where *ln* is the natural log, *W*1 is starting biomass, *W*3 is total biomass on day 3 and ∆*T* is the length of time. As biomass was removed throughout each experiment to maintain a constant plant density, this formula was only used to calculate the *RGR* up to the first instance of removal (day 3). The total increase in mass over the course of the experiment is presented as the yield.

#### 2.3.2. Chlorophyll *a* Fluorescence

Chlorophyll *a* fluorescence measurements were taken for randomly selected plants on days 0 and 7, using a pulse amplitude modulated fluorometer (WALZ Imaging fluorometer, Effeltrich, Germany). The procedure that was followed is detailed in Walsh et al. [32].

#### 2.3.3. Total Nitrogen and Total Phosphorous Analysis

A sample of medium was taken for total nitrogen (TN) and total phosphorous (TP) analysis on the initial and final days of each experiment. For TN analysis, Hach test LCK138 was used with a Hach DR3900 spectrophotometer. Firstly, the sample was digested with peroxo-disulphate for one hour at 100 ◦C causing inorganically and organically bonded nitrogen to oxidise to nitrate (Koroleff digestion). The resulting oxidised nitrate was then analysed photometrically in a reaction with 2,6-dimethylphenol. For TP analysis, Hach test LCK348 was used. Firstly, the medium was digested using the persulphate digestion method for one hour at 100 ◦C. The resulting solution was then analysed photometrically through the ascorbic acid/phosphomolybdenum blue method.

#### 2.3.4. Protein Analysis

*Lemna minor* samples, taken on the final day of experiments, were kept at −20 ◦C until used for protein extraction and analysis. Protein was extracted using 50 mM potassium phosphate buffer (pH 7, containing 0.1 mM polyvinylpyrrolidone (PVP) and 0.1 mM EDTA). Between 50–80 mg of fresh plant material was homogenised in cold potassium phosphate buffer (1 mL of buffer to 80 mg of plant sample). The homogenised sample was then centrifuged at 20,000× *g* for 30 min at 4 ◦C [48]. The resulting supernatant was used for protein analysis using the Bradford method with bovine serum albumin as a standard [49]. For absorbance measurements, 5 µL of sample was added to 1 mL of Bradford reagent in a cuvette and left for five minutes in dark conditions. Absorbance was measured at 595 nm using a spectrophotometer (UV-160A Shimadzu). In order to calculate the proportion of protein based on dry plant biomass, 4% dry weight content of fresh duckweed weight was used [28].

#### *2.4. Data Analysis*

Statistical analyses were conducted using R (version 3.4.3 [50]). One- and two-way ANOVAs were used to analyse differences between treatments for the measured parameters. Post hoc Tukey tests were used for pairwise comparisons of treatment groups. Normality was assessed through a graphical assessment of the distribution of the residual values for data points (i.e., histogram). Homoscedasticity was assessed with 'residuals vs. predicted values' plots as well as Fligner-Killeen and Levene's tests.

#### **3. Results**

#### *3.1. Stationary Remediation Experiment 1: Growth and Remediation at Variable Plant Densities*

The absolute plant biomass yield (g) did not significantly vary over the course of the experiment although the general trend of the average yield increased with increasing density (one-way ANOVA: F(7) = 1.57, *p* = 0.174; Figure 2a). SGR (d−1 ) exhibited the opposite trend; rates decreased as density increased (one-way ANOVA: F(7) = 8.357, *p* < 0.001; Figure 2b). The overall removal of TN (mg) from synthetic dairy wastewater increased as plant density increased (one-way ANOVA: F(7) = 2.574, *p* < 0.05; Figure 2c). However, when TN removal was expressed per frond surface area (mg N m−2 day−1 ), the rate decreased as density increased (one-way ANOVA: F(7) = 9.287, *p* < 0.001; Figure 2d). A similar pattern was found for TP removal in which the overall removal of TP (mg) from synthetic dairy wastewater increased as plant density increased (one-way ANOVA: F(7) = 5.11, *p* < 0.001; Figure 2e). While the TP removal rate per frond surface area (mg P m−2 day−1 ) decreased as density increased (one-way ANOVA: F(7) = 8.158, *p* < 0.001; Figure 2f). There was no difference in protein content (% dry duckweed mass) detected in relation to plant density (one-way ANOVA: F(7) = 0.334, *p* = 0.933; Figure 2g).

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**Figure 2.** Mean (± SE) values with natural log trendline of (**a**) yield (g), (**b**) specific growth rate (SGR, d−1), (**c**) total nitrogen (TN) removal (mg N), (**d**) TN removal rate per frond surface area (mg N m−2 d−1), (**e**) total phosphorous (TP) removal (mg P), (**f**) TP removal rate per frond surface area (mg P m−2 d−1) and (**g**) % protein content based on fresh duckweed biomass, FW, and dry duckweed biomass, DW, for *L. minor* grown on synthetic wastewater over 7 days under a range of plant surface covers (10–80%). Points that do not share the same letter significantly differ from one another, as per the Tukey post hoc test, *p* < 0.05. **Figure 2.** Mean (±SE) values with natural log trendline of (**a**) yield (g), (**b**) specific growth rate (SGR, d−1 ), (**c**) total nitrogen (TN) removal (mg N), (**d**) TN removal rate per frond surface area (mg N m−2 d −1 ), (**e**) total phosphorous (TP) removal (mg P), (**f**) TP removal rate per frond surface area (mg P m−2 d −1 ) and (**g**) % protein content based on fresh duckweed biomass, FW, and dry duckweed biomass, DW, for *L. minor* grown on synthetic wastewater over 7 days under a range of plant surface covers (10–80%). Points that do not share the same letter significantly differ from one another, as per the Tukey post hoc test, *p* < 0.05.

#### *3.2. Stationary Remediation Experiment 2: Growth and Remediation at Low and High Density 3.2. Stationary Remediation Experiment 2: Growth and Remediation at Low and High Density*

On both half-strength Hutner's and synthetic wastewater *L. minor* grown at a lower density (25% plant surface coverage) displayed a higher absolute yield (two-way ANOVA: F(1) = 46.607, *p* < 0.001; Figure 3a) than plants grown at the higher density condition (60% plant surface coverage). The same was found for SGR (two-way ANOVA: F(1) = 45.994, *p* < 0.001; Figure 3b). TN removal (mg) was not significantly affected by density (two-way ANOVA: F(1) = 3.642, *p* = 0.0805; Figure 3c), nor was TN removal rate per frond area (mg N m−2 d−1) (two-way ANOVA: F(1) = 3.154, *p* = 0.101; Figure 3d), although average values were lower at the higher density. Density condition did not significantly affect TP removal (mg) (two-way ANOVA: F(1) = 2.592, *p* = 0.136; Figure 3e) or TP removal rate per frond area (mg P m−2 d−1) (two-way ANOVA: F(1) = 2.293, *p* = 0.158; Figure 3f). On both half-strength Hutner's and synthetic wastewater *L. minor* grown at a lower density (25% plant surface coverage) displayed a higher absolute yield (two-way ANOVA: F(1) = 46.607, *p* < 0.001; Figure 3a) than plants grown at the higher density condition (60% plant surface coverage). The same was found for SGR (two-way ANOVA: F(1) = 45.994, *p* < 0.001; Figure 3b). TN removal (mg) was not significantly affected by density (two-way ANOVA: F(1) = 3.642, *p* = 0.0805; Figure 3c), nor was TN removal rate per frond area (mg N m−2 d −1 ) (two-way ANOVA: F(1) = 3.154, *p* = 0.101; Figure 3d), although average values were lower at the higher density. Density condition did not significantly affect TP removal (mg) (two-way ANOVA: F(1) = 2.592, *p* = 0.136; Figure 3e) or TP removal rate per frond area (mg P m−2 d −1 ) (two-way ANOVA: F(1) = 2.293, *p* = 0.158; Figure 3f).

**Figure 3.** Mean (± SE) (**a**) yield (g), (**b**) SGR (d−1), (**c**) TN removal (mg N), (**d**) TN removal rate per frond surface area (mg N m−2 d−1), (**e**) TP removal (mg P) and (**f**) TP removal rate per frond surface area (mg P m−2 d−1), for *L. minor* grown on synthetic wastewater or half-strength Hutner's medium at two plant surface covers (low, 25% and high, 60%) over 7 days. A hash symbol (#) denotes an effect of density for *p* < 0.05, as per the two-way ANOVA. **Figure 3.** Mean (±SE) (**a**) yield (g), (**b**) SGR (d−1 ), (**c**) TN removal (mg N), (**d**) TN removal rate per frond surface area (mg N m−2 d −1 ), (**e**) TP removal (mg P) and (**f**) TP removal rate per frond surface area (mg P m−2 d −1 ), for *L. minor* grown on synthetic wastewater or half-strength Hutner's medium at two plant surface covers (low, 25% and high, 60%) over 7 days. A hash symbol (#) denotes an effect of density for *p* < 0.05, as per the two-way ANOVA.

#### *3.3. Chlorophyll a Fluorescence 3.3. Chlorophyll a Fluorescence*

Chlorophyll *a* fluorescence measurements were taken on the initial day of stationary experiment 2 (day 0, data not shown). They showed that plants grown in both halfstrength Hutner's and synthetic wastewater displayed similar values for a range of chlorophyll fluorescence parameters: Fv/Fm, Y(II), Y(NPQ) and Y(NO) (one-way ANOVAs across all treatments: F(1) = 1.006, 0, 0.049, 0.076, *p* = 0.354, 0.986, 0.833, 0.792, respectively). Chlorophyll fluorescence measurements taken on the final day (day 7) of the experiment revealed some differences between treatments. Similar to day 0 values, mean Fv/Fm stayed largely constant between 0.68 and 0.8, and was not significantly affected by density (twoway ANOVA: F(1) = 2.038, *p* = 0.179; Figure 4a) or medium (two-way ANOVA: F(1) = 0.911, *p* = 0.359; Figure 4a). However, measurements taken on day seven showed that higher plant density in both media resulted in a lower Y(II) (two-way ANOVA: F(1) = 34.054, *p* < 0.001; Figure 4b). This effect was strongest in plants grown on synthetic wastewater (post hoc Tukey test low:high density in synthetic wastewater: *p* < 0.001; Figure 4b). Medium alone did not significantly affect Y(II) (two-way ANOVA: F(1) = 0.907, *p* = 0.360; Figure 4b). There was, however, a significant interaction between density and medium (two-way ANOVA interaction 'density\*medium': F(1) = 7.369, *p* < 0.05; Figure 4b). Y(NPQ) was not significantly affected by density (two-way ANOVA: F(1) = 0.594, *p* = 0.456; Figure 4c) or medium (two-way ANOVA: F(1) = 1.953, *p* = 0.188; Figure 4c). Nor was Y(NO) significantly affected by density (two-way ANOVA: F(1) = 2.016, *p* = 0.181; Figure 4d) or medium (two-way ANOVA: F(1) = 2.631, *p* = 0.131; Figure 4d). Chlorophyll *a* fluorescence measurements were taken on the initial day of stationary experiment 2 (day 0, data not shown). They showed that plants grown in both half-strength Hutner's and synthetic wastewater displayed similar values for a range of chlorophyll fluorescence parameters: Fv/Fm, Y(II), Y(NPQ) and Y(NO) (one-way ANOVAs across all treatments: F(1) = 1.006, 0, 0.049, 0.076, *p* = 0.354, 0.986, 0.833, 0.792, respectively). Chlorophyll fluorescence measurements taken on the final day (day 7) of the experiment revealed some differences between treatments. Similar to day 0 values, mean Fv/Fm stayed largely constant between 0.68 and 0.8, and was not significantly affected by density (twoway ANOVA: F(1) = 2.038, *p* = 0.179; Figure 4a) or medium (two-way ANOVA: F(1) = 0.911, *p* = 0.359; Figure 4a). However, measurements taken on day seven showed that higher plant density in both media resulted in a lower Y(II) (two-way ANOVA: F(1) = 34.054, *p* < 0.001; Figure 4b). This effect was strongest in plants grown on synthetic wastewater (post hoc Tukey test low:high density in synthetic wastewater: *p* < 0.001; Figure 4b). Medium alone did not significantly affect Y(II) (two-way ANOVA: F(1) = 0.907, *p* = 0.360; Figure 4b). There was, however, a significant interaction between density and medium (two-way ANOVA interaction 'density\*medium': F(1) = 7.369, *p* < 0.05; Figure 4b). Y(NPQ) was not significantly affected by density (two-way ANOVA: F(1) = 0.594, *p* = 0.456; Figure 4c) or medium (two-way ANOVA: F(1) = 1.953, *p* = 0.188; Figure 4c). Nor was Y(NO) significantly affected by density (two-way ANOVA: F(1) = 2.016, *p* = 0.181; Figure 4d) or medium (two-way ANOVA: F(1) = 2.631, *p* = 0.131; Figure 4d).

