12 Nature-based solutions have been proven in recent decades as a reliable and cost-effective technology for the 13 treatment of wastewater. They are widely used in several countries, mainly as secondary or tertiary biological 14 treatment. Such systems rely on the ability of photosynthetic organisms to assimilate and remove, to a certain 15 extent, nutrients valuable for their own growth. Different plant species have been studied for this purpose, but 16 particular attention has been given to duckweeds, the smallest flowering plant in the world. These plants have been 17 proven to be highly efficient for wastewater treatment given their rapid growth, natural abundance among 18 macrophytes, and the quality of the biomass produced. However, despite being considered a seemingly simple 19 technology, the performance of treatment systems based on duckweed is dependent on environmental and 20 operational conditions not very well understood. While there have been many studies on growth of duckweed for 21 wastewater treatment, the difference in species, systems, variables, scales and reporting units make it very difficult 22 to draw comparisons across studies. This study employs a systematic review approach to conduct a meta-analysis 23 of the effect of temperature, light, and nutrient availability on duckweed growth by means of standardized IQ-scores. 24 The analysis of the results considered the duckweed species being used and the interaction between these 25 parameters. The results suggest that daily light integral (DLI) is a useful parameter to assess the overall effect of 26 light (photoperiod and intensity) on duckweed growth and that the effect of nitrogen and phosphorus supply should 27 consider the nitrogen species available for plant growth and its ratio to phosphorus concentrations. By establishing 28 the optimal range of culture conditions for duckweed , this study provides important insights for optimizing 29 wastewater treatment systems that rely on duckweed for nutrient control and recovery, which is primarily mediated 30 by duckweed growth. 31 Keywords: Duckweed, Light intensity, Nutrient control, Temperature, Wastewater, meta-analysis 32 Abbreviations: NBS, Nature-based solutions; WWT, Wastewater treatment; RGR, Relative growth rate; TP, total phosphorus; 33 TN, Total nitrogen; DLI, Daily light integral 34 Highlights 35 • Comparing duckweed growth studies in varying experimental conditions is challenging. 36 • A meta-analysis with standardised scores can overcome this limitation. 37 • Different genera have different temperature optima. 38 • The daily light integral is a useful parameter for assessing the impact of light on duckweed growth. 39 2 • There is no clearly defined preference for N source. 40 • The ratio of N:P has important effects on growth rates. 41 42 1. INTRODUCTION 43 Nature-based solutions (NBS) which harness the growth of photosynthetic organisms in 44 wastewater are being thoroughly investigated as a cost-effective method for decentralized wastewater 45 treatment. Among the NBS, treatment systems based on aquatic plants – e.g., macrophytes – have 46 been widely used to remove pollutants from water, as a tool for proper wastewater management and 47 disposal. For more than forty years, these systems have been implemented in Europe and North 48 America for nutrient control and recovery from wastewater at low loading rates – i.e., wastewater 49 treatment units for polishing final effluents (Brix, 1994; Donde et al., 2018). Today, macrophytes are 50 increasingly being used worldwide to treat different types of effluent, including municipal and industrial 51 wastewaters, acid mine drainage, agricultural and livestock wastes, and leachate from landfills, among 52 others. In rural areas and developing countries, macrophyte-based systems play a vital role in the 53 treatment of municipal wastewater from small and decentralised systems, where energy intensive 54 treatment units are not suitable due to technical or economic constrains (Upadhyay et al., 2016). 55 Aquatic macrophytes act as a biological filter, taking up nutrients from polluted waters to support 56 biomass production, while fixing atmospheric carbon dioxide. The great diversity of macrophytes has 57 resulted in a wide variety of systems being used for wastewater treatment, ranging from systems using 58 large aquatic plants like water hyacinth, to very small plants like duckweed. The latter have proven to 59 be efficient in removing nutrients, organic matter, and toxic substances from water. The success of such 60 treatment systems is based on their adaptability and fast-growing capacity. Furthermore, wastewater 61 treatment systems using duckweeds have proven their ability to perform well in both urban and rural 62 settings, and strong environmental credentials due to their low energy consumption and operational 63 costs (Brix, 1994). 64 Duckweeds have been tested for a wide range of wastewater treatment conditions (El-Shafai et al., 65 2004; Hassan & Edwards, 1992). These plants grow very rapidly and remove nutrients at a higher rate 66 than other aquatic macrophytes (Oron et al., 1988). Under optimal growth conditions, including nutrient 67 bioavailability, light intensity and water temperature, they can double their weight every 2 or 3 days 68 (Rusoff et al., 1980). This reproduction rate is greater than that of any other higher plant, resulting in 69 the formation of dense mantles over the surface of water bodies, especially when the concentrations of 70 nitrogen and phosphorous in the water column correspond to mesotrophic/eutrophic environments 71 (Portielje & Roijackers, 1995). 72 Despite the multiple benefits of duckweed-based systems for wastewater treatment, some 73 limitations associated with their engineering design and operation persist. For instance, the efficiency 74 of treatment processes is seasonal, in response to changing environmental conditions and free surface 75 area available to support biomass growth and photosynthesis. These conditions have a direct effect on 76 the ability of duckweed to take up and metabolise nutrients, which ultimately affect the quality of the 77 3 final effluent. For this reason, it is necessary to firstly appraise the performance of duckweed-based 78 systems for wastewater treatment under a range of culture conditions, typical to the corresponding 79 application (i.e., nutrient loading rates, flow rates, retention times, climate conditions, etc.,) 80 As other photosynthetic organisms, duckweeds require a supply of macronutrients (carbon, 81 nitrogen and phosphorus) and trace nutrients to grow. These nutrients are all present in wastewaters, 82 either in mineral or organic form, hence the potential for using wastewater as a medium to support 83 duckweed biomass growth. Apart from the concentration of nutrients in the growth medium (i.e., 84 wastewaters), culture conditions such as temperature, pH, initial mantle density, surface area 85 availability, photoperiod and light intensity, have a significant influence on duckweed growth and nutrient 86 uptake. 87 Moreover, to successfully improve the quality of wastewater effluents, we need to be aware that not 88 all duckweed species are equally effective at taking up nutrients and hence, biomass productivity and 89 composition vary. For that reason, process performance is highly dependent on duckweed strains which 90 may be well or poorly adapted to specific operation and/or environmental conditions (Bergmann et al., 91 2000; Cheng & Stomp, 2009). In this sense, appropriate selection of duckweed strains to work with 92 must be undertaken. 93 Overall, reported outcomes on how environmental and operational conditions impact growth and 94 nutrient uptake by duckweeds are highly variable in published literature. Therefore, it is very difficult to 95 extract meaningful comparisons for such diverse studies which use different duckweed species, 96 different growth media or effluents, different culture setups and controlled or naturally varying 97 photoperiod and temperature. To try to synthesise this information and draw meaningful conclusions a 98 systematic review was undertaken, and a meta-analysis applied to the data from the retrieved 99 publications. This approach has its origins in medical studies where there are often small sample sizes 100 and confounding variables, but the methodology is much more generally applicable (Page et al., 2021). 