KOH for 6 h at RT. Plastic beads were gently flushed and rinsed with millipore water through a vacuum system to remove the degraded biofilm and were allowed to dry at room temperature for 1 h. Samples were then sonicated three times for 20 min with 5 mL of dichloromethane, HPLC grade, and the supernatant was collected after each sonication step. Extracts were pre-concentrated by a Rotavapor system and analysed using the following HRGC/HRMS methods: USEPA Method 1668B, USEPA Method 8290A and USEPA Method 1614A for polychlorinated biphenyls, dioxins and brominated flame retardants, respectively, using a GC–MS/MS ; and the following GC-ECD method for chlorinates pesticides: USEPA Method 508.1 using a GC–MS . Isotope labelled PCB mixture 77/101/141/178; 1,2,3,4,6,7,8- heptabromodibezofuran and Aldrin 13C12 were added as internal standards. The limit of quantification was set to three times the value of limit of detection .For the harbour, the two fish farms and the positive control, the levels of the majority of pesticides and half of the novel brominated flame retardants analysed were also below the detection limit in all polymer types. Hence, only data for dioxins and PCBs were used in the PERMANOVA analyses. The concentrations of all studied POPs sorbed onto the four MP polymer types are shown in Supplementary Table S7. The levels of POPs sorbed to the MPs were quantitatively assessed to understand whether MPs can bind POPs associated with fish farming. PERMANOVA was used to compare the composition of POPs sorbed to MPs from the different sites and the positive control . The results of the PERMANOVA analysis showed that the composition of POPs in MPs from the harbour was statistically different from the composition of POPs in MPs from FF1 and FF2,flood tray and the positive control, . However, there were no significant differences in the composition of POPs sorbed to MPs from FF1, FF2 and the positive control. To evaluate whether binding affinities of POPs differed among the studied MP polymers, a Kruskal-Wallis test was done for each dioxin congener and each PCB Aroclor . Since this study focuses on the sorption of POPs from fish farming, only data of dioxins and PCBs sorbed to MPs from the two fish farms and the positive control were used. Positive control was included in this analysis because no differences were found in the previous analysis in the composition of POPs of these MPs with the ones of the fish farms. Data of dioxins and PCBs sorbed onto the MPs placed at the harbour and the reference station were excluded because different cocktail of pollutantsmight result in different sorption affinities.

Chemical properties of the analysed dioxins and PCBs are shown in Supplementary Table S8. This study suggests that MPs can sorb POPs associated with Atlantic salmon farming, and documents that such pollutants have polymerspecific binding affinities. Polymer type and pollutant type, in addition to background pollution in the water, are therefore determining factors that should be considered when assessing the potential role of MPs as vectors of pollutants from aquaculture in the marine environment. In this study, four types of MP polymers were either incubated for three days with fish feed or placed in the sea for three months next to two marine fish farms, in an urban harbour and in a non-polluted fjord. MPs collected from the two fish farms, the harbour and the lab control sorbed dioxins, PCBs and some brominated flame retardants , whereas pesticides were barely detected. In contrast, MPs placed in the non-polluted fjord did not have quantifiable levels of any of the analysed POPs. It is well known that MPs can sorb pollutants in the environment. At the beginning of this century, plastic pellets sampled in waters from all five continents were reported to be polluted with PCBs and pesticides . Since then, several studies have reported the presence of POPs bound to MPs in the marine environment . The main mechanisms by which MPs sorb POPs include hydrophobic and electrostatic interactions, although multiple mechanisms are often cooccurring in complex natural environments. Chemicals characterised by high hydrophobicity, which are defined by high octanol/water partition coefficients , are expected to be more easily sorbed by plastic particles .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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