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**Figure 4.** Mean (± SE) (**a**) Fv/Fm, (**b**) Y(II), (**c**) Y(NPQ) and (**d**) Y(NO), for *L. minor* grown on either synthetic wastewater or half-strength Hutner's medium at two plant surface covers (low, 25% and high, 60%) over 7 days. A hash symbol (#) denotes an effect of density for *p* < 0.05, and a star symbol (\*) denotes an interactive effect between density and medium for *p* < 0.05, as per the two-way ANOVA. **Figure 4.** Mean (±SE) (**a**) Fv/Fm, (**b**) Y(II), (**c**) Y(NPQ) and (**d**) Y(NO), for *L. minor* grown on either synthetic wastewater or half-strength Hutner's medium at two plant surface covers (low, 25% and high, 60%) over 7 days. A hash symbol (#) denotes an effect of density for *p* < 0.05, and a star symbol (\*) denotes an interactive effect between density and medium for *p* < 0.05, as per the two-way ANOVA.

#### *3.4. Re-Circulating Remediation System: Growth and Remediation at Variable Plant Densities 3.4. Re-Circulating Remediation System: Growth and Remediation at Variable Plant Densities*

The yield of *L. minor* in the re-circulating experiment was not significantly affected by the plant density (one-way ANOVA: F(2) = 3.238, *p* = 0.087; Figure 5a). However, the average biomass yield was lowest at the 20% plant density and increased to a plateau at 50% (post hoc Tukey: *p* = 0.076; Figure 5a). SGR (d−1) steadily decreased with increasing density (one-way ANOVA: F(2) = 5.143, *p* = 0.032; post hoc Tukey 20–80: *p* < 0.05; Figure 5b). Overall, TN removal (mg) from synthetic wastewater was not significantly impacted by density (one-way ANOVA: F(2) = 0.698, *p* = 0.525; Figure 5c). However, TN removal rate (mg N m−2 day−1) decreased significantly as density increased (one-way ANOVA: F(2) = 5.701, *p* < 0.05; Figure 5d). TN removal rate values dropped from around 2500 mg N m−2 day−1 at 20% plant surface cover to around 500 mg N m−2 day−1 at 80% (post hoc Tukey: *p* < 0.05; Figure 5d). Overall, mean TP removal remained at around 17 mg across the three plant densities (one-way ANOVA: F(2) = 0.419, *p* = 0.670; Figure 5e). TP removal rate (mg P m−2 day−1) decreased with increasing density conditions (one-way ANOVA: F(2) = 29.240, *p* < 0.001; Figure 5f). Mean TP removal rate dropped from 300 mg P m−2 day−1 at 20% to 75 mg P m−2 day−1 at 80% (post hoc Tukey: *p* < 0.001; Figure 5f). Protein content (% protein of dry duckweed mass) did not vary between plant densities, with mean values remaining between 17–20% (one-way ANOVA: F(2) = 0.225, *p* = 0.803; Figure 5g). The yield of *L. minor* in the re-circulating experiment was not significantly affected by the plant density (one-way ANOVA: F(2) = 3.238, *p* = 0.087; Figure 5a). However, the average biomass yield was lowest at the 20% plant density and increased to a plateau at 50% (post hoc Tukey: *p* = 0.076; Figure 5a). SGR (d−1 ) steadily decreased with increasing density (one-way ANOVA: F(2) = 5.143, *p* = 0.032; post hoc Tukey 20–80: *p* < 0.05; Figure 5b). Overall, TN removal (mg) from synthetic wastewater was not significantly impacted by density (one-way ANOVA: F(2) = 0.698, *p* = 0.525; Figure 5c). However, TN removal rate (mg N m−2 day−1 ) decreased significantly as density increased (one-way ANOVA: F(2) = 5.701, *p* < 0.05; Figure 5d). TN removal rate values dropped from around 2500 mg N m−2 day−1 at 20% plant surface cover to around 500 mg N m−2 day−1 at 80% (post hoc Tukey: *p* < 0.05; Figure 5d). Overall, mean TP removal remained at around 17 mg across the three plant densities (one-way ANOVA: F(2) = 0.419, *p* = 0.670; Figure 5e). TP removal rate (mg P m−2 day−1 ) decreased with increasing density conditions (oneway ANOVA: F(2) = 29.240, *p* < 0.001; Figure 5f). Mean TP removal rate dropped from 300 mg P m−2 day−1 at 20% to 75 mg P m−2 day−1 at 80% (post hoc Tukey: *p* < 0.001; Figure 5f). Protein content (% protein of dry duckweed mass) did not vary between plant densities, with mean values remaining between 17–20% (one-way ANOVA: F(2) = 0.225, *p* = 0.803; Figure 5g).

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**Figure 5.** Mean (± SE) (**a**) yield (g), (**b**) SGR (d−1), (**c**) TN removal (mg N), (**d**) TN removal rate per frond surface area (mg N m−2 d−1), (**e**) TP removal (mg P), (**f**) TP removal rate per frond surface area (mg P m−2 d−1) and (**g**) % protein content based on fresh duckweed biomass, FW, and dry duckweed biomass, DW, for *L. minor* grown on synthetic wastewater in a recirculating system at three plant surface covers (20, 50 and 80 %) over 5 days. Points that do not share the same letter significantly differ from one another, as per the Tukey post hoc test, *p* < 0.05. **Figure 5.** Mean (±SE) (**a**) yield (g), (**b**) SGR (d−1 ), (**c**) TN removal (mg N), (**d**) TN removal rate per frond surface area (mg N m−2 d −1 ), (**e**) TP removal (mg P), (**f**) TP removal rate per frond surface area (mg P m−2 d −1 ) and (**g**) % protein content based on fresh duckweed biomass, FW, and dry duckweed biomass, DW, for *L. minor* grown on synthetic wastewater in a re-circulating system at three plant surface covers (20, 50 and 80 %) over 5 days. Points that do not share the same letter significantly differ from one another, as per the Tukey post hoc test, *p* < 0.05.

#### **4. Discussion 4. Discussion**

[36,37].

#### *4.1. L. minor Growth on Synthetic Wastewater 4.1. L. minor Growth on Synthetic Wastewater*

RGR values recorded in the stationary experiments (based on the first 3-days of growth; 0.03–0.15 d−1) are at the lower end of the range found in the literature for duckweed grown on wastewater (0.04–0.3 d−1) [24,51,52]. They are also lower than those found for duckweed grown on an optimised medium (0.153–0.519 d−1) [26]. This may, in part, be due to the composition of the medium, as duckweed cultivated on wastewater often results in lower growth rates than obtained on optimised growing medium (compare growth rates in Ziegler et al. [26] with Al-Nozaily et al. [51]). As well as this, the relatively low light intensity of 50 µmol m−2 s −1 used for the stationary experiments may have also contributed to reduced growth rates [53]. Nevertheless, it has been shown that the light saturation point is around 50 µmol m−2 s −1 for *L. minor* grown on synthetic dairy wastewater [32], although the light saturation point for duckweed on more optimised media is 400–600 µmol m−2 s −1 [44,54]. Commonly used experimental light intensities range from 85 to 130 µmol m−2 s −1 [26,52,55], which are in line with OECD guidelines for duckweed toxicity growth inhibition tests [56]. Although, a higher light intensity of 300 µmol m−2 s −1 was used in the re-circulating experiment this only led to marginally improved growth rates. In both the stationary and re-circulating experiments, SGR decreased with increasing density, which corresponds with general trends noted within the literature RGR values recorded in the stationary experiments (based on the first 3-days of growth; 0.03–0.15 d−1 ) are at the lower end of the range found in the literature for duckweed grown on wastewater (0.04–0.3 d−1 ) [24,51,52]. They are also lower than those foundfor duckweed grown on an optimised medium (0.153–0.519 d−1 ) [26]. This may, in part, bedue to the composition of the medium, as duckweed cultivated on wastewater often results in lower growth rates than obtained on optimised growing medium (compare growthrates in Ziegler et al. [26] with Al-Nozaily et al. [51]). As well as this, the relatively low light intensity of 50 µmol m−2 s −1 used for the stationary experiments may have also contributed to reduced growth rates [53]. Nevertheless, it has been shown that the light saturation point is around 50 µmol m−2 s −1 for *L. minor* grown on synthetic dairy wastewater [32], although the light saturation point for duckweed on more optimised media is 400–600 µmol m−2 s −1 [44,54]. Commonly used experimental light intensities range from 85 to 130 µmol m−2 s −1 [26,52,55], which are in line with OECD guidelines for duckweed toxicity growth inhibition tests [56]. Although, a higher light intensity of 300 µmol m−2 s −1 was used in the re-circulating experiment this only led to marginally improved growth rates. In both the stationary and re-circulating experiments, SGR decreased with increasing density, which corresponds with general trends noted within the literature [36,37].

## *4.2. Exploring the Mechanism Underlying Density Dependent Changes in Growth Using Chlorophyll Fluorometry*

The negative effect of high density on growth may be a result of greater competition for nutrients [57], a quicker depletion of nutrients [58], or self-shading between colonies [59]. Yet, plant-plant competition has also been related to plant neighbour detection, including plant responses such as shade avoidance, root foraging, and use/induction of chemical defences [60,61]. Therefore, it is possible that *L. minor* senses the closeness of other plants and switches to a more defensive growth strategy, the trade-off of which is a reduction in growth rate. To explore the mechanism underlying the observed decrease in SGR with increasing plant surface density, plants were grown at lower and higher plant surface densities, but with identical biomass per medium volume (stationary experiment 2). This experiment confirmed the impediment of growth rate at higher plant surface densities. However, as plants at each surface density had access to the same volume of medium, the data imply that the growth impediment was due to factors other than nutrient depletion. Indeed, this point is further confirmed by the observation that similar growth impediments occurred at high plant density irrespective of whether nutrient rich Hutner's medium or more oligotrophic dairy wastewater was used.

Analysis of photosynthetic parameters in stationary experiment 2, measured after seven days of growth, showed that at high density the photosynthetic quantum yield of PSII, Y(II), was significantly depressed. A reduction in Y(II) for *L. minor* grown at high density means that the plants were using light energy less efficiently compared to those at a lower plant surface density [62]; an effect which was stronger for plants grown on synthetic wastewater. Y(II), together with Y(NPQ) and Y(NO) account for the partitioning of absorbed light energy in PSII [63], the sum of which equals 1 [64]. Accordingly, a reduction in Y(II) implies a concurrent increase in Y(NPQ) and/or Y(NO). The data reveal non-significant increases in Y(NPQ) at higher plant densities for both media, indicating minor increases in the amount of light energy dissipated in a regulated manner, i.e., through thermal dissipation [63], at these higher densities. Thus, a key finding is a densitydependent decrease in Y(II) which is not matched by clear significant parallel increases in Y(NO) and Y(NPQ), nor a clear effect on Fv/Fm.