101 By drawing on many studies, patterns or trends emerge which are not visible in individual studies. 102 This meta-analysis study focuses on establishing the influence that temperature, light and Nitrogen 103 and Phosphorus have on duckweed biomass growth and nutrient uptake, considering tested natural 104 and engineered environments, that will support the importance of selecting suitable duckweed isolates 105 and species for process development studies and engineering applications. By comparing different 106 outcomes under a standardized methodology, it is possible to plan and design more reliable, robust, 107 and resilient duckweed-based systems for wastewater treatment and nutrient recovery. The goal is to 108 offer an integrated analysis of the dynamics involved in nutrient reclamation and biomass production by 109 duckweeds. 110 111 112 113 4 2. METHODS 114 2.1. Literature search 115 The data for the present meta-analysis study was put together from three different peer-116 reviewed literature databases (PubMed, Web of Science and Scopus) following the Prisma guidelines 117 (http://www.prisma-statement.org/) (Figure 1). All scientific articles published prior to June 2021 were 118 retrieved using the advanced search tool from each database. Different keywords and synonyms were 119 grouped into five topics to be searched using the following Boolean operation: TITLE-ABSTRACT-120 KEYWORDS - (growth OR composition) AND (duckweed) AND (nutrient OR reclamation). 121 2.2. Inclusion and exclusion criteria 122 Article titles and abstracts were manually screened to exclude studies not related to the topic. 123 Only studies in wastewater treatment and nutrient recovery using different species of duckweed were 124 included in the analysis. In a further step, relevant articles were examined to determine fit to the eligibility 125 criteria of this review. 126 The exclusion criteria included the following: 127 (1) Toxicological studies using duckweeds: Studies assessing the potential of plants for emerging 128 contaminants remediation or the ecotoxicological effect of pollutants on duckweed growth. These 129 studies were excluded as the use of standard culture conditions for the cultivation of duckweeds 130 was limited to the control experiment. 131 (2) Review papers: Publications collecting and reviewing data from other authors already included 132 within the database or papers presenting the state of art of duckweeds in wastewater treatment. 133 (3) Not enough data of interest: Papers in which either the relative growth rate of plants or the data/plots 134 required for its calculation is not presented. 135 (4) Different research question: Scientific reports whose objective was other than assessing the effect 136 of temperature, light, and nutrient availability on the growth of duckweeds. 137 (5) Non-retrieved papers: Papers that cannot be found using selected databases or without any 138 response from contacted authors. 139 (6) Language: Papers published in a language other than English or without any English translation 140 available. 141 142 5 143 Figure 1. Identification of studies via databases and registers. This PRISMA flow diagram (Moher et al., 2010) 144 shows the literature search results, highlighting the main exclusion criteria used in the screening stage, of peer-145 reviewed papers published in English prior June 2021. 146 147 2.3. Data extraction 148 All data retrieved from the studies included in the review are available in Supplementary 149 Material S1. From each study, the following data was extracted: (1) authors and year of publication, (2) 150 test species, (3) culture conditions, (4) culture media characteristics, and (5) observed response in the 151 treatments. A summary of extracted variables and their respective units is presented in Table 1. 152 Whenever provided, data on the characteristics of duckweed studied, such as genus, species, 153 collection reference number and country of origin, were included. Culture conditions tested in each of 154 the studies were collected and classified either as environmental or simulated culture conditions. When 155 provided, the volume and total surface area of cultivation, initial stocking density, temperature, 156 photoperiod, and light intensity were noted as in the original publication. In some cases, surface area 157 was calculated upon the dimensions of the containers in which the experiments were done. The initial 158 stocking density, or mat density, was calculated as the amount plant material, in fresh or dry basis, per 159 unit of surface area at the beginning of the experiment. Where experiments were conducted under 160 ambient/outdoor culture conditions, and data on temperature, photoperiod and light intensity were not 161 reported, these data were retrieved from the Photovoltaic Geographical Information System from the 162 European Commission (https://re.jrc.ec.europa.eu/pvg_tools/en/) for the location. Normal direct 163 irradiance values were converted to Photosynthetically Active Radiation (PAR) using a conversion 164 factor of 4.6 (Langhans & Tibbitts, 1997). 165 Records identiﬁed from:Databases (n = 3)Registers (n = 661)166 PubMed321 Web of Science194 ScopusRecords screened(n = 431)Records removed before screening:Duplicate records removed (n = 250)Records excluded based on review oftitle/abstract (n = 100)Reports excluded: 240Toxicological studies (n = 85)Review papers (n = 10)No data of interest (n = 109)Diﬀerent research question (n = 28)Papers no retrieved (n = 5)Language other than English (n = 3)Records assessed for eligibility(n = 331)IdentificationScreeningIncludedStudies included in review(n = 91) 6 Table 1. Variables and reported units extracted from independent experiments in reviewed reports. 166 No. Reports: 91 No. Experiments: 220 No. Datapoints: 920 Duckweed Culture conditions Culture media Responses Genera Species Clone Origin Real / Simulated Stocking density Coverage Temperature Photoperiod Light intensity - (mg m-2) (%) (°C) (Light hours) (µmol m-2 s-1) Real / Synthetic Nitrogen source Total N Ammonium Nitrate Total P Orthophosphate - - (mg N L-1) (mg NH4-N L-1) (mg NO3-N L-1) (mg P L-1) (mg PO4-P L-1) RGR BC EC N removal rate P removal rate (d-1) (% dw) (% dw) (mg N L-1 d-1) (mg P L-1 d-1) RGR = Relative growth rate, BC = Biochemical composition (protein, lipid, starch), EC = Elemental composition (C, H, O, N) 167 168 In addition to this, some characteristics of the culture media in which plants were grown were 169 recorded. Medium was classified as synthetic or real, based on the methods described by the authors. 170 Total Nitrogen (TN) was noted along with the initial concentration of both ammonium and nitrate in the 171 media, all expressed as mg N L-1. Total Phosphorus (TP) and phosphate concentrations are reported 172 as mg P L-1. 173 Finally, duckweed growth parameters like biomass productivity, relative growth rate (RGR) and 174 doubling time were taken out from the screened literature. When data was not provided, RGR was 175 calculated as RGR = Ln (Xf / Xi) / t, with Xf and Xi either the dry biomass, wet biomass, number of fronds 176 or total fronds area at the end and start of the experiment respectively, and t the cultivation time in days. 177 In cases where biomass growth was presented in time course plots, the corresponding RGR was 178 calculated by fitting growth curves data to the differential form of the equation dX/dt = RGR x t. 179 When possible, data were extracted from tables and text of the publication; however, when 180 results were presented only on graphs, they were retrieved by reversing data visualizations using the 181 software WebPlotDigitizer (https://automeris.io/WebPlotDigitizer/). To facilitate the analysis, all data 182 collected for each variable were converted to the same units (as per Table 1) using relevant conversion 183 factors. 184 2.4. Data analysis 185 Data obtained from the literature search was catalogued and curated using Microsoft Excel 186 software; data analysis and visualisation was conducted using R software. Statistical analysis of RGR 187 values included one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance 188 criterium was defined as p value < 0.05. Z-scores were used to standardize the size effect of culture 189 conditions on response variables, to a same scale, to make them comparable. For each independent 190 experiment, Z-values for any response variable were obtained as 𝑍=(𝑥−𝜇)/𝜎, where 𝑥 corresponds 191 to the value of the response variable at any given culture condition within the experiment; µ is the mean 192 response, and σ the standard deviation. A further transformation was performed on the data to avoid 193 7 negative values at the time of obtaining regression curves. For this, Z-values were adjusted to have a 194 mean of 100 and a standard deviation of 15. The new values, so called IQ-scores, were calculated as 195 𝐼𝑄=𝑍∗15+100. 