Overall, the data indicate that duckweed density can affect aspects of the plant's metabolism (e.g., carbon assimilation or nitrogen metabolism), rather than having a direct effect on PSII activity (i.e., Fv/Fm). As such, this would indirectly reduce photosynthetic yield (Y(II)) and therefore biomass growth [65]. Previously, Kufel et al. [66] described complex changes in plant morphology in *L. minor* grown at different plant densities. While Zhang et al. [67] described morphological responses of *Spirodela polyrhiza* to population density that included decreased frond and root size, as well as increased frond thickness. It is possible that shading is a driver of these changes and induces a shade avoidance response in plants at higher densities [60,61]. Nevertheless, acclimation to shade typically results in increased Y(II) and decreased Y(NPQ) at low measuring light intensities [68,69]. However, the chlorophyll fluorescence data in this study show decreased Y(II) at higher plant densities where shading might potentially have been an issue. Therefore, neither a lack of nutrient nor light supply, two well-advocated explanations, adequately explain the high-density induced impediment of growth. Rather, the data point to plant neighbour detection between *Lemna*-colonies as the most likely explanation of this [60,61]. At present, touch, volatile organic compounds, chemical exudates and possibly even acoustic signals, have all been associated with neighbour detection [70]. Although it is not known to what extent these apply to duckweed, which tend to produce dense mats in natural habitats [35]. Jang et al. [71] reported that *Lemna japonica* may release interfering chemicals via its root systems, although the effects were interspecific. Similarly, Bich and Kato-Noguchi [72] reported on interspecific allelopathic signals from *Lemna minor*. It is less clear how allelopathic signals manifest between neighbouring duckweed colonies of the same species or clone. In some duckweed species high density, or 'overcrowding', has been associated with the production of ethylene, a possible early signal for an increasing lack

of space which can inhibit growth [73]. Ethylene has been shown in other plant species to an inhibitor of plant growth [74]. Further studies have shown that the overcrowdingstimulated production of ethylene in duckweed is a Ca2+ and phytochrome-dependent process [75,76]. A transient increase in cytoplasmic Ca2+ is followed by an increase in ethylene production [75]. Nevertheless, further exploration is required to understand how plant signals, such as ethylene and other potentially unknown signals, influence *L. minor* growth and metabolism in high density conditions.

#### *4.3. L. minor Biomass Yield and Protein Content under Variable Density and System Conditions*

Given its high protein content [29,77], and usefulness as a biofuel and a source of phytochemicals [28,78], *Lemna minor* biomass is an important by-product of the wastewater remediation process. As such, the absolute biomass yield is an important parameter, as it relates directly to the amount of plant mass available for further use [27]. Previously, high duckweed densities of 60–80% plant coverage (around 160–280 g m−2 ) have been reported to result in maximum yields, in combination with different harvesting regimes [39,79,80]. Data from stationary experiment 1 and the re-circulating experiment show that at higher plant densities the logarithmic relationship between plant density and yield will plateau. Consequently, the yield increment becomes smaller. However, significant differences between density treatments were not found. Therefore, a clear benefit of higher plant density for biomass yield was not detected. The second stationary experiment shows the opposite trend between density and yield. In this experiment the use of the same plant biomass, but with different container surface areas, led to both a higher growth rate and overall yield at low density. Thus, the use of a shallower/wider container for a volume of wastewater would improve duckweed yield, i.e., surface area space is an important factor in duckweed yield. However, consideration would have to be given to the impact of algae on this result, as less duckweed cover tends to increase the light availability to algae [81].

Duckweed density did not affect protein content in the stationary or re-circulating systems. Nevertheless, the protein content of 30–35% on a dry weight basis found in this study compares favourably with literature sources, where duckweed protein contents up to 45% of dry weight have been reported [44]. Although, more commonly reported values are between 20–35% [28,82]. Furthermore, this compares relatively well with the commonly used high-protein feed, soybean (33–49% [83]). It should be noted that the use of the Bradford assay with BSA as a standard can underestimate plant protein content when compared to other techniques [84,85], which may affect comparisons with literature sources.

The protein content detected for plants grown in the re-circulating system ranged from 17.5–20% of dry weight, which can be considered a low protein content relative to the published range. This demonstrates that scaling up, and the use of circulatory, non-axenic systems may have unexpected consequences. Starch content was not measured in this study, but, as has been observed in some studies, a lower protein content can result in, or be a result of, higher starch content [27]. If low protein content is a consistent problem for duckweed grown on a large-scale, there are established alternative uses for the biomass that do not depend on the protein content, such as biofuel production [86].

#### *4.4. Remediation of TN and TP by L. minor from Synthetic Wastewater*

The trend of decreasing growth with increasing density was reflected in the relationship between TN/TP removal rate and density. In both stationary and re-circulating experiments, the lower removal rates of TN and TP per plant surface area (g m−2 d −1 ) at higher density conditions show that each duckweed colony is removing less nutrients at higher plant densities than those kept at lower densities. The TN and TP removal rates found in the stationary experiments were on the lower end of the wide range of values recorded in the literature: 124–4400 mg N m−2 d −1 and 14–590 mg P m−2 d −1 [57,82,87–90]. This can be explained by the lower growth rates observed in these experiments [91], which are likely to be in part due to the low light intensity used [32], the specific medium [23], as well as density effects on *L. minor* discussed previously. Both TN and TP removal rates

from the non-axenic, re-circulating experiment compare well with values from non-axenic systems in literature sources [57,91]. Higher removal rates found in the re-circulating experiment compared to stationary may be mostly explained by the presence of algae and microorganisms, which can account for up to 50–70% of nutrient removal in non-axenic systems [57,88], but also by improved mixing of the medium.

Another important criterium for analysing the effectiveness of a remediation system is the absolute amount of nutrients removed from the remediated wastewater. In stationary experiment 1, a higher density of plants per surface area more effectively takes up nutrients, although the growth and nutrient removal rates per frond are significantly lower [36,37]. As the removal of nutrients started to plateau at higher densities of 50–80%, this suggests that 50–80% represents an optimal duckweed density for dairy wastewater phytoremediation.

There were additional issues encountered in a non-axenic re-circulating system, in which the absolute removal of TN and TP was relatively flat across the three densities. The exact cause of this is uncertain. However, microorganisms were present in substantial amounts at all plant densities in this non-axenic system. Previously, algae were shown to significantly contribute to nutrient removal [57,81], as well as having negative effects on duckweed such as increased decomposition rates [92]. At both high and low density, a high proportion of the absolute removal of TN and TP may be attributed to microorganisms, breaking the direct link between plant growth and nutrient removal (e.g., poor plant growth, but high nutrient removal). Considering wastewater remediation alone, whether remediation is fuelled primarily by duckweed or algae, nutrient removal is the desired outcome. However, if duckweed biomass is to be generated as a valuable by-product for further use, then strong competition for nutrients with non-utilised microorganisms is undesirable. Ideally, the duckweed should be taking up the majority of the nutrients. One way to achieve this is by maximising the duckweed surface density, thereby decreasing algal growth [93]. Overall, these results show that upscaling from laboratory-based, stationary systems to larger scale, recirculatory systems is complex, and that simple extrapolations are not necessarily correct. Accordingly, management of duckweed incubators will need to be informed by the effects of plant density on biomass yield, TN and TP removal, as well as competition with algal species.

#### **5. Conclusions**

*Lemna minor* can be successfully grown on synthetic dairy processing wastewater, opening the perspective to both remediate and valorise such waste, in accordance with the principles of the circular economy. *Lemna minor* has been shown to produce the best remediation at higher densities (50–80%), even though growth rates and nutrient uptake rates were slowest at these densities. The decrease in growth at high density was linked to a decrease in photosynthetic yield, rather than competition for light or nutrients, which points towards signals from neighbouring colonies as the potential cause of growth restrictions. However, in non-axenic, scaled-up conditions that better reflect an industrial duckweedbased remediation system, the benefits of high density were not as clear. High algal presence led to suppressed duckweed yield and static nutrient removal. Thus, despite the suitability of *L. minor* for valorisation of dairy processing waste, management of wastewater is subject to both interactions between plant density, yield and nutrient removal, as well as complex upscaling effects.

**Author Contributions:** É.W., H.K. and M.A.K.J. contributed to the study conception and design. Material preparation, data collection and analysis were performed by É.W., H.K. and S.O.; É.W., N.E.C., S.O., M.A.K.J. and H.K. contributed to the interpretation of results. The first draft of the manuscript was written by É.W.; É.W., N.E.C., S.O., M.A.K.J. and H.K. contributed to writing and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was funded under the EPA Research Programme 2014–2020 (grant 2016- W-LS-11). The EPA Research Programme is a Government of Ireland initiative funded by the Department of Communications, Climate Action and Environment. It is administered by the Environmental Protection Agency, which has the statutory function of co-ordinating and promoting environmental research.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets used in the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** MAKJ acknowledges support by WoB.

**Conflicts of Interest:** The authors have no relevant financial or non-financial interests to disclose.

### **References**

## *Article* **Cultivation of Lemna Minor on Industry-Derived, Anaerobically Digested, Dairy Processing Wastewater**

**Rachel O'Mahoney 1,2, Neil E. Coughlan 1,2 , Éamonn Walsh 1,2 and Marcel A. K. Jansen 1,2,\***

**Abstract:** The growth and nutrient uptake capacity of a common duckweed (Lemnaceae) species, Lemna minor "Blarney", on dairy processing wastewater pre-treated by an anaerobic digester (AD-DPW) was explored. *L. minor* was cultivated in small stationary vessels in a controlled indoor environment, as well as in a semi-outdoor 35 L recirculatory system. The use of AD-DPW as a cultivation medium for *L. minor* offers a novel approach to dairy wastewater treatment, evolving from the current resource-intensive clean-up of wastewaters to duckweed-based valorisation, simultaneously generating valuable plant biomass and remediating the wastewater.

**Keywords:** anaerobic digestate; biomass generation; circular economy; Lemnaceae; nutrient recovery; phytoremediation

## **1. Introduction**

Clean water is fundamental for life on Earth. However, by 2025 more than 40% of people worldwide will live under conditions of water insecurity [1]. Such a serious level of insecurity calls for advances in the efficiency of water use, for example, through water conservation and improved wastewater remediation [2]. Currently, food production industries account for a large portion of water use and wastewater production in many parts of the world [1,3,4]. Accordingly, there is considerable scope to develop circular economy approaches for improved water use efficiency and wastewater valorisation within many industry settings. For example, the dairy industry produces a considerable amount of wastewater during the production process and is in many countries considered the most significant source of wastewater [5]. It has been approximated that up to 11 litres of wastewater are produced for every one litre of milk processed, depending on the dairy product being made (e.g., drinking milk, cheese, butter, yogurt) [5,6]. Consequently, a dairy processing plant can produce upwards of 550 million litres of wastewater per year [6]. At present, dairy industries in most countries operate extensive wastewater treatment plants cleaning wastewater so that release on local surface waters is permissible [7]. This amounts to a considerable economic cost for the industry. Yet, nutrient-rich dairy processing wastewater (DPW) contains potentially valuable resources that can be valorised in line with the principles of circular economy [8].

A range of sequential biological and physicochemical techniques are used in DPW treatment facilities to minimise the concentrations of environmentally damaging pollutants [9], which include high biological oxygen demand (BOD) and chemical oxygen demand (COD) together with considerable amounts of nitrogen and phosphorus [5,10]. Anaerobic digestion is a widely employed method, in which microbial organisms, such as *Bacteria*, *Archaea*, and protozoa, metabolise organic compounds primarily into methane [11]. Anaerobic digestion can substantially decrease BOD and COD but is less effective in reducing nitrogen and phosphorus content [12]. Therefore, following biological treatment,

**Citation:** O'Mahoney, R.; Coughlan, N.E.; Walsh, É.; Jansen, M.A.K. Cultivation of Lemna Minor on Industry-Derived, Anaerobically Digested, Dairy Processing Wastewater. *Plants* **2022**, *11*, 3027. https://doi.org/10.3390/ plants11223027

Academic Editors: Viktor Oláh, Klaus-Jürgen Appenroth and K. Sowjanya Sree

Received: 26 August 2022 Accepted: 4 November 2022 Published: 9 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

physicochemical treatments serve to filter, precipitate, coagulate, flocculate or even electrochemically process nitrogen and phosphorus, as well as any remaining suspended fats, lactose, and other organic matter contents [13]. Typically, nitrogen is dissipated in the form of nitrogen–gas through the nitrification–denitrification cycle, whilst phosphorus is chemically precipitated, often using aluminium. While this remediates water and prevents eutrophication, the aforementioned nutrients are not reused. It has been argued that recycling of nutrients, particularly the phosphate, nitrate, and ammonium content of DPW, can help generate value from the waste and diminish the need to exploit limiting resources of rock phosphate as well as the need for fossil fuel-based urea production. Essentially, through the cultivation of plant biomass on wastewater, nutrients can be captured and recycled [14,15].

Duckweeds (Lemnaceae) are a family of small, free-floating aquatic macrophytes, of which there are five genera and 36 species dispersed throughout much of the world [16]. Lemnaceae are the fastest-growing angiosperms in the plant kingdom with growth often described as doubling times [17]. Lemnaceae are particularly apt for wastewater remediation due to their rapid growth, high nutrient uptake rate, broad-ranging tolerance of growing conditions, and overall ease of harvesting [17–19]. Lemnaceae preferentially take up ammonium over nitrate, which is paramount in wastewater remediation, in which ammonium is the dominant form of N [20,21]. Value from Lemnaceae's high biomass production can be found in use as biofuel, fertiliser, and/or feed [21,22]. In essence, Lemnaceae can clean water, while simultaneously producing a valuable protein-rich biomass that embodies the closed-loop, circular economy approach of retaining value through the recovery of resources from waste [21,22]. Thus, the integration of Lemnaceae cultivation into the wastewater purification processes can be advantageous to the long-term sustainability of the dairy industry.

In the present study, it was assessed to what extent a common Lemnaceae species, *Lemna minor*, can be grown on DPW pre-treated by an anaerobic digester (AD-DPW) with consideration of both biomass generation and wastewater remediation. The organic load of DPW is high and facilitates strong microbial growth, at the expense of duckweed growth. Therefore, pre-treatment of DPW is required to lower COD and BOD and to facilitate duckweed growth. Previous research has focussed on dairy wastewater pre-processed through small-batch microbial digesters, linked to the production of bioplastics [23]. Here, we pioneered *L. minor* growth on industry-derived, anaerobically digested, dairy processing wastewater (AD-DPW) in small, stationary vessels in a controlled indoor environment, as well as in a semi-outdoor, 35-litre recirculatory system. It was hypothesised that there would be a clear concentration dependence of growth, with lower concentrations of AD-DPW being sub-optimal, and higher concentrations of AD-DPW being super-optimal for key plant nutrients.

#### **2. Results**

#### *2.1. Physicochemical Analysis of AD-DPW*

Physicochemical analysis revealed the concentrations of compounds and elements present in 100% AD-DPW (Table 1). BOD and COD were present at 28.1 and 117 mg O2 L −1 , respectively, while the concentration of total solids amounted to 3.29 g L−1 . The total nitrogen (TN) concentration was 105 mg L−1 , of which 95 mg N L−1 was ammonia and less than 0.01 mg L−1 was nitrate. Thus, the total ammonia concentration in AD-DPW was close to the maximum tolerated by duckweed, although at the applied pH (pH = 6) concentrations of toxic ammonia would have been very low. The total phosphorus (TP) concentration was 27.7 mg P L−1 of which 24.8 mg L−1 was orthophosphate. Thus, the orthophosphate concentration in the medium was close to optimal for duckweed growth. Other plant nutrients were also present in AD-DPW at concentrations close to optimal, such as potassium, calcium, magnesium, iron, zinc, and copper. Chloride is present at a high concentration, but within the limits typically tolerated by duckweed species.

**Table 1.** Concentration of compounds and elements present in AD-DPW c , along with duckweed's required, tolerated, and optimal concentrations.

a [15], b ND—not determined, c AD-DPW—anaerobically digested dairy processing wastewater, d see [24], e prior to experiments the pH of AD-DPW was reduced to pH 6.0.

### *2.2. The Effect of Varying AD-DPW Concentration on L. minor Growth*

Plants displayed modest growth on all concentrations of AD-DPW, with RGR values reaching up to 0.13 d−1 . Overall, significant differences were observed between treatments (ANOVA: χ 2 = 12.788, df = 6; *p* = 0.03: Figure 1) although, the post hoc analysis did not reveal any specific differences between concentrations. Plants cultivated on 10% AD-DPW had the highest RGR and appeared to have the healthiest colour and frond-to-colony ratio compared to all other AD-DPW concentrations. Frond size on 50% and 100% AD-DPW concentrations appeared reduced in comparison to 10% AD-DPW. Plants kept on 100% AD-DPW also displayed fragmented colonies and death (chlorosis).

#### *2.3. Growth of L. minor on Varying Concentrations of AD-DPW over a Six-Week Period*

For this experiment, the highest RGR value found was 0.35 d−1 (Figure 2). In some cases (50% and 100% AD-DPW) no growth was observed. The RGR differed significantly for *L. minor* grown on different AD-DPW concentrations over a six-week period (ANOVA: χ 2 = 303.94, df = 35; *p* < 0.0001, Figure 2). Concentration had a significant effect on RGR (*p* < 0.0001), while the week had no effect (*p* > 0.05). A significant interaction effect on RGR was detected for concentration and week (*p* < 0.0001). For individual weeks, RGR was greater for plants grown on the control (half-strength Hutner's medium), 1%, 5%, and 10% concentrations than for duckweed cultivated on 50% and 100% concentrations. The 50% and 100% concentrations gave the least growth, with no survival occurring except for that of the 50% in week 1. Overall, as the concentration of AD-DPW increased, the

*Plants* **2022**, *11*, x FOR PEER REVIEW 4 of 14

RGR decreased. However, the distribution of *L. minor* growth over the successive weeks followed a different trajectory pattern for each concentration.

**Figure 1.** Mean (±SE) RGR (d−1) of *Lemna minor* cultivated on AD-DPW at 1%, 5%, 10%, 50%, and 100% concentrations in one-week experiments, as well as a control treatment. No significant differ-**Figure 1.** Mean (±SE) RGR (d−1 ) of *Lemna minor* cultivated on AD-DPW at 1%, 5%, 10%, 50%, and 100% concentrations in one-week experiments, as well as a control treatment. "a": Denoting no significant difference was detected through post hoc analysis.

#### ence was detected through post hoc analysis. *2.4. Growth of L. minor in Semi-Outdoor, Recirculating Systems*

*2.3. Growth of L. minor on Varying Concentrations of AD-DPW over a Six-Week Period*  For this experiment, the highest RGR value found was 0.35 d−1 (Figure 2). In some cases (50% and 100% AD-DPW) no growth was observed. The RGR differed significantly for *L. minor* grown on different AD-DPW concentrations over a six-week period (ANOVA: χ2 = 303.94, df = 35; *p* < 0.0001, Figure 2). Concentration had a significant effect on RGR (*p* < 0.0001), while the week had no effect (*p* > 0.05). A significant interaction effect on RGR was detected for concentration and week (*p* < 0.0001). For individual weeks, RGR was Plants displayed modest growth on both 5% and 10% AD-DPW, with RGR values ranging from 0.07 to 0.1 d−1 . The RGR significantly differed for *L*. *minor* cultivated on different concentrations of AD-DPW in the tiered tank system (ANOVA: χ 2 = 24.287, df = 2; *p* < 0.0001, Figure 3). Concentration had a significant effect on RGR (*p* < 0.0001), while tank position in the tiered system had no effect (*p* > 0.05). No interaction effect on RGR was detected for concentration and tank (*p* > 0.05). Plants grown on 10% AD-DPW had a higher RGR than *L*. *minor* cultivated on either the control medium (*p* < 0.0001) or 5% AD-DPW (*p* < 0.001). The RGR (d−1 ) of plants grown on 5% AD-DPW was similar to that of plants grown on the control medium (*p* > 0.05).

#### greater for plants grown on the control (half-strength Hutner's medium), 1%, 5%, and 10% concentrations than for duckweed cultivated on 50% and 100% concentrations. The 50% *2.5. Nutrient Depletion and Removal Rates in the Semi-Outdoor, Recirculatory System*

and 100% concentrations gave the least growth, with no survival occurring except for that of the 50% in week 1. Overall, as the concentration of AD-DPW increased, the RGR decreased. However, the distribution of *L. minor* growth over the successive weeks followed a different trajectory pattern for each concentration. Total nitrogen (TN) and total phosphorus (TP) concentrations in the medium were determined at the start (day 0) and the end (day 7) of the 7-day experiments growing *L. minor* in the tiered tank system, using different media. Total nitrogen concentrations (TN) at day 7 were significantly lower than initial TN at day 0 for both 5% and 10% AD-DPW (both *p* < 0.0001).

TN was found to significantly differ between treatments and time-points (GLM: χ 2 = 85.757, df = 3; *p* < 0.0001, Figure 4). The tested media (5% and 10% AD-DPW) and sampling day (day 0 or day 7) were found to have a significant effect on the TN concentration (both *p* < 0.0001). A significant interaction effect was also detected for the media and day (*p* < 0.0001).

**Figure 2.** Mean (±SE) RGR (d⁻ 1 ) of *Lemna minor* growth for the control treatment (panel A), as well as on 1%, 5%, 10%, 50%, and 100% AD-DPW (panel B, C, D, E and F, respectively) over the course of the six successive weeks. Different letters within each panel denote statistical differences between weeks. For individual weeks, RGR was greater for plants grown on the control (half-strength Hutner's medium), 1%, 5%, and 10% concentrations than for duckweed cultivated on 50% and 100% concentrations: week 1 (all *p* < 0.05), week 2 (all *p* < 0.05), week 3 (all *p* < 0.01; except the 10% solution, *p* > 0.05), week 4 (all *p* < 0.01), week 5 (all *p* < 0.01), week 6 (all *p* < 0.01). **Figure 2.** Mean (±SE) RGR (d−1 ) of *Lemna minor* growth for the control treatment (panel (**A**)), as well as on 1%, 5%, 10%, 50%, and 100% AD-DPW (panel (**B**–**F**), respectively) over the course of the six successive weeks. Different letters within each panel denote statistical differences between weeks. For individual weeks, RGR was greater for plants grown on the control (half-strength Hutner's medium), 1%, 5%, and 10% concentrations than for duckweed cultivated on 50% and 100% concentrations: week 1 (all *p* < 0.05), week 2 (all *p* < 0.05), week 3 (all *p* < 0.01; except the 10% solution, *p* > 0.05), week 4 (all *p* < 0.01), week 5 (all *p* < 0.01), week 6 (all *p* < 0.01).

The concentration of total phosphorus (TP) within the tiered tank system was significantly reduced between day 0 and day 7, in both 5% and 10% AD-DPW (both *p* < 0.001). Similar to TN, TP was found to significantly differ across treatments and time-points (GLM: χ 2 = 29.233, df = 3; *p* < 0.0001, Figure 4). The tested media (5% and 10% AD-DPW) and sampling day (day 0 or day 7) were found to have a significant effect on TP concentration (*p* < 0.05 and *p* < 0.0001, respectively). No significant interaction effect was detected.

The removal rate of TN and TP from AD-DPW by *L. minor* was determined by calculating the difference between the initial (day 0) and final (day 7) nutrient concentrations and expressing it per m−2 duckweed per day. The 5% AD-DPW had nutrient removal rates of 43.72 mg TN m−2d −1 and 53.76 mg TP m−2d −1 while 10% AD-DPW had higher nutrient removal rates of 248.46 mg TN m−2d −1 and 126.54 mg TP m−2d −1 .

*2.4. Growth of L. minor in Semi-Outdoor, Recirculating Systems*

ranging from 0.07 to 0.1 d−1

< 0.001). The RGR (d−1

grown on the control medium (*p* > 0.05).

Plants displayed modest growth on both 5% and 10% AD-DPW, with RGR values

ferent concentrations of AD-DPW in the tiered tank system (ANOVA: χ2 = 24.287, df = 2; *p* < 0.0001, Figure 3). Concentration had a significant effect on RGR (*p* < 0.0001), while tank position in the tiered system had no effect (*p* > 0.05). No interaction effect on RGR was detected for concentration and tank (*p* > 0.05). Plants grown on 10% AD-DPW had a higher RGR than *L*. *minor* cultivated on either the control medium (*p* < 0.0001) or 5% AD-DPW (*p*

. The RGR significantly differed for *L*. *minor* cultivated on dif-

) of plants grown on 5% AD-DPW was similar to that of plants

**Figure 3.** Mean (±SE) RGR (d⁻ 1 ) for *Lemna minor* grown on the control medium (half-strength Hutner's medium), 5% and 10% AD-DPW in the semi-outdoor, recirculating system. Bars that do not **Figure 3.** Mean (±SE) RGR (d−1 ) for *Lemna minor* grown on the control medium (half-strength Hutner's medium), 5% and 10% AD-DPW in the semi-outdoor, recirculating system. Bars that do not share the same letter significantly differ from one another for *p* < 0.001, as per the post hoc test. *Plants* **2022**, *11*, x FOR PEER REVIEW 7 of 14

*p* < 0.0001). A significant interaction effect was also detected for the media and day (*p* < 0.0001). **Figure 4.** Comparison of the total nitrogen (TN) and the total phosphate (TP) concentration (mg L⁻1 ) on day 0 and day 7 of *L. minor* growth, when cultivated on 5% or 10% AD-DPW (mean ±SE). For all treatments, the total nitrogen or total phosphate concentration was significantly lower on day 7 than on day 0 (*p* < 0.05). **Figure 4.** Comparison of the total nitrogen (TN) and the total phosphate (TP) concentration (mg L−1 ) on day 0 and day 7 of *L. minor* growth, when cultivated on 5% or 10% AD-DPW (mean ± SE). For all treatments, the total nitrogen or total phosphate concentration was significantly lower on day 7 than on day 0 (*p* < 0.05).