196 197 3. RESULTS AND DISCUSSION 198 3.1. Study sample and experimental design characteristics 199 This review identified 661 studies that met the inclusion criteria, see Figure 1 for the PRISMA 200 flow diagram summarizing the study selection process. Out of these studies, 91 provided sufficient 201 information to be included in the final quantitative analysis. 202 Lemna seems to be the most studied duckweed genus as 62% of the studies included in the 203 review had this genus as a study subject (Figure 2A). L. minor and L. gibba were the species for which 204 most experimental data was available, from 39 and 23 publications respectively. The fact that L. minor, 205 also known as common duckweed, is the most widespread duckweed species and broadly used in 206 toxicity testing (Moody & Miller, 2005; OECD, 2002) makes it the most extensively studied of all 207 duckweed species (Ceschin et al., 2016; Wang, 1986). Of the 14 species discovered for this genus, 208 data were available for 8 of them. The next most studied duckweed genera were Spirodela and 209 Landoltia, of which the same number of studies (16) was available for the species S. polyrrhiza and L. 210 punctata. Finally, Wolffia and Wolffiela are the least studied duckweed genera. Recognised as rootless 211 duckweed, both genera contribute only 11% of the papers selected for analysis. Being studied in 6 of 212 the selected publications, W. arrhiza is the species with the highest representation of this group. Overall, 213 our database has a good representation of the different duckweed species (19 out of 36 species 214 discovered so far) for each of the genera and, the variability of climates from which each species is 215 representative (61% from temperate climate locations – including China, 24% from tropical climate 216 regions, and 15% from subtropical climate areas). 217 In terms of culture conditions, data related to temperature, photoperiod and light intensity was 218 collected, and classified according to the degree of control over the experiments (Figure 2B). As per 219 the density plots, 50% of the selected experiments were carried out at temperatures between 23 and 220 26°C, with a median value of 24°C, under controlled cultured conditions. The temperature range 221 increased when cultures were carried out under ambient/outdoor conditions (from 19 to 27°C) due to 222 seasonality in temperate climate regions. In the latter case, the distribution of data is multimodal, with 223 peaks at 20, 26 and 39°C. 224 Regarding photoperiod, under controlled culture conditions, the preference was to carry out 225 trials under simulated long daylight conditions (16h of light for 50% of the data), while under ambient 226 conditions, most of the studies were carried out under natural light, with photoperiods varying between 227 8.5 and 14 hours of light per day, with a median of 12h of light per day. Perhaps the biggest difference 228 between data collected at ambient and controlled growing conditions concerns light intensity. At the 229 former condition, several authors used sunlight as a source of energy radiation. Light intensity values 230 8 were normally distributed, with most of the tests carried out between 270 and 450 µmol m-2 s-1, 231 consistent with average values for solar PAR radiation in countries with climates ranging from temperate 232 to subtropical (Global Solar Atlas, n.d.; Wang et al., 2013). In contrast, data from experiments performed 233 in controlled environments present a right-skewed distribution, and only 25% of the data exceeds 150 234 µmol m-2 s-1. 235 236 Figure 2. Summary of selected dataset descriptors grouped by variable category. (A) Number of experiments 237 per duckweed Genus and species included in the review, (B) Density plots showing the data distribution of 238 environmental factors studied across different studies performed under controlled and uncontrolled culture 239 conditions, (C) Density plots showing the data distribution for Total Nitrogen and nitrogen species concentration 240 from papers using real and synthetic wastewater as culture media, (D) Density plots showing the data distribution 241 for Total Phosphorus and Phosphate concentration from papers using real and synthetic wastewater as culture 242 media. 243 9 When considering the characteristics of the culture medium, whether synthetic or real 244 wastewater, there are differences in the composition of the different phosphorus and nitrogen species. 245 As far as nitrogen is concerned, there are two main species that contribute to the total nitrogen content 246 of the culture medium, nitrate and ammonium. Figure 2C shows the number of publications studying 247 the effect of either nitrogen species or total nitrogen on duckweed growth. In works using urban 248 wastewater, the major input of total nitrogen comes from ammonium resulting from the decomposition 249 of organically bound nitrogen. In this sense, the plots show how the number of studies on ammonium 250 significantly influences the results that can be found when total nitrogen is the variable of study. The 251 nitrogen concentration ranges used in the studies on ammonium and total nitrogen removal/uptake 252 were distinct from each other. Half of the research on ammonium utilized concentrations ranging from 253 11 to 51 mg N L-1. Meanwhile, around 50% of the studies on total nitrogen employed concentrations 254 between 25 to 70 mg N L-1. These ranges are similar to what is typically observed in urban wastewater 255 (Ma et al., 2016; Metcalf et al., 2004). Higher ammonia concentrations tested by authors correspond to 256 the use of wastewater from sources other than urban areas. In contrast, studies on ammonium carried 257 out with synthetic media barely exceeded 8 mg L-1 (just 25% of the data). 258 In general, experiments carried out using nitrate as the sole nitrogen source in both synthetic 259 and real wastewater showed that the concentrations of nitrate were usually lower than the total nitrogen 260 concentrations tested in other studies. Whereas nitrate concentration varied between 0 – 8 mg N L-1 in 261 75% of cases, higher concentrations were used only in synthetic media, where the nitrogen source was 262 adjusted in such a way as to match the total nitrogen values normally found in wastewater (25% of the 263 test run in synthetic media had a nitrate concentration over 18 mg N L-1). 264 Finally, data distribution regarding total phosphorus (TP) and phosphate (PO4-P) concentration 265 in synthetic and real wastewater is reported in (Figure 2D). For both types of culture media (real and 266 synthetic media), there is a correlation between the phosphate concentration and the total phosphorus 267 values tested. Regardless of the type of culture media, half of the experiments tested phosphorus 268 concentrations below 6 mg P L-1, which was reported either as phosphate or total phosphorus. 269 Moreover, authors using synthetic media in their studies tended to cover a wider range of phosphate 270 concentration to assess scenarios mimicking repleted and depleted nutrient conditions. 271 272 3.2. Effect of environmental factors on plant growth 273 In recent years an increasing number of researchers have focused on understanding how global 274 climate is changing and the corresponding impacts on life on earth. It is undeniable that any change in 275 environmental conditions has direct repercussions on living organisms, consequently influencing their 276 metabolism (e.g., growth rates) and performance in engineering applications. For biomass growth, 277 duckweeds use light and nutrients to carry out photosynthesis. While growing, these aquatic plants also 278 produce and accumulate metabolic products, of which relative amounts in biomass depend upon the 279 specific species studied, and environmental conditions tested (i.e., Light intensity and photoperiod, 280 10 temperature, and availability of nutrients). To have a comprehensive understanding on the effect of 281 these parameters on the growth of duckweeds, it is necessary to critically assess the existing literature. 