#### The concentration of total phosphorus (TP) within the tiered tank system was signif-**3. Discussion**

**3. Discussion**

icantly reduced between day 0 and day 7, in both 5% and 10% AD-DPW (both *p* < 0.001). Similar to TN, TP was found to significantly differ across treatments and time-points (GLM: χ2 = 29.233, df = 3; *p* < 0.0001, Figure 4). The tested media (5% and 10% AD-DPW) and sampling day (day 0 or day 7) were found to have a significant effect on TP concentration (*p* < 0.05 and *p* < 0.0001, respectively). No significant interaction effect was detected. Multiple factors determine whether AD-DPW is a suitable growth medium for *L. minor*,and hence suitable for the process of phytoremediation. These include the presence of essential macro- and micronutrients in suitable quantities; low enough BOD and COD levels to avoid excessive microbial growth; a favourable combination of pH and ammonia for plant growth; and a suitable calcium-to-magnesium ratio for growth.

#### The removal rate of TN and TP from AD-DPW by *L. minor* was determined by calculating the difference between the initial (day 0) and final (day 7) nutrient concentrations *3.1. Presence in Appropriate Quantities of All Essential Macro- and Micronutrients*

and expressing it per m−2 duckweed per day. The 5% AD-DPW had nutrient removal rates of 43.72 mg TN m−2d−1 and 53.76 mg TP m−2d−1 while 10% AD-DPW had higher nutrient removal rates of 248.46 mg TN m−2d−1 and 126.54 mg TP m−2d−1 . Various studies have shown that highly concentrated wastewater inhibits duckweed growth, which means that dilutions are necessary to prevent phytotoxic effects [20]. The present study shows that *L. minor* grows best on lower concentrations of AD-DPW, under both stationary indoor as well as recirculatory semi-outdoor conditions. The RGR of

Multiple factors determine whether AD-DPW is a suitable growth medium for *L. mi-*

Various studies have shown that highly concentrated wastewater inhibits duckweed growth, which means that dilutions are necessary to prevent phytotoxic effects [20]. The present study shows that *L. minor* grows best on lower concentrations of AD-DPW, under both stationary indoor as well as recirculatory semi-outdoor conditions. The RGR of *L. minor* was greater for plants cultivated on 10% AD-DPW compared to 5% which suggests that the nutrient composition of 5% AD-DPW is sub-optimal for growth, potentially with various nutrients becoming limiting. Conversely, reduced growth on 100% AD-DPW (compared to 10% AD-DPW) indicates some form of toxicity. To understand why *L. minor* grew slower or not at all at low (<5%) as well as high (>50%) concentrations of AD-DPW,

Duckweed prefers total nitrogen concentrations of between 2.8 and 350 N mg−1 L−1 and generally takes up N in the form of ammonium or nitrate (Gil-Pulido et al. 2018). The concentration of total ammonia on full-strength AD-DPW is above optimal, and this may contribute to the lower growth of *L. minor* on full-strength AD-DPW compared to dilutions [24]. However, there are also other elements present in the medium that may cause growth impediments at higher concentrations. For example, chloride (Cl) levels in 100%

levels to avoid excessive microbial growth; a favourable combination of pH and ammonia

nutrient requirements of *L. minor* were considered.

for plant growth; and a suitable calcium-to-magnesium ratio for growth.

*3.1. Presence in Appropriate Quantities of All Essential Macro- and Micronutrients*

*L. minor* was greater for plants cultivated on 10% AD-DPW compared to 5% which suggests that the nutrient composition of 5% AD-DPW is sub-optimal for growth, potentially with various nutrients becoming limiting. Conversely, reduced growth on 100% AD-DPW (compared to 10% AD-DPW) indicates some form of toxicity. To understand why *L. minor* grew slower or not at all at low (<5%) as well as high (>50%) concentrations of AD-DPW, nutrient requirements of *L. minor* were considered.

Duckweed prefers total nitrogen concentrations of between 2.8 and 350 N mg−1 L −1 and generally takes up N in the form of ammonium or nitrate (Gil-Pulido et al., 2018). The concentration of total ammonia on full-strength AD-DPW is above optimal, and this may contribute to the lower growth of *L. minor* on full-strength AD-DPW compared to dilutions [24]. However, there are also other elements present in the medium that may cause growth impediments at higher concentrations. For example, chloride (Cl) levels in 100% AD-DPW are at 1319 mg L−1 , well above the optimal range of 0.035–350 mg L−1 [25,26]. Similarly, sodium levels are higher than those tolerated by duckweed. Salinity-induced osmotic stress of *L. minor* inhibits vegetative growth [25,26]. Thus, it is concluded that to facilitate *L. minor* growth and remediation on AD-DPW, it needs to be substantially diluted. In contrast, concentrations of several cations (Mn, Zn, Fe, and K) are close to optimum in undiluted AD-DPW [26–28] and dilutions may cause deficiencies. Similarly, orthophosphate concentrations in undiluted AD-DPW are considered optimal, being between 0.3 and 54.2 P mg−1 L −1 [26,29]. So, phosphate will easily become a limiting factor at the lower concentrations tested here (i.e., 1% and 5% dilution), especially after a period of duckweed growth [30]. The present study shows that duckweed removed 46% of the TN and 90% of the TP present in the 10% concentration of AD-DPW, under semi-realistic conditions of medium velocity and outdoor light conditions. Thus, the low concentration of TP remaining in both 5% and 10% AD-DPW may in turn have hindered further growth and, thereby, uptake of TN, and explain both the relatively low RGR (d−1 ) values and low TN removal. This observation underlines important issues concerning the management of wastewater and triggers the question of whether supplementation of wastewater-derived media with specific elements (i.e., phosphate in this case) is required to maintain sufficient *L. minor* growth and consequent parallel TN and TP removal.

#### *3.2. Low BOD and COD Levels to Avoid Excessive Microbial Growth*

Raw DPW contains a high load of BOD and COD and is not suitable for *L. minor* growth due to the rapid growth of a microbial scum on top of the lactose-rich medium (Walsh et al. in press). AD-DPW has already much of its organic components removed [31]. A physicochemical analysis of the raw non-diluted AD-DPW used by the present study (i.e., 100% concentration), confirms that both BOD and COD levels in 100% AD-DPW are unlikely to impede *L. minor* growth (see Table 1).

#### *3.3. A Favourable Combination of pH and Ammonia for Plant Growth*

The addition of H2SO4 to decrease the pH of the AD-DPW prevented the negative effects of a high pH on growth as observed in a pilot experiment. This is most likely due to avoidance of the combination of an alkaline pH with high ammonia concentrations. Total ammonia in solution consists of ionised ammonium (NH4 + ) and un-ionised ammonia (NH3), with the relative proportion of each being pH-dependent. The main toxic form is un-ionised ammonia, with a more minor contribution of ammonium. So, a high total ammonia concentration is predominantly toxic at a higher pH [24,32]. For example, in the case of 100% AD-DPW, ammonia levels of 95 mg L−1 were present. At the native pH of 8, 3–4 percent of the total ammonia will have occurred as toxic un-ionised ammonia (i.e., ~3 mg L−1 ). The latter concentration of un-ionised ammonia is reflected in the negative growth observed in the pilot experiment, in accordance with the isopleth maps generated by Körner et al. [24] for *L. gibba*. In contrast, at pH 6 less than 0.2 mg L−1 of un-ionised ammonia will have been present in 100% AD-DPW, but still, a modest growth inhibition can be predicted to occur due to ionised ammonium [24]. Körner et al. [24] found a maximum

RGR was achieved with 10 mg L−1 total ammonia at pH 6.8, resulting in a combination of 9.97 mg L−1 ionised ammonium (NH4 + ) and 0.03 mg L−1 un-ionised ammonia (NH3). This explains why the best growth results were obtained using dilutions of around 10% of AD-DPW, in combination with a lowering of the pH to around 6.

#### *3.4. A Suitable Calcium-to-Magnesium Ratio for Growth*

The raw AD-DPW solution contained both Ca and Mg, both within the optimal range outlined for duckweed growth parameters. Recent studies have demonstrated the importance of the calcium-to-magnesium ratio for good growth of *L. minor* [15]. The 100% AD-DPW solution contained 112 mg L−1 of calcium and 11.6 mg L−1 of magnesium which translates to a molar Ca:Mg ratio of 5.6:1.0. This ratio aligns with the work demonstrated by Walsh et al. [15] which details the growth effect of a skewed Ca:Mg ratio in wastewater and the importance of a ratio which favours calcium over magnesium for *L. minor* growth.

It may be hypothesised that long-term cultivation of *L. minor* on AD-DPW will highlight both deficient and toxic phenomena, which may not be noticed in short-term experiments. However, over a six-week period, no negative effects were noted. It is concluded that diluted AD-DPW is suitable as a cultivation medium for *L. minor*. This leads to a subsequent question, namely, what is the nutrient uptake capacity of *L. minor* when grown on AD-DPW? TN and TP removal rates were assessed on 5% and 10% AD-DPW. At both dilutions decreases in TN and TP were measured. The nutrient depletion and removal rates of *L. minor* in the 10% conditions were greater in comparison with the lower concentration of 5%, which matches the established relationship between plant growth and nutrient uptake [33]. The TN removal rate achieved within the recirculating semi-outdoor setting was 248.46 mg TN m−2d −1 for the 10% concentration. This value is at the lower end of the removal rate range reported in the literature, which varies from 124 to 4400 mg TN m−2d −1 [34,35]. This may be explained by the relatively low growth rate, which may be associated with relatively low concentrations of phosphate, zinc, iron, potassium, and manganese at that dilution. The TP removal rate was 126.54 mg TP m−2d −1 for the 10% concentration which is also within the removal rate range commonly reported in the literature as between 14 and 590 mg TP m−2 d −1 [20,34]. The relatively low TN removal rate indicates that AD-DPW is not a particularly good medium for duckweed growth, as also indicated by the relatively low RGR values (Figures 1–3). Nevertheless, duckweed effectively lowered TN and TP concentrations in the AD-DPW. The EU Urban Wastewater Treatment Directive (98/15/EC) states TN and TP allowable limits for release into sensitive receiving waters as 10–15 mg L−1 and 1–2 mg L−1 , respectively. A comparison of the allowable release limits with the nutrient concentrations of AD-DPW shows that remediation of 10% AD-DPW lowered the mean TN content from 10.5 mg L−1 to 5.7 mg L−1 by day 7. Based on the measured nitrogen removal rate of 0.248 g TN m−2 d −1 , a system of six stacked recirculatory systems [36], each comprised of 10 m2 duckweed per m2 of the floor, would be capable of removing a total of 15 g TN d−1 , and clean 10% AD-DPW to releasable rates, thus indicating the potential for commercial applications. Nevertheless, expanding the scale beyond that of the present study requires critical consideration of scaling-up issues and adaption to operation within a commercial setting. Of particular interest is how the long-term operation of a remediation system will impact the concentrations of salts other than N and P: Will there be depletion, or rather a build-up of high concentrations of salts, such as manganese, iron, and zinc in the medium? The answer to this question is important to determine the viability of large-scale, long-term, duckweed flow-through systems.