282 3.2.1. Temperature 283 Temperature is probably the most important environmental factor regulating duckweed growth, 284 composition, and nutrient uptake. As for other aquatic organisms, water temperature controls the rate 285 at which biochemical reactions take place, including duckweed’s photosynthesis, metabolism and 286 catabolism. These plants can grow in a broad range of temperatures, subject to species and isolate, 287 acclimation, and seasonal ambient conditions. The relationship between temperature and duckweed 288 growth can be described by the Arrhenius equations, previously used in kinetic models for other 289 photosynthetic organisms (Feng et al., 1990; Goldman & Carpenter, 1974). It is assumed that duckweed 290 growth rate continuously increases with temperature increments up to a point in which growth rate 291 decreases, i.e., optimal temperature. Therefore, to have a more accurate representation of the 292 relationship between growth rate and temperature data, the thermal performance model curve (TPC), 293 described by the Hinshelwood equation was employed (Hinshelwood, 1947). The Hinshelwood thermal 294 model assumes that the rate of biomass growth is proportional to the overall enzyme activity and the 295 kinetic growth rate constant. It also assumes that changes in the kinetic growth rate constant as a 296 function of temperature can be described by the Arrhenius equation. The model predicts a unimodal 297 relationship between biomass growth rate and temperature, with an optimal temperature at which the 298 rate is at its maximum. 299 Based on the data obtained from published literature (Figure 3), the tested ambient air temperatures 300 ranged from 5 to 40°C, within which the actual relative growth rate (RGR) varied between 0.0 and 0.41 301 d-1; the highest RGR value correspond to Lemna minor cultured in synthetic media under controlled 302 culture conditions (Lasfar et al., 2007). In general, it is found that temperature affects duckweed 303 biomass growth in similar ways in the different studies analysed. The growth rate increases as the 304 temperature rises from 5°C and reaches a maximum at around 25ºC. Above this temperature, plants 305 become stressed and reduce their growth rate. This behaviour follows well known fundamental 306 principles of plant growth and experimental results from other species of aquatic plants and microalgae 307 used for wastewater treatment (Carr et al., 1997; Ras et al., 2013). Outside the optimal temperature 308 range (20-30°C), or even at extreme temperature values, the plants do not grow as fast or simply die. 309 This fact explains why L. minor and L. minuta do not survive over winter in uncontrolled outdoor 310 experiments (Paolacci et al., 2018). Duckweeds sense environmental conditions and when these are 311 not favourable, most of them can enter a dormant state by turion formation (Appenroth, 2002; Kuehdorf 312 & Appenroth, 2012). This ability allows these plants to survive in environments with seasonal climatic 313 variability. 314 11 315 Figure 3. Effect of temperature on the relative growth rate (RGR) of different duckweed genera. 316 Thermal performance curves for Landoltia, Lemna and Spirodela were fitted to datasets from 3, 15 and 4 317 independent experiments respectively, using the Hinshelwood model. In all cases, RGR is expressed as the IQ 318 scores from each independent experiment. Dashed lines represent the RGRIQ-score baseline (= 100). Coefficients 319 and standard errors for the fitted curves are given in Supporting Information, Table S2. 320 321 When analysing the variation of RGR IQ-scores with respect to temperature, a region is 322 revealed, above the baseline (IQ-score = 100), where plants growth is better than the average from all 323 the experiments included in the review (average growth rate = 0.119 d-1). Based on the thermal 324 performance curves resulting from the datasets for different genera of duckweed (Figure 3), we found 325 that the temperature range within which the area of optimal growth is contained varied between genera. 326 Lemna species can cope better with extreme temperatures and exhibit a good growth performance in 327 a wide range of temperatures (11.4 to 38.1°C) while the range of temperatures for optimal growth was 328 narrower for Landoltia and Spirodela species (18.1 to 32.3°C, and 19.0 to 29.2°C, respectively). In the 329 case of Lemna, plants that are grown outside the optimal temperature range end up having a RGR 55% 330 lower than the average RGR above the IQ-scores baseline. These results highlight the importance of 331 choosing the most suitable duckweed species according to local temperature conditions. 332 The temperature range for RGR IQ-scores higher than 100% is of great importance for the 333 development of duckweed-based processes for wastewater treatment. If temperature alone is 334 considered, a treatment system operated at ambient conditions will be reliable if the selected duckweed 335 species perform well within the local temperature variations. Thus, the implementation of duckweed-336 based systems for wastewater treatment in regions with tropical or subtropical climates is favoured due 337 to narrow temperature variations from optimal duckweed growth conditions throughout the year. In 338 cool/cold temperature regions actions need to be taken to engineered wastewater treatment systems 339 to avoid temperature falling below the optimal range. 340 341 342 LandoltiaLemnaSpirodela 10203040102030401020304080100120 Temperature (°C)RGRIQ - SCORE 12 3.2.2. Light 343 Light is an essential factor for plant growth as it is the energy source for photosynthesis, which 344 enables plants to fix atmospheric inorganic carbon and turn it into organic compounds. When referring 345 to light during the cultivation of duckweeds, three factors must be considered: light intensity, light/dark 346 cycles or photoperiod, and light spectral composition. All factors affect duckweed biomass growth 347 through their impact on photosynthesis. In terms of light intensity, it has been found that the growth rate 348 of aquatic plants and microalgae increases with increasing light intensity, up to a maximum RGR value 349 when light saturation conditions are reached (Madsen & Sand-Jensen, 1994; Sorokin & Krauss, 1958). 350 Further light intensity increments above this point reduce plant growth rates and may even inhibit 351 photosynthesis (photo-inhibition). However, results may vary depending on the species and isolates 352 studied, as well as on photoadaptation processes that improve the photosynthetic efficiency of the 353 organisms. This includes changes in chlorophyll content and ratios, number of chloroplasts and 354 respiration patterns (Lichtenthaler et al., 1981). 355 Although light intensity plays a fundamental role in photosynthesis, the time during which the 356 radiation is incident on the plants must also be considered. At low light intensities the RGR of 357 duckweeds increases with longer day conditions, but at high light intensities longer photoperiods 358 negatively impact plants growth rate (Lasfar et al., 2007; Yin et al., 2015). In this sense, it is necessary 359 to consider the total amount of radiation that reaches the plants while they are exposed to the light (e.g., 360 daily light integral – DLI) to avoid photosystem inhibition and damage, so that photosynthesis can 361 continue (Sundby et al., 1993). 362 363 Light intensity 364 The relative growth rate of different duckweed species increases with increasing light intensity, 365 reaching maximum biomass growth at around 200 µmol m-2 s-1 (Figure 4); further increases in light 366 intensity do not significantly affect plant growth (even up to 800 µmol m-2 s-1, not shown in the figure). 367 The change in IQ-scores of the selected duckweed species with respect to light intensity was fitted to a 368 Monod-like model widely used for microalgae (Béchet et al., 2013). From the results, it can be 369 established that overall, for all duckweed species, light saturation is reached at around 100 µmol m-2 s-370 1. One exception is L. aequinoctialis grown in continuous light, which is saturated by light at an intensity 371 of 50 µmol m-2 s-1. Furthermore, within the range of reported light intensities (0 – 800 µmol m-2 s-1), no 372 evidence of photoinhibition can be seen. Similar results were reported by Wedge et. al (1982), who 373 found that, depending on the temperature, light saturation in Lemna minor plants occurs between 300 374 – 600 µmol m-2 s-1 and that there is no photoinhibition unless the light intensity is greater than 1200 375 µmol m-2 s-1. 376 13 377 Figure 4. Effect of light intensity on the relative growth rate (RGR) of duckweeds cultivated under different 378 photoperiods (light hours: dark hours). Curves represent the general trend of the data for different duckweed 379 species upon parametric fitting of datasets to Monod-like equations. The numbers above the boxes represent the 380 number of hours light: dark per day. In all cases, RGR is expressed as the IQ scores from each independent 381 experiment. Dashed line represents the RGRIQ-score baseline (= 100). Data for L. aequinoctialis, L. minor, S. 382 polyrrhiza and La. Punctata was retrieved from 4, 4, 1 and 2 independent experiments respectively. 383 384 Although the overall effect of light intensity on the RGR of the plants is the same (direct 385 increment until saturation), the magnitude of the effect varies according to the photoperiod and 386 duckweed species in question. On the one hand, the positive effect of increasing light intensity on RGR 387 is compromised as the length of light hours increases. In the case of L. aequinoctialis, there is no 388 significant effect of increasing the photoperiod from 12 to 16 hours, but an additional increase of 8 hours 389 reduces the RGR by 24% (Yin et al., 2015). On the other hand, when grown at the same day length (16 390 h) and below light saturation condition, the RGR of different duckweeds species improves differently for 391 each unit by which the light intensity is increased. As an example, an increment of 50 µmol m-2 s-1 392 improves the RGR of L. minor, L. aequinoctialis, La. punctata and S. polyrrhiza by 23.1, 31.9, 31.2 and 393 33.3% respectively (Y. Li et al., 2016; Walsh et al., 2021; Z. Zhao et al., 2014). 394 395 Photoperiod 396 When it comes to photoperiod, two different trends are discernible when analysing the growth 397 of L. minor under constant light intensity (Figure 5A). When the light intensity at which duckweeds grow 398 is higher than 300 µmol m-2 s-1, the RGR increases from 0.01 to 0.43 day-1 when the light exposure is 399 increased from 0 to 12 hours a day. Longer photoperiods reduce the rate at which the plants grow. In 400 this case there is a region above the IQ-scores baseline, between 7 and 18 hours of day length, in 401 which the RGR of duckweeds is greater than the average RGR of all the retrieved data. This range can 402 be defined as the optimal photoperiod range for duckweed growth. At low light intensities (e.g., 156 403 12:1216:824:0 80100120 Light intensity(µmol m-²s-¹)RGRIQ - SCOREDuckweed speciesL. aequinoctialisL. minorS. polyrhizaLa. punctata000505050100100100150150150200200200 14 µmol m-2 s-1) the effect of photoperiod on the RGR of L. minor is the same as at high intensities, 404 however, the optimal range in which plants can grow is extended by 7 hours. In this case, we found that 405 the effect of photoperiod on the RGR was the same despite the difference in culture temperature 406 between the experiments (Lasfar et al., 2007; Paterson et al., 2020). 407 408 409 Figure 5. Effect of photoperiod on the relative growth rate (RGR) of duckweeds under natural and controlled 410 culture conditions. (A) Duckweed cultivated in controlled environments at two different constant light intensities; 411 (B) Duckweed cultivated in real environments with varying photoperiod and light intensities (ranging between 100 412 – 400 µmol m-2 s-1). Curves represent the general trend of the data upon non-parametric fitting of datasets. In all 413 cases, RGR is expressed as the IQ scores from each independent experiment. Dashed line represents the RGR 414 IQ-score baseline (= 100). Data for L. minor, L. japonica and L. minuta was retrieved from 3, 1 and 1 independent 415 experiments respectively. 416 417 A particular case is that of L. aequinoctialis, whose RGR increases with longer day length, 418 reaching a maximum under continuous light independently of the light intensity (ranging from 20 to 400 419 µmol m-2 s-1, data not shown) (Yin et al., 2015). The difference between both Lemna species highlights 420 the importance of species selection in the design of wastewater treatment systems. L. minor copes 421 better with daylength changes in open air treatment systems, while L. aequinoctialis can be used in 422 engineered indoors systems with a continuous supply of light. When analysing the effect of the 423 photoperiod on the RGR of plants that were grown outdoors under variable light intensity conditions 424 (Figure 5 B), it is observed that the data follows similar trends to those of plants cultivated under 425 constant light intensities, but in a narrower range. For light intensities varying between 100 and 300 426 µmol m-2 s-1 the optimal photoperiod range for three different duckweed species is reduced to 7 hours 427 only, between 9 and 15h of day length on average. 428 Although the results do not establish a direct relationship between photoperiod and other 429 duckweed genera and species (not enough data from data collection process), they do lead to the 430 conclusion that photoperiod and light intensity should be considered together when analysing the effect 431 of light on plant growth. 432 433 LI = 155 µmol m-²s-¹ LI = 371 µmol m-²s-¹Constant light intensity 00661212181824248080100100120120 Day length (hours)Day length (hours)RGRIQ - SCORE RGRIQ - SCOREAVariable light intensityDuckweed speciesL. japonicaL. minorL. minutaB 15 Combined effect of light intensity and photoperiod – The daily light integral concept 434 By integrating the light intensity at which the plants are grown together with the time at which 435 they are exposed to light, it is possible to analyse the combined effect of those two variables on the 436 relative growth rate of duckweeds (Figure 6). This combined variable is named as daily light integral 437 (DLI), which describes the number of photosynthetically active radiation (PAR) measured as photons 438 (individual particles of light in the 400-700 nm range) that are delivered to a specific area over a period 439 of time (mol m-2). When the two effects of light intensity and exposure are integrated into the DLI 440 variable, it was found that a biomass growth rate increases with DLI until reaching a maximum at at 15 441 mol m-2, corresponding to the light saturation value. Below this value, plant growth declines rapidly 442 because they do not receive enough energy to efficiently carry out photosynthesis and therefore there 443 is no cell reproduction. On the other hand, when a DLI of 24 mol m-2 is reached, the effect of 444 photosystem inhibition becomes significant and the RGR falls below the IQ-scores baseline. 445 446 Figure 6. Daily light integral (DLI) as a parameter to assess the effect of light on the relative growth rate 447 (RGR) of Lemna species. Curves represent the general trend of the data upon non-parametric fitting of datasets. 448 In all cases, RGR is expressed as the IQ scores from each independent experiment. Dashed line represents the 449 RGR IQ-score baseline (= 100). Data for L. aequinoctialis, L. japonica, L. minor and L. minuta was retrieved from 450 10, 2, 7 and 1 independent experiments respectively. 451 452 In this regard, further studies testing the turnover of D1 protein of photosystem II in varying DLI 453 values need to be addressed to confirm the extent to which the damage in the photosystem affect the 454 RGR in duckweeds (Aro et al., 1993). In the past, it has been proven that DLI not only affects plant 455 growth but many other plant traits (Poorter et al., 2019). In general, it was found in the literature that 456 plant growth is limited below a DLI of 5 mol m-2, whereas saturation of most traits occurs beyond 20 mol 457 m-2. The fact that the reported data fell within this range supports the idea that there is little difference 458 in plasticity with respect to DLI between different plant species. 459 L. minorL. minutaL. aequinoctialisL. japonica 0102030 010203080100120 80100120 DailyLightIntegral(molm-2)RGRIQ-score 16 The analysis of the DLI as a control variable, suggest that the effect of light on RGR of 460 duckweed is independent of the duckweed species (different Lemna species in this case) being tested. 