#### **4. Materials and Methods**

#### *4.1. Stock Cultivation and Anaerobically Digested DPW (AD-DPW) Source*

The duckweed strain used in this study was *Lemna minor* L. "Blarney", number 5500 in the Rutgers Duckweed Stock Cooperative database. A stock of *L. minor* was maintained indoors at a light intensity of 50 µmol m−2 s −1 PAR (photosynthetically active radiation), at a temperature of 20 ± 2 ◦C with a 16:8 h light:dark photoperiod. Axenic stock cultures

were maintained on a two-part commercial medium that consisted of pH Perfect Grow (2 mL L−1 ) and pH Perfect Micro (2 mL L−1 ) (Advanced Nutrients) [37].

The AD-DPW medium was sourced from a large-scale commercial dairy processing plant producing milk, cheese, and yogurt products in Ireland. In this dairy processing plant, wastewater is treated in an anaerobic digester-producing biogas from the organic load present in the DPW. The effluent released from the digester (i.e., AD-DPW) is low in BOD and COD and normally feeds a biological nutrient removal system [31].

For a full physicochemical assessment of the AD-DPW medium, samples of the wastewater were analysed by a GLP-certified laboratory (Aquatic Services Unit, Cork, Ireland). BOD, COD, total solids, total nitrogen (TN), and total phosphorus (TP) were measured on whole, unfiltered, wastewater samples, as per standard methods for wastewater analysis [38,39]. Wastewater was filtered (0.45 µm) to determine the dissolved concentrations of ammonia, nitrate, nitrite, and orthophosphate using the Lachat QuikChem 8000 by Zeilweger Analytics, Inc., Milwaukee, USA (QuikChem Methods 10-107-06-3-D, 10- 107-04-1-C, 10-107-04-1-C, and 10-115-01-1-B, respectively). Sodium, potassium, calcium, magnesium, zinc, and iron were measured in filtered wastewater using a flame AAS (Varian Australia Ply Ltd., 1989). Copper and manganese were measured using a graphite furnace AAS (Varian Australia Ply Ltd., Mulgrave, Australia, 1989). Chloride was measured using the ferricyanide method on filtered wastewater [39].

#### *4.2. Experimental Designs*

### 4.2.1. The Effect of Varying AD-DPW Concentrations on *L. minor* Growth

*Lemna minor* was cultivated on AD-DPW at five different concentrations (1%, 5%, 10%, 50%, or 100%), in Magenta vessels with vented lids (GA-7, 7.7 cm length × 7.7 cm width × 9.7 cm height). The concentrations were obtained by diluting the AD-DPW with distilled water. Control treatment used half-strength Hutner's medium [40] since fullstrength Hutner's medium is too concentrated for Lemnaceae [26]. At each treatment concentration, the initial pH was recorded using a pH meter (Hanna instruments). Typically, the pH of AD-DPW was pH 8. This was adjusted to pH 6 via the addition of sulfuric acid (H2SO4), thus reducing the pH to near-optimal values for the growth of *L. minor* [41]. A pilot experiment had shown that there was a significant effect of controlling the pH, with *L. minor* survival and growth occurring only on the pH-controlled medium. In each replicate experiment, three colonies of four fronds from the axenic *L. minor* stock culture were grown on 100 mL of AD-DPW in individual Magenta vessels. All replicates were kept under a light regime of 16:8 h light:dark photoperiod at a light intensity of 100 µmol m−2 s −1 PAR, and a temperature of 20 ± 2 ◦C for a period of seven days. The initial biomass of *L. minor* per Magenta was recorded based on an average of representative samples. At the end of the seven days, the fresh weight of each sample was determined. All treatments were replicated three times (*n* = 3).

#### 4.2.2. Growth of *L. minor* on Varying Concentrations of AD-DPW over a Six-Week Period

*Lemna minor* was cultivated on five concentrations of AD-DPW (1%, 5%, 10%, 50%, or 100%) set up in Magenta vessels as described above. Initially, three colonies of four fronds from an axenic stock culture were added to each of the Magentas and an average representative initial biomass of *L. minor* was recorded (*n* = 5). At the end of each week, three colonies of four fronds were extracted from each Magenta vessel and placed on 100 mL of fresh AD-DPW media, at the same initial concentration as before. For the duration of the experiment, the media were based on the same AD-DPW stock. The experiment was maintained for six weeks.

#### 4.2.3. Growth of *L. minor* in Semi-Outdoor Recirculating Systems

*Lemna minor* was cultivated in non-sterile, semi-outdoor, recirculating systems (*n* = 3). Each system consisted of five tiered tanks (21.5 cm length × 15.5 cm width × 11 cm height) and a lower sump tank (37 cm × 29 cm × 52 cm) with a capacity of 35 L per system

(Figure 5). The five tanks had a combined total surface area for duckweed growth of 0.165 m2 . The growing medium was pumped from the sump tank to the highest-tiered tank (1.1 L h−1 : JYC-2000 Jiayaocheng, Ltd., Foshan, China) at a slow pace to minimise plant disruption [37]. All other tanks were sequentially gravity fed from the top tank and effluent from the lowest tank was returned to the sump tank. Filters inserted into the outlet pipe of each tank prevented movement of *L. minor* between tanks, whilst allowing the continued circulation of the medium. A muslin cloth cover was suspended above the three recirculating systems to provide 50% shading from natural sunlight. A preliminary assessment indicated that *L. minor* grew better at a reduced light intensity rather than in direct sunlight. The mean (± SE) light intensity experienced by *L. minor* between dawn and dusk was 139.9 ± 11.9 µmol m−2 s −1 , with a min–max of 2.9–463.2 µmol m−2 s −1 (measured using HOBO MX2202, Onset). m2 . The growing medium was pumped from the sump tank to the highest-tiered tank (1.1 L h−1: JYC-2000 Jiayaocheng, Ltd.) at a slow pace to minimise plant disruption [37]. All other tanks were sequentially gravity fed from the top tank and effluent from the lowest tank was returned to the sump tank. Filters inserted into the outlet pipe of each tank prevented movement of *L. minor* between tanks, whilst allowing the continued circulation of the medium. A muslin cloth cover was suspended above the three recirculating systems to provide 50% shading from natural sunlight. A preliminary assessment indicated that *L. minor* grew better at a reduced light intensity rather than in direct sunlight. The mean (± SE) light intensity experienced by *L. minor* between dawn and dusk was 139.9 ± 11.9 µmol m−2 s −1, with a min–max of 2.9–463.2 µmol m−2 s −1 (measured using HOBO MX2202, Onset).

**Figure 5.** Schematic of the semi-outdoor, 35 L, recirculatory cultivation system. **Figure 5.** Schematic of the semi-outdoor, 35 L, recirculatory cultivation system.

In the recirculating system, *L. minor* was cultivated on 5% or 10% AD-DPW dilutions for 7 days. A two-part, non-axenic commercial hydroponics solution of FloraGrow (0.25 mL L−1 ) and FloraMicro (0.25 mL L−1 ) (GH Inc.) was used as a control medium (*n* = 3). The initial pH of the prepared solution in each sump tank was measured using a handheld pH meter and standardised to pH 6 by the addition of H2SO4. In the recirculating system, *L. minor* was cultivated on 5% or 10% AD-DPW dilutions for 7 days. A two-part, non-axenic commercial hydroponics solution of FloraGrow (0.25 mL L−1 ) and FloraMicro (0.25 mL L−1 ) (GH Inc.) was used as a control medium (*n* = 3). The initial pH of the prepared solution in each sump tank was measured using a handheld pH meter and standardised to pH 6 by the addition of H2SO4.

At the start of the experiment, the five tanks of each replicate system (excluding the sump tank) were seeded to achieve 60% duckweed surface cover, using stock plants cultivated in the same semi-outdoor setting. Preliminary imaging analysis using Easy Leaf Area software established that 12.75 g (fresh weight) of *L. minor* per tank gave 60% plant surface cover. Each treatment was left for a seven-day growth period. At the end of each experiment, plant biomass was dried using absorbent tissue paper to remove excess water before being weighed. A representative, unfiltered, water sample was collected from the sump tank on day 0 and day 7 for each replicate and refrigerated at 4 °C until analysed. At the start of the experiment, the five tanks of each replicate system (excluding the sump tank) were seeded to achieve 60% duckweed surface cover, using stock plants cultivated in the same semi-outdoor setting. Preliminary imaging analysis using Easy Leaf Area software established that 12.75 g (fresh weight) of *L. minor* per tank gave 60% plant surface cover. Each treatment was left for a seven-day growth period. At the end of each experiment, plant biomass was dried using absorbent tissue paper to remove excess water before being weighed. A representative, unfiltered, water sample was collected from the sump tank on day 0 and day 7 for each replicate and refrigerated at 4 ◦C until analysed.

#### *4.3. Data Collection*

*4.3. Data Collection* For experiments in Magenta vessels, the total colony number and the total alive frond number were visually assessed and recorded. Where *L. minor* survived, the final fresh For experiments in Magenta vessels, the total colony number and the total alive frond number were visually assessed and recorded. Where *L. minor* survived, the final fresh

biomass was recorded. Total growth for each replicate was determined by subtracting the

initial fresh biomass (*W*1) from that of the final fresh biomass (*W*2).

biomass was recorded. Total growth for each replicate was determined by subtracting the initial fresh biomass (*W*1) from that of the final fresh biomass (*W*2).

$$RGR = \frac{\ln \frac{W\_2}{W\_1}}{\Delta T} \tag{1}$$

where *ln* is the natural log, *W*1 is the initial biomass, *W*2 is the final biomass and ∆*T* is the length of the experiment in days.

Total nitrogen (TN) and total phosphorus (TP) concentrations were measured in water samples taken on day 0 and day 7 of the recirculating system experiment. TN was determined using the LCK138 Laton testing kit (1–16 mg L−1 TN). TP was determined using the LCK348 Phosphate testing kit (0.5–5.0 mg L−1 PO4-P). These concentration measurements were used to calculate the net TN and TP depletion per system, over the seven-day period and based on the 35 L volume of one individual system. Mean TN and TP removal rates were calculated based on the total available growing area per system. The calculated nutrient removal rates provide an estimate for N and P removed per m2 of *L. minor* per day (mg TN m−2 d −1 ; mg TP m−2 d −1 ).

#### *4.4. Data Analysis*

Statistical analyses were conducted using R software (R Core Team 2021; R 4.1.2). All data were assessed for normality of residual distributions (Shapiro–Wilk test: library *psych*) and homoscedasticity of variances (Levene's test: library *car*). Where data were found to be normally distributed (*p* > 0.05) with homoscedastic residuals (*p* > 0.05), general linear models (ANOVA) were used to analyse differences in RGR and nutrient depletion of the media. Logistic regression in the form of generalised linear models (GLM: *car*) was employed for non-normal data and/or heteroscedastic residuals (*p* < 0.05). A stepwise depletion approach was used to remove non-significant terms if required, while the overall model significance was determined using likelihood ratio tests in all cases (*lmtest*). Where *p*values were significant (α < 0.05), a Tukey adjustment for the multiple pairwise comparison was used for post hoc analysis (*emmeans*; Lenth 2020).

#### **5. Conclusions**

AD-DPW can support the cultivation of *L. minor*. In particular, plants can be cultivated in semi-outdoor, recirculatory systems. Plant growth and nutrient removal rates from the medium are modest, but this is compensated by the double benefit of simultaneous water remediation and generation of protein-rich biomass. These data indicate a new perspective on AD-DPW treatment, whereby the emphasis moves from resource-intensive clean-up of AD-DPW to duckweed-based valorisation.