461 The finding is useful for the design of engineered treatment systems based on duckweed biomass and 462 using either natural or artificial light. In the former case, the DLI supports the potential use of solar 463 energy as a source of radiation, thus reducing energy costs and dependence on fossil fuels. 464 Furthermore, DLI monitoring would allow prediction of biomass growth in environments where the 465 intensity and amount of light is not constant during the system operation period, making it possible to 466 get more reliable systems for engineering applications (e.g., wastewater treatment). Moreover, the 467 analysis of the data reveals that after exceeding a threshold value in DLI (7.5 mol m-2), there is no major 468 gain in terms of RGR so that energy savings can be considered during the design of the treatment 469 process. For instance, by doubling the DLI from 7.5 to 15 mol m-2, the energy cost doubles in a system 470 of constant area while the RGR of the plants improves by only 5.8%. 471 The spectral composition of light, or the specific wavelengths of light that are present, is another 472 parameter that can affect the growth of duckweed. Different pigments in the plant absorb different 473 wavelengths of light, which can stimulate or inhibit growth. Studies have concluded that red and blue 474 light are the most effective in promoting growth and increasing biomass production. Duckweed grown 475 in either blue or red light resulted in 10% and 31% increase in dry weight, respectively, in comparison 476 to cultures under cool white light (Q. Li et al., 2022). Moreover, the combination of red and blue light at 477 different ratios does not significantly impact duckweed growth but influences the accumulation of starch 478 (Q. Li et al., 2022; Petersen et al., 2022). These findings have important implications for optimizing 479 duckweed cultivation for applications such as wastewater treatment with resource recovery (i.e., 480 production of starch-rich duckweed biomass for animal feed). 481 482 3.3. Effect of nutrient supply on duckweed growth 483 In addition to light, plants need the right combination of nutrients to live, grow and reproduce. 484 Both excess and deficiency of nutrients can cause problems to plant growth. Among the elements that 485 plants need in relatively high amounts, macronutrients like phosphorus and nitrogen are of most interest 486 due to their low bioavailability in aquatic environments (Vitousek et al., 2010). Although both nutrients 487 are abundant in agricultural runoff and wastewater discharges, their presence in aquatic ecosystems is 488 undesirable due to the potential development of anoxia and eutrophication in surface waters. As we 489 well know, nitrogen and phosphorus fertilizers are usually added to soils to ensure that plants have 490 adequate access to these essential nutrients. In plants, both nutrients are present either as ionic species 491 or as constituents of biomolecules of great importance for the plant (Maathuis, 2009). Like terrestrial 492 plants, duckweeds can acquire significant amounts of inorganic nutrients through their root system 493 (Cedergreen & Vindbadk, 2002; Ying Fang et al., 2007). However, due to their aquatic nature and the 494 fact that the fronds float directly on the surface of the water, nutrient absorption is mostly carried out 495 from the underside of the frond (Ice & Couch, 1987; Oron, 1994). The extent to which duckweeds growth 496 17 is affected by nitrogen and phosphorus supply is reviewed in the context of their use for wastewater 497 treatment. 498 3.3.1. Nitrogen 499 Nitrogen (N) is involved in the synthesis of amino acids (the building blocks of proteins), 500 chlorophyll and nucleic acids (DNA, RNA). It promotes the photosynthetic capacity and the growth of 501 plant tissue, making it an important performance factor (Barker & Bryson, 2006; Novoa & Loomis, 1981). 502 N is present in wastewater in mineral (N-NO3 and N-NH4) or organic forms, but it is mainly absorbed by 503 duckweeds in mineral form like ammonium and /or nitrate (Ding et al., 2018; Joy, 1969). Both nitrogen 504 deficiency and excess affect plant growth, but the extent to which it is affected depends on the species 505 of nitrogen used for cultivation. 506 507 Nitrogen species 508 The forms of nitrogen in wastewater vary depending on the type of wastewater, pH and 509 temperature (Caicedo et al., 2000). As a result of different biological and chemical processes, the main 510 nitrogen compounds in wastewater are ammonium, nitrate and nitrite. Among them, ammonium is the 511 main chemical specie, as it originates from the decomposition of organic matter. However, significant 512 amounts of nitrate can be found in wastewaters and runoff resulting from industrial or farming activities 513 requiring significant amounts of nitrate-based chemicals or fertilisers. 514 515 516 Figure 7. Differences in the relative growth rate (RGR) of duckweeds grown at different ammonium to 517 nitrate ratios. Violin plots represent the distribution of datapoints, and box plots represent the median, the 25th 518 and 75th percentiles, minimum, maximum and outlying points. Black points mark the average RGR value for each 519 NH4 - NO3 ratio. Only data for different species of the genus Lemna are presented. The number of observations 520 per group (n) is presented on top of each plot. Lower case letters represent statistical significance (p < 0.5). 521 522 In the Lemnaceae family, the preference for ammonium over nitrate is still a subject of discussion. Most 523 of authors have stated that ammonium is adopted as the first source of nitrogen by duckweed, because 524 it is important for the synthesis of amino acids and proteins, and there is an associated saving of energy 525 for the assimilation process (Oron, 1994; Porath & Pollock, 1982). However, ammonium assimilation is 526 temperature sensitive and occurs only at pH values between 6 and 8 (Caicedo et al., 2000). It has also 527 aabbn = 51n = 9n = 57 0.00.20.40.6 100 − 050 − 500 − 100Ammonium - Nitrate ratio (%)RGR(day-1) 18 been pointed out that ammonium is a limited source of nitrogen due to its toxicity to plants (Joy, 1969). 528 When both nitrogen sources are available in the medium, the plant prefers to absorb ammonium, but 529 can take nitrate when it is the only nitrogen source (Ying Fang et al., 2007). When a wider range of pH 530 values is considered, some duckweeds species have shown predilection for nitrate over ammonium 531 while the absorption of other macronutrients (P) was enhanced (Paterson et al., 2020). 532 533 The possibility of using both ionic species as a source of nitrogen to grow duckweeds is 534 reflected in the number of publications studying the effect of different ammonium to nitrate ratios on the 535 RGR of plants. In the case of Lemna species, when considering the sole effect of the nitrogen source 536 on duckweeds RGR, it was found that there is no statistically significant difference (p > 0.05) between 537 mean RGR values when using ammonia, nitrate or both nitrogen species in the culture medium (Figure 538 7). The differences between the culture conditions employed in studies considered in the analysis reveal 539 that the preferred nitrogen source for each duckweed species is species-dependent and may be 540 determined by the acclimatisation of plants to the growing conditions, the nitrogen concentration and 541 the N:P ratio. 542 543 Nitrogen concentration 544 Total nitrogen concentration in domestic wastewater varies between 20 and 80 mg N L-1. 545 Ammonia is the major contributor to total nitrogen (~ 60%), followed organic nitrogen and nitrate (Henze 546 et al., 2002). In some cases, total nitrogen concentration can be as high as 200 mg N L-1, especially in 547 wastewater from industries like aquaculture, and run-off water from agriculture (Korner et al., 2003). If 548 duckweeds are intended to remediate wastewater from aquaculture, it is necessary not only to 549 understand the effect of the nitrogen species present in the effluent of this industry, but also the effect 550 of the concentration of nitrogen on the potential of these plants to grow. 551 552 Studies have shown that duckweeds have a wide range of tolerance to nitrogen concentrations, 553 and the optimum concentration may vary depending on the species, growing conditions and nitrogen 554 source (Figure 8). In general, when nitrate is the only nitrogen source, duckweed growth is supported 555 at moderate concentrations (between 2-70 mg N L-1, Figure 8-A). In presence of ammonium duckweed 556 growth is supported at lower concentrations (between 5-15 mg N L-1, Figure 8-B). However, higher 557 nitrogen concentrations beyond RGR maxima are detrimental to duckweed growth in both cases. In 558 addition to the above, when comparing the kinetic curves obtained with respect to N concentrations and 559 the RGRIQ-score baseline, the RGR of the Lemna species is higher than the average RGR value in 560 cultures grown with nitrate (2 – 195 mg N L-1) than those grown in ammonium (5 – 60 mg N L-1). 561 The fact that the RGR response curves to different nitrogen concentrations follow the same 562 trend for nitrate and ammonium suggests that duckweed does not have a particular preference for a 563 specific nitrogen source, since, under certain conditions, both nitrogen sources benefit plant growth. 564 What the results suggest is that to some extent ammonium has greater inhibitory effects on the growth 565 of L. gibba and L. minor than nitrate. This can be explained due to potential ammonium toxicity. 566 19 567 Figure 8. Effect of the supply of different nitrogen species on the relative growth rate (RGR) of duckweeds. 568 (A) duckweeds grown in media with nitrate as only source of nitrogen; (B) duckweeds grown in media with 569 ammonium as only source of nitrogen. Curves represent the general trend of the data upon parametric fitting of 570 datasets to a substrate-inhibition kinetic mode (Haldane, 1965). In all cases, RGR is expressed as the IQ scores 571 from each independent experiment. Dashed line represents the RGRIQ-score baseline (= 100). Data for L. 572 aequinoctialis, L. gibba, L. minor, L. minuta and L. trisulca was retrieved from 1, 2, 3, 1 and 1 independent 573 experiments respectively. 574 575 There are two main mechanisms by which ammonium is toxic to plants. The first derives from 576 the ease at which ammonium is transported across the cell membrane and the second from changes 577 in pH as a result of ammonium uptake (Britto & Kronzucker, 2002). Both ammonium (NH4+) and its non-578 ionised form, ammonia (NH3), are transported into the membrane by low affinity transporters, which 579 activity is upregulated at high external nitrogen concentrations, resulting in increased influx of nitrogen 580 (Cerezo et al., 2001; M. Y. Wang et al., 1993). As the ammonium uptake rate of the plant exceeds the 581 assimilation rate or the storage capacity, the plant will actively transport ammonium back to the exterior 582 (Hecht & Mohr, 1990; Husted et al., 2000). As a result, the energy demand for this process (Britto et 583 al., 2001), together with a reduced influx of other cations (e.g., K+, Mg+2, Ca+2) and increased uptake of 584 anions (Cl-, SO4=) may limit overall plant growth (Gerendás et al., 1997; Roosta & Schjoerring, 2007; 585 Van Beusichem et al., 1988). A recent study on Landoltia punctata has shown that the coordination of 586 carbon and nitrogen metabolism in duckweeds may act as ammonium detoxification mechanism, 587 making duckweeds more tolerant to ammonium than other higher plants (Tian et al., 2021). 588 80100120 0100200300Nitrate(mgNL-1)RGRIQscoreDuckweed speciesL. aequinoctialisL. gibbaL. minorL. minutaL. trisulcaA 80100120 0255075100Ammonium(mgNL-1)RGRIQscoreDuckweed speciesL. gibbaL. minorB 20 The second proposed mechanism by which ammonium is toxic to plants relates to external and 589 internal pH changes (McQueen & Bailey, 1990; Schubert & Yan, 1997). Ammonium uptake by higher 590 plants is linked to a cation counter-phase, to compensate for the charges on the cell membrane 591 potential. This effect occasionally leads to the acidification of the culture medium in which the plant is 592 growing (Brix et al., 2002; Ruan et al., 2007; Schubert & Yan, 1997). Moreover, nitrate reduction in 593 plants is considered a sink for excess NADPH production by photosynthesis. When an already reduced 594 source of nitrogen is supplied, like ammonium, the accumulation of NADPH can indirectly affect the 595 internal cell pH by altering the reactive oxygen species and enzymes involved in maintaining the pH 596 balance (Guo et al., 2007). In duckweeds, it has been found that the optimum pH value for growth is 597 around 7 (Caicedo et al., 2000; Jones et al., 2023; McLay, 1976), so that, in cases where ammonium 598 is the only available source of nitrogen, there is a double stress factor that reduces plant growth. 599 600 3.3.2. Phosphorus 601 Phosphorus (P) is a cellular constituent and an energy carrier. It is a component of the 602 phospholipids that make up cell membrane and DNA, RNA, and ATP molecules (Maathuis, 2009). As 603 a cellular constituent, P supports plant growth, particularly in the development of roots that have several 604 adaptive responses to acquire P from the soil and aquatic environments. P also promotes flowering, 605 fruit setting and seed formation (Maathuis, 2009). In wastewaters, phosphorus can be found in mineral 606 form, mainly as orthophosphates (PO43-, HPO42-, H2PO4-,) and, in a smaller amount, in organic form. 607 The form at which mineral phosphorus can be found strongly depends on water temperature and pH. 608 Furthermore, phosphorus is a non-renewable resource, that is unevenly distributed in the world, hence 609 the importance of its recovery and reuse from waste streams (Slocombe et al., 2020). 610 611 Phosphorus concentration 612 The occurrence of phosphorus in wastewater is closely related to the sources of phosphorus. 613 Industrial, agricultural and household activities have the greatest impact on the amount of phosphorus 614 found in wastewater. As such, phosphorus concentration can be relatively low, as in domestic 615 wastewater (0.2 – 20 mg P L-1) or high, as in effluents from intensive crop and livestock production (12 616 – 780 mg P L-1) (Carrillo et al., 2020). A particular case is that of aquaculture where the large volumes 617 of water used for fish production dilute the phosphorus concentration to values below 1 mg P L-1. In 618 aquatic environments (fresh waters), phosphorus is usually considered as the limiting nutrient 619 controlling growth of photosynthetic organisms. Therefore, the effect of low phosphorus concentrations 620 on duckweed growth needs to be assessed. 621 Our results show that increasing phosphorus concentration of the culture medium improves 622 duckweed relative growth rate, however, how this occurs depends on the nitrogen source used for the 623 culture (Figure 9). On one hand, when ammonium is used as the sole source of nitrogen, L. minuta 624 reaches a maximum growth rate at a phosphorus concentration of 1.5 mg P L-1. Thereafter, higher P 625 21 concentrations reduce the rate at which the plant grows (Figure 9A). On the other hand, in the presence 626 of nitrate, the RGR of different duckweed species reaches a maximum at a phosphorus concentration 627 of 1 mg P L-1. In this case, the growth rate is not affected by further increases in phosphorus supply, 628 remaining always above the RGRIQ-score baseline (Figure 9B). 629 630 631 Figure 9. Effect of phosphorus supply on the relative growth rate (RGR) of duckweed in media with different 632 nitrogen source. (A) duckweeds grown in media with ammonium as only source of nitrogen (median N:P ratio = 633 7.0); (B) duckweeds grown in media with nitrate as only source of nitrogen (median N:P ratio = 6.5). Curves 634 represent the general trend of the data upon non-parametric fitting of datasets. In all cases, RGR is expressed as 635 the IQ scores from each independent experiment. Dashed line represents the RGR IQ-score baseline (= 100). Data 636 for L. aequinoctialis, L. japonica, L. minor and L. minuta was retrieved from 10, 2, 7 and 1 independent experiments 637 respectively. 638 639 In higher plants, phosphorus uptake and relocation are carried out by phosphorus transporter 640 proteins (PHT) (Młodzińska & Zboińska, 2016). There is evidence that PHT proteins can be induced 641 either at low (high-affinity) or high (low-affinity) external phosphorus concentration (Bayle et al., 2011). 