**Author Contributions:** Conceptualization, R.O., N.E.C., É.W. and M.A.K.J.; methodology, N.E.C., É.W. and M.A.K.J.; validation, R.O., N.E.C. and M.A.K.J.; formal analysis, R.O. and N.E.C.; investigation, R.O. and N.E.C.; resources, M.A.K.J.; data curation, N.E.C. and M.A.K.J.; writing—original draft preparation, R.O. and N.E.C.; writing—review and editing, R.O., N.E.C., É.W. and M.A.K.J.; supervision, N.E.C. and M.A.K.J.; project administration, M.A.K.J.; funding acquisition, M.A.K.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the European Regional Development Fund through the Ireland Wales Cooperation Programme (82078), and the Environmental Protection Agency (EPA)(2016-W-LS-11). The EPA Research Programme is a Government of Ireland initiative funded by the Department of Communications, Climate Action, and Environment. It is administered by the EPA, which has the statutory function of co-ordinating and promoting environmental research.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** M.A.K.J. acknowledges support from WoB.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

## *Article* **Integrated Multitrophic Aquaculture; Analysing Contributions of Different Biological Compartments to Nutrient Removal in a Duckweed-Based Water Remediation System**

**Simona Paolacci 1,\* , Vlastimil Stejskal 1,2 , Damien Toner 3 and Marcel A. K. Jansen 1,4**

**Abstract:** Duckweed (Lemnaceae) can support the development of freshwater aquaculture if used as extractive species in Integrated MultiTrophic Aquaculture (IMTA) systems. These aquatic plants have the advantage of producing protein-rich biomass that has several potential uses. On the contrary, other biological compartments, such as microalgae and bacteria, present in the water and competing with duckweed for light and nutrients cannot be harvested easily from the water. Moreover, as phytoplankton cannot easily be harvested, nutrients are eventually re-released; hence, this compartment does not contribute to the overall water remediation process. In the present study, a mesocosm experiment was designed to quantify the portion of nutrients effectively removed by duckweed in a duckweed-based aquaculture wastewater remediation system. Three tanks were buried next to a pilot-scale IMTA system used for the production of rainbow trout and perch. The tanks received aquaculture effluents from the adjacent system, and 50% of their surface was covered by duckweed. Daily water analyses of samples at the inlet and outlet of the mesocosm allowed quantification of the amount of nutrients removed in total. The portion removed by duckweed was determined by examining the nutrient content in the initial and final biomass. The portion of nutrients removed by other compartments was similarly estimated. The results show that duckweed is responsible for the removal of 31% and 29% of N and P, respectively. Phytoplankton removed 33% and 38% of N and P, respectively, while the biofilm played no major role in nutrient removal. The remainder of the removed nutrients were probably assimilated by bacteria or sedimented. It is speculated that a higher initial duckweed density can limit phytoplankton growth and, therefore, increase the portion of nutrients removed by the duckweed compartment.

**Keywords:** Lemnaceae; aquaculture effluents; IMTA; RAS; phytoplankton; bacteria; phytoremediation

## **1. Introduction**

Demand for protein is rising sharply, with worldwide shortages of quality protein expected in the nearby future [1]. Aquaculture has the potential to contribute substantially to the production of protein required to feed an increasing world population [2]. Freshwater aquaculture can be a local source of protein, even in regions distant from the coast, where supply of marine seafood would involve food miles, and associated carbon emissions [3].

The development of freshwater aquaculture is sometimes impeded by concerns of negative environmental impacts. Consequently, the development of innovative, sustainable approaches to aquaculture is increasingly recognized as central to accelerate growth of the sector [4]. Such sustainable aquaculture should focus on high yields of quality produce, as

**Citation:** Paolacci, S.; Stejskal, V.; Toner, D.; Jansen, M.A.K. Integrated Multitrophic Aquaculture; Analysing Contributions of Different Biological Compartments to Nutrient Removal in a Duckweed-Based Water Remediation System. *Plants* **2022**, *11*, 3103. https://doi.org/10.3390/ plants11223103

Academic Editors: Viktor Oláh, Klaus-Jürgen Appenroth and K. Sowjanya Sree

Received: 19 October 2022 Accepted: 12 November 2022 Published: 15 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

well as minimise negative effects on the environment. Two negative effects of traditional freshwater aquaculture relate to water-use and eutrophication. Intensive aquaculture generates effluents rich in dissolved inorganic nutrients such as ammonia, and phosphate. If discharged without treatment, these effluents can have strong negative impacts on the water quality of receiving waterbodies. Eutrophication can lead to excessive growth of phytoplankton leading to algal blooms may, in turn, cause hypoxia which can affect a broad spectrum of organisms ranging from invertebrates to fish [5]. Moreover, the use of large volumes of freshwater for traditional aquaculture can exert pressure on local water resources in drier regions [6].

Recirculating Aquaculture Systems (RAS) can reduce the amount of water necessary to farm fish and prevent the negative effects of nutrients released by aquaculture on aquatic ecosystems [7]. In such recirculating systems, water is partially reused after undergoing remediation treatment using algae [8], bacteria [9] or aquatic plants [10]. Recently, several papers have described the use of duckweed as part of RAS system [11–13]. The use of duckweed in RAS has a promising future as it addresses two separate problems simultaneously; (1) it reduces the impact of aquaculture by capturing plant nutrients and (2) it converts a waste product into a resource that has an economic value [11].

The term duckweed refers to a group of freshwater free-floating plants belonging to the family of Lemnaceae. These plants are characterized by high growth rate and high protein content [14]. Thanks to their opportunistic nature, these plants thrive in eutrophic environments [15] and their use for the treatment of wastewater has been amply demonstrated, with early papers going back as far as 1973 [16]. In recent years, there has been a renewed interest in these species and their use for the treatment of a range of different agri-food wastewaters [17–19]. Rapid growth is associated with a high capacity to extract nutrients from water. Rapid growth similarly results in a high capacity to generate valuable biomass. Due to their high protein content, the use of these plants has been suggested as an alternative source of protein for livestock [20]. Alternatively, these plants can substitute conventional synthetic fertilizers [21] or be used as a biofuel [22].

A duckweed-based remediation system is characterized by a dynamic balance between the main compartments: duckweed, phytoplankton, biofilm, and other photosynthetic bacteria. Each group forms a functional compartment that affects the other two, and the co-existence of the three compartments determines the remediation efficiency of the whole system. The balance between duckweeds, algae and bacteria changes seasonally [12]. A reduced mat of duckweed in winter and spring is associated with a relative increase in phytoplankton. In contrast, during the summer, rapid growth of duckweed results in the formation of a conspicuous duckweed mat on the water surface, resulting in the shading of the underlying water column [12]. This, in turn, will impede algal photosynthesis, and growth, and ultimately the sequestration of nutrients from the water by algae [13,23]. Understanding of the relative proportion of nutrients removed by different taxa is important in order to make accurate models on water quality, and to inform IMTA management. Moreover, the removal of nutrients by taxa other than duckweed, will affect the portion of nutrients effectively recovered from the aquaculture system, as only duckweed can be readily harvested. In turn, this means that the competition for nutrients will also determine the yield of valuable biomass, and therefore the commercial outcome from a duckweed-based IMTA [23].

In the present study, an experiment was designed to quantify the relative uptake rate of nitrogen and phosphorus by duckweed species, algae and bacteria, in a duckweed-based aquaculture wastewater restoration system. The experiment aims to improve understanding of the balance between the different biological compartments in order to develop best practices for the management of these systems. The knowledge produced will help to optimize water restoration and maximize biomass production.

#### **2. Materials and Methods 2. Materials and Methods**

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The experiment was carried out at an Integrated Multitrophic Aquaculture (IMTA) fish farm (Co. Offaly Ireland, coordinates 53.275555, −7.208392) where Oncorhynchus mykiss and Perca fluviatilis are farmed. The aquaculture effluents produced in four fish ponds are sent to a system of canals where the duckweed species *Lemna gibba* and *Lemna minor* are used to remove plant nutrients from the water. After treatment by duckweed, the water is returned to the fishponds. The technical details of this IMTA system have been extensively described by [13]. The experiment was carried out in August 2020, when suitable conditions for duckweed growth were present, and the effluent treatment canals were abundantly covered in *L. gibba* and, to a lesser extent, *L. minor*. The experiment was carried out at an Integrated Multitrophic Aquaculture (IMTA) fish farm (Co. Offaly Ireland, coordinates 53.275555, −7.208392) where Oncorhynchus mykiss and Perca fluviatilis are farmed. The aquaculture effluents produced in four fish ponds are sent to a system of canals where the duckweed species *Lemna gibba* and *Lemna minor* are used to remove plant nutrients from the water. After treatment by duckweed, the water is returned to the fishponds. The technical details of this IMTA system have been extensively described by [13]. The experiment was carried out in August 2020, when suitable conditions for duckweed growth were present, and the effluent treatment canals were abundantly covered in *L. gibba* and, to a lesser extent, *L. minor*.

develop best practices for the management of these systems. The knowledge produced

will help to optimize water restoration and maximize biomass production.

#### *2.1. Experimental Set-Up 2.1. Experimental Set‐Up*

A small model of a duckweed-based effluent treatment system was set up next to the full-scale system. This mesocosm consisted of three independent tanks (i.e., three replicates) into which fresh aquaculture effluent was pumped. A small model of a duckweed‐based effluent treatment system was set up next to the full‐scale system. This mesocosm consisted of three independent tanks (i.e., three replicates) into which fresh aquaculture effluent was pumped.

Prior to the experiment, six tanks (area 35 × 60 cm, depth 45 cm) were submerged in one of the duckweed-covered canals for seven days (Figure 1) to establish a biofilm of microorganisms and sediment on the internal surface of the tanks. Out of these six tanks, three tanks were subsequently used as part of the experimental set-up, while a further three tanks were used to analyze the biofilm present at the start of the experiment. After having been submerged for seven days in a canal, the latter three tanks were air-dried for three days, and the dry biofilm was used to determine the organic fraction and Total Nitrogen (TN) and Total Phosphorous (TP) contained within the biofilm at the start of the experiment. Prior to the experiment, six tanks (area 35 × 60 cm, depth 45 cm) were submerged in one of the duckweed‐covered canals for seven days (Figure 1) to establish a biofilm of microorganisms and sediment on the internal surface of the tanks. Out of these six tanks, three tanks were subsequently used as part of the experimental set‐up, while a further three tanks were used to analyze the biofilm present at the start of the experiment. After having been submerged for seven days in a canal, the latter three tanks were air‐dried for three days, and the dry biofilm was used to determine the organic fraction and Total Nitrogen (TN) and Total Phosphorous (TP) contained within the biofilm at the start of the experiment.

**Figure 1.** Tanks (circled in red) were submerged for a week in one of the duckweed‐covered canals prior to being used for the mesocosm experiment. The pre‐treatment resulted in the establishment of a biofilm on the internal surface of the tanks. **Figure 1.** Tanks (circled in red) were submerged for a week in one of the duckweed-covered canals prior to being used for the mesocosm experiment. The pre-treatment resulted in the establishment of a biofilm on the internal surface of the tanks.

The experimental tanks were buried next to one of the IMTA treatment canals. Just 5 cm of the side of the tank was left above ground level. At the start of the experiment, the tanks were filled with 70 L of aquaculture effluent, and 40% of their surface area was covered with duckweed, both taken from the neighboring IMTA system. An equal amount of duckweed biomass was collected, weighed, dried at 60 °C for three days, and analyzed for TP and TN. These values of TP and TN represent the initial P and N content in duckweed biomass. The experimental tanks were buried next to one of the IMTA treatment canals. Just 5 cm of the side of the tank was left above ground level. At the start of the experiment, the tanks were filled with 70 L of aquaculture effluent, and 40% of their surface area was covered with duckweed, both taken from the neighboring IMTA system. An equal amount of duckweed biomass was collected, weighed, dried at 60 ◦C for three days, and analyzed for TP and TN. These values of TP and TN represent the initial P and N content in duckweed biomass.

A small pump was placed inside each tank in order to generate internal effluent recirculation. The flow rate was 490 L·h −1 , a value that is similar to the flow conditions in the IMTA canals (Figure 2.1). Every 12 h, a sample of water was collected from each replicate mesocosm tank, after which the tanks were gently drained without disruption of the biofilm or the duckweed biomass (Figure 2.2). The water removed was immediately replaced with fresh aquaculture effluent, pumped in from the adjoining canal (Figure 2.3). A sample of effluent pumped in from the canal was also collected for analysis in order to quantify the initial nutrient concentration in the water before being treated. The collected water samples were filtered to separate phytoplankton from the remainder of the water sample. TP and TN were quantified both in filtered water and in phytoplankton. Furthermore, algal chlorophyll content, cyanobacteria, and turbidity (expressed in Formazin Turbidity Unit, FTU) were measured both in the tanks and in the canal using an AlgaeTorch produced by Bbe-Modanke. A Seneye online system (Seneye Ltd., Norwich, UK) is present in the system to monitor pH and temperature. mesocosm tank, after which the tanks were gently drained without disruption of the bio‐ film or the duckweed biomass (Figure 2.2). The water removed was immediately replaced with fresh aquaculture effluent, pumped in from the adjoining canal (Figure 2.3). A sam‐ ple of effluent pumped in from the canal was also collected for analysis in order to quan‐ tify the initial nutrient concentration in the water before being treated. The collected water samples were filtered to separate phytoplankton from the remainder of the water sample. TP and TN were quantified both in filtered water and in phytoplankton. Furthermore, algal chlorophyll content, cyanobacteria, and turbidity (expressed in Formazin Turbidity Unit, FTU) were measured both in the tanks and in the canal using an AlgaeTorch pro‐ duced by Bbe‐Modanke. A Seneye online system (Seneye Ltd., Norwich, UK) is present in the system to monitor pH and temperature.

A small pump was placed inside each tank in order to generate internal effluent re‐ circulation. The flow rate was 490 L∙h−1, a value that is similar to the flow conditions in the IMTA canals (Figure 2.1). Every 12 h, a sample of water was collected from each replicate

*Plants* **2021**, *10*, x FOR PEER REVIEW 4 of 18

**Figure 2.** Experimental design. Tanks are buried next to a canal with aquaculture effluent. The tank is filled with aquaculture effluent (**1**). Twice a day, the tank is emptied without disrupting the bio‐ film or duckweed mat (**2**) and re‐filled with fresh aquaculture effluent from the nearby canal (**3**). **Figure 2.** Experimental design. Tanks are buried next to a canal with aquaculture effluent. The tank is filled with aquaculture effluent (**1**). Twice a day, the tank is emptied without disrupting the biofilm or duckweed mat (**2**) and re-filled with fresh aquaculture effluent from the nearby canal (**3**).

The mesocosm experiment was terminated when the duckweed achieved a tank sur‐ face coverage close to 90%; this happened after eight days. At this stage, the duckweed biomass was harvested, dried at 60 °C for 3 days, and analyzed for TP and TN. The dif‐ ference in the total amount of nutrients in the final duckweed biomass versus the initial duckweed biomass (i.e., plant concentration times biomass at start and finish) represents the TN or TP removed by duckweed. The mesocosm experiment was terminated when the duckweed achieved a tank surface coverage close to 90%; this happened after eight days. At this stage, the duckweed biomass was harvested, dried at 60 ◦C for 3 days, and analyzed for TP and TN. The difference in the total amount of nutrients in the final duckweed biomass versus the initial duckweed biomass (i.e., plant concentration times biomass at start and finish) represents the TN or TP removed by duckweed.

Once the duckweed was removed, the tanks were emptied and air‐dried for three days. The internal biofilm was gently removed and weighed. Half the biofilm biomass was used for the quantification of TP and TN, while the other half was retained to deter‐ mine the organic and inorganic components. The difference between the total nutrient content in the final biofilm versus the initial biofilm (i.e., concentration times biomass at start and finish) represents the TN or TP removed from the water by bacteria, periphytic and epipelic algae, and other biofilm constituents. The experimental protocol is summa‐ rised in Table S1 of the Supplementary Material. Once the duckweed was removed, the tanks were emptied and air-dried for three days. The internal biofilm was gently removed and weighed. Half the biofilm biomass was used for the quantification of TP and TN, while the other half was retained to determine the organic and inorganic components. The difference between the total nutrient contentin the final biofilm versus the initial biofilm (i.e., concentration times biomass at start andfinish) represents the TN or TP removed from the water by bacteria, periphytic and epipelic algae, and other biofilm constituents. The experimental protocol is summarised in Table S1 of the Supplementary Material.

Half of the volume of water sampled was analyzed without filtering it, while the other half was filtered using a vacuum pump and a 5 μm filter. The phytoplankton bio‐ mass present in each sample was assessed by weighing the clean filter and then weighing it again after water sample filtration. Half of the volume of water sampled was analyzed without filtering it, while the other half was filtered using a vacuum pump and a 5 µm filter. The phytoplankton biomass present in each sample was assessed by weighing the clean filter and then weighing it again after water sample filtration.

In the present paper, the term "duckweed" refers to the plant and its microbiome, and the term "biofilm" is used to indicate the mix of microorganisms (algae, protista, bac‐ teria, and otherwise) and inorganic particles that build up on the internal surface of the tanks and contribute to nutrients sequestration. The term "phytoplankton" is used to in‐ dicate all the organisms with a diameter greater than 5 μm that live in the water column. In the present paper, the term "duckweed" refers to the plant and its microbiome, and the term "biofilm" is used to indicate the mix of microorganisms (algae, protista, bacteria, and otherwise) and inorganic particles that build up on the internal surface of the tanks and contribute to nutrients sequestration. The term "phytoplankton" is used to indicate all the organisms with a diameter greater than 5 µm that live in the water column. Samples of biofilm and phytoplankton were observed under a Leica DM500 light microscope, and the microalgae were identified at the genus level using the dichotomic key presented in [24].

#### *2.2. Analytical Methodology*

TN in the unfiltered water sample was determined using an automated colorimetric method involving digestion of the unfiltered sample with potassium persulphate and boric acid in an alkaline solution in an autoclave at 121 ◦C for 30 min [25]. TP in the unfiltered water was determined using a modified Molybdate—Ascorbic Acid method following digestion of the unfiltered sample with persulphate and sulphuric acid in an autoclave at 121 ◦C for 30 min [26].

The other half of the samples were filtered and the dry weight of phytoplankton (mg·L −1 ) was determined. Total Dissolved Nitrogen (TDN) and Total Dissolved Phosphorus (TDP) were determined in the filtered effluent sample using the same method as detailed above. The difference between TN and TDN was taken as the total nitrogen present in phytoplankton. TP in phytoplankton was determined using the same approach. The amount of nutrients removed by phytoplankton was estimated by calculating the difference between TN and TP in the phytoplankton contained in the inlet and outlet water.

Plant samples, dried at 60 ◦C for 48 h and milled, were digested with concentrated sulphuric acid and Kjeltab Cu/3.5 in a TECATOR 2040 Digestor at 420 ◦C for 1 h. Digested samples were diluted to 250 mL using deionised water. The Total Kjeldahl Nitrogen was analysed using QuickChem IC + FIA flow injection analyzer (8000 series) manufactured by Lachat Instrument. For the determination of TP, the samples were acid digested and analysed using the ammonium molybdate method (Murphy and Riley, 1962). Absorbance was measured using a UV-Visible Recording Spectrophotometer manufactured by SHI-MADZU Corporation (Model: SHIMADZU UV-160A). The same procedures were used for the analyses of TN and TP in the biofilm.

#### *2.3. Duckweed Growth Analysis*

A photograph of the duckweed mat was taken every morning and analysed using the imaging software ImageJ to determine the growth in the duckweed covered tank surface area. The Relative Growth Rate (RGR) was calculated using the formula by [27]:

$$\text{RGR} = \ln(\text{Yf}/\text{Yi})/\text{t}$$

where Yi is the initial area of duckweed cover, Yf is the final area, t is the time in days and ln is the natural logarithm.

#### *2.4. Calculations*

The total amount of nitrogen removed from the water during the 8 days of experiments is expressed with the formula:

$$\text{TNr} = \text{TNb} + \text{TNb} + \text{TNp} + \text{TNo}$$

where:

TNr = Total Nitrogen removed from the wastewater

TNd = Total Nitrogen removed by duckweed

TNb = Total Nitrogen removed by the biofilm

TNp = Nitrogen removed by phytoplankton

TNo =Nitrogen removed by other processes/organisms

The variables of this equation were calculated as follows:

$$\text{TNr} = \sum\_{day}^{day} \left( \text{CN}\_{\text{i}} \times \text{V} \right) - \left( \text{CN}\_{\text{0}} \times \text{V} \right)$$

CNi = concentration of nitrogen in the inflow (filtered water pumped in from the canal) expressed in mg·L −1

CNo = concentration of nitrogen in the outflow (water in the tank after 12 h) expressed in mg·L −1

V = volume of water treated in 12 h expressed in l

$$\text{TNd} = (\text{CNd}\_{\text{f}} \times \text{dw} \text{d}\_{\text{f}}) - (\text{CNd}\_{\text{i}} \times \text{dw} \text{d}\_{\text{i}})$$

CNdf = concentration nitrogen in final duckweed biomass wdf = final duckweed weight CNdi concentration of nitrogen in initial duckweed biomass wdi = initial duckweed weight

$$\text{TNb} = \text{(CNb}\_{\text{f}} \times \text{dw} \text{b}\_{\text{f}}) - \text{(CNb}\_{\text{i}} \times \text{dw} \text{b}\_{\text{i}})^2$$

CNbf = concentration of nitrogen in the final biofilm

dwbf = final biofilm dry weight

CNbi = concentration of nitrogen in the initial biofilm

Dwbi = initial biofilm weight

$$\text{TNp} = \sum\_{day}^{day} \, ^8(\text{CNp}\_{\text{f}} \times \text{wp}\_{\text{f}}) - (\text{CNp}\_{\text{i}} \times \text{wp}\_{\text{i}})\, ^4$$

CNpf = concentration of nitrogen in phytoplankton in the outflowing water wpf = final phytoplankton weight

CNpi = concentration of nitrogen in phytoplankton in the inflowing effluent wpi = initial phytoplankton weight

After determining the variables described above, TNo was determined as a difference between the TNr and TN extracted by the other compartments:

$$\text{TNo} = \text{TNr} - (\text{TNd} + \text{TNp} + \text{TNb})$$

The total amount of phosphorus removed from the water during the eight days of experiments was expressed with the same formula described above for the total amount of nitrogen removed.

### *2.5. Statistical Analyses*

R studio version 4.1.1. was used for the statistical analysis. The correlation between the area of the duckweed mat and N and P removed from the tanks was analyzed with the non-parametric Kendall Tau test. A t-test was used to analyze differences in nutrient removal between night and day.

#### **3. Results**

#### *3.1. Water Parameters*

During the experiment, the water temperature varied between 11 and 19 ◦C.

The pH of the aquaculture effluent varied between 7.55 and 8.53 during the 8-day experiment, while the turbidity varied between 20.9 and 24.6 FTU (Figure 3).

The total dissolved nitrogen and phosphorus concentrations in the incoming aquaculture effluent were quantified twice a day, i.e., every time the water in the tanks was replaced. TN varied between 2.97 and 4.3 mg·L −1 , while TP varied between 0.43 and 0.72 mg·L −1 (Figure 4).

The concentration of algal chlorophyll and cyanobacteria in the incoming effluent was also measured twice a day. The chlorophyll concentration in the water varied between 251.5 and 342.2 µg·L −1 . The concentration of cyanobacteria was extremely high on the first day of the experiment (66.1 µg·L −1 ), but it decreased during the following days, reaching a low of 21.1 µg·L −1 toward the final days of the experiment (Figure 5).

*Plants* **2021**, *10*, x FOR PEER REVIEW 7 of 18

**Figure 3.** pH and turbidity in the aquaculture wastewater measured at 8:00 am and 8:00 pm during the eight days of the experiment. FTV = Formazin Turbidity Unit. **Figure 3.** pH and turbidity in the aquaculture wastewater measured at 8:00 am and 8:00 pm during the eight days of the experiment. FTV = Formazin Turbidity Unit. culture effluent were quantified twice a day, i.e., every time the water in the tanks was replaced. TN variedbetween 2.97 and 4.3 mg∙L−1, while TP varied between 0.43 and 0.72 mg∙L−1 (Figure 4).

**Figure 4.** Total Nitrogen and Total Phosphorus concentrations in the aquaculture effluent measured at 8:00 am and 8:00 pm over the eight days of the experiment. **Figure 4.** Total Nitrogen and Total Phosphorus concentrations in the aquaculture effluent measured at 8:00 am and 8:00 pm over the eight days of the experiment.

at 8:00 am and 8:00 pm over the eight days of the experiment.

**Figure 4.** Total Nitrogen and Total Phosphorus concentrations in the aquaculture effluent measured

The concentration of algal chlorophyll and cyanobacteria in the incoming effluent was also measured twice a day. The chlorophyll concentration in the water varied be‐ tween 251.5 and 342.2 μg∙L−1. The concentration of cyanobacteria was extremely high on the first day of the experiment (66.1 μg∙L−1), but it decreased during the following days,

reaching a low of 21.1 μg∙L−1 toward the final days of the experiment (Figure 5).

**Figure 5.** Algal chlorophyll and cyanobacteria concentration in the aquaculture wastewater meas‐ ured