642 In a recent study, 73 PHT highly conserved genes have been identified in different duckweed species 643 (X. Zhao et al., 2021). Within these, 21 belong to the PHT1 subfamily, responsible for P acquisition from 644 the environment, suggesting that P uptake by duckweed follows similar mechanisms to those previously 645 reported in terrestrial plants. In general, an excess supply of phosphorus does not negatively affect 646 plant growth, unless the concentration of phosphorus in the plant tissues exceeds 1% of the plant dry 647 weight, a phenomenon known as Pi toxicity (Marschner, 1996; Takagi et al., 2020). The estimated 648 Michaelis-Menten constant (Km) for low-affinity and high-affinity PHT transporters suggest that 649 saturation condition is reached at external P concentration of 0.1 and 1.5 mg P L-1 respectively 650 (Nussaume et al., 2011). Also, there is a close link between nitrogen and phosphorus uptake, in which 651 PHT proteins interact with nitrogen transport proteins to maintain nutrient balance in the plant (H. Feng 652 et al., 2017). As a result, high external phosphorus concentrations induce higher P uptake and 653 consequently higher N uptake, meaning that plant growth would not be affected by phosphorus but by 654 the concentration and species of nitrogen being taken up. If ammonium is the nitrogen source (as in 655 NH4 80100120 02468TotalPhosphorus(mgPL-1)RGRIQscoreANO3 80100120 02468TotalPhosphorus(mgPL-1)RGRIQscoreDuckweed speciesL. aequinoctialisL. gibbaL. minorL. minutaL. trisulcaB 22 Figure 9-A) we have that, at constant N:P ratios, the concentration of nitrogen can be such that 656 duckweed growth is inhibited, as explained in the previous section. 657 658 Nitrogen and phosphorus supply balance 659 The nitrogen to phosphorus supply ratio (N:P ratio) is important for plants nutriiton as it is a 660 parameter that indicates the availability of phosphorus and nitrogen for plant growth. The assessment 661 of the N:P ratio allows to establish the condition in which plant growth can be limited by low availability 662 of a nutrient, or the appropriate proportion of nutrients for biomass production. The optimal N:P ratio for 663 plant growth can vary depending on the plant species and the environmental conditions. 664 665 666 Figure 10. Nitrogen and phosphorus balance affect the relative growth rate (RGR) of duckweeds. Nitrogen 667 to phosphorus ratio (N:P ratio) was calculated only for those experiments carried out between 25 – 27°C using 668 nitrate as the sole source of nitrogen. The curve represents the general trend of the data upon non-parametric 669 fitting of datasets. RGR is expressed as the IQ scores from each independent experiment. Dashed line represents 670 the RGR IQ-score baseline (= 100). Data for L. minor and L. trisulca was retrieved from 2 and 1 independent 671 experiments, respectively. 672 673 In the case of two different Lemna species, we found that the optimal N:P supply ratio that 674 maximises plant growth is 15:1 (Figure 10). At lower N:P ratios (the nutrient imbalance causes plants 675 to undergrow due to lack of nitrogen, and at higher ratios the lack of phosphorus and excess nitrogen 676 cause plant growth to be limited or inhibited. It has previously been reported that the optimal N:P molar 677 ratio for plant growth is 15:1 (7:1 masss ratio) (Koerselman & Meuleman, 1996) which is consistent with 678 that found for Lemna species. Similar values were also found for grain legumes (Sadras, 2006) and 679 microalgae (Liu et al., 2011). In wastewater, the molar N:P ratio varies on the type of wastewater and 680 usually fluctuates between 11:1 and 22:1 (5:1 – 10:1, mass ratio) (de Godos et al., 2016; L. Wang et 681 al., 2010), suggesting that wastewater can be used for duckweed cultivation without the need for 682 additional nutrient supply. In conventional wastewater treatment nutrient balance is also an important 683 parameter as it influences microbial activity responsible for the removal of organic matter and oxygen 684 consumption. 685 8090100110 101001000N:Pratio(molNmolP-1)RGRIQscoreDuckweed speciesL. minorL. trisulca 23 4. LIMITATIONS OF THE STUDY 686 The limitations of this study are noteworthy, particularly concerning the absence of data on 687 additional factors influencing duckweed growth beyond temperature, light, and nutrient availability. 688 Firstly, there is a scarcity of studies addressing variables such as pH, plant mat density, interactions 689 with other microorganisms, and others, making it challenging to draw meaningful comparisons and 690 conclusions. Secondly, the difficulty in extracting quantitative data from existing literature can be 691 attributed to a lack of standardization in result presentation, complicating the calculation of comparison 692 indicators for a more comprehensive analysis. Lastly, the focus of the study was been deliberately 693 narrowed to ensure a more in-depth analysis of the specified variables, sacrificing a broader 694 understanding of the multifaceted aspects affecting duckweed growth. Consequently, these limitations 695 emphasize the need for future research to explore the interplay of a wider array of factors to enhance 696 the comprehensiveness of findings in the field of duckweed cultivation and its applicability for 697 wastewater treatment. 698 699 5. CONCLUSIONS 700 Duckweed-based systems for wastewater treatment and nutrient recovery have the potential to 701 provide sustainable and cost-effective solutions for water pollution and nutrient management. However, 702 careful consideration must be given to various factors that affect the growth and nutrient uptake of 703 duckweeds, such as temperature, light, and nutrient supply. These factors can be controlled and 704 optimized through proper design, construction and operation of duckweed-based systems. 705 Temperature is a critical factor that affects the growth and development of duckweeds, and the selection 706 of the appropriate duckweed species for the local climate is essential. While temperature controls the 707 rate of chemical reactions and influences the growth rate of duckweeds, light is the primary energy 708 source for photosynthesis. Light intensity and photoperiod are crucial in regulating the total amount of 709 radiation that reaches the plants and understanding the effect of these factors on duckweed growth can 710 help optimize cultivation conditions and inform new technology developments, particularly for indoor 711 cultivation using artificial light. Nutrient supply, especially nitrogen and phosphorus, significantly affects 712 duckweed growth and nutrient uptake. Nitrogen plays a crucial role in the growth and development of 713 duckweeds, while phosphorus is an essential component of cellular structure and an important energy 714 carrier in plants. The concentration of these nutrients in wastewater can vary depending on the source 715 of the wastewater; careful control of nutrient supply is essential for optimal duckweed growth, but typical 716 N:P ratios in wastewater are sufficient to support duckweed growth. The recovery of nitrogen and 717 phosphorus from wastewater is particularly crucial due to its non-renewable nature and P uneven 718 distribution in the world. Duckweed-based systems can provide a sustainable solution for nutrient 719 recovery from wastewater and overall nutrient management in catchments if use as an alternative for 720 wastewater remediation. With the right design, construction and operation, duckweed-based systems 721 can offer a cost-effective and sustainable alternative to conventional wastewater treatment methods. 722 Overall, the implementation of duckweed-based systems for wastewater treatment and nutrient 723 recovery requires a comprehensive understanding of the various factors that affect duckweed growth 724 24 and nutrient uptake. By considering temperature, light, and nutrient supply in the planning and design 725 of these systems, sustainable and cost-effective solutions can be developed for water pollution and 726 nutrient management. 727 728 Declaration of Competing Interest 729 The authors declare that they have no known competing financial interests or personal 730 relationships that could have appeared to influence the work reported in this paper. 731 732 Data availability 733 Data is available as part of supplementary materials. 734 735 Acknowledgements 736 This work was funded by the School of Civil Engineering, University of Leeds, through a PhD 737 scholarship granted to Mr Pason-Panqueva; the UKRI funded GCRF Water Security and Sustainable 738 Development Hub (ES/S008179/1); and the Colombia’s Ministry of Science, Technology and Innovation 739 (MINCIENCIAS) for international doctoral scholarship number 860-2019 (contract no.106124). 740 741 Supplementary materials 742 Supplementary material associated with this article can be found, in the online version when published. 743 744 745 746 747 748 749 750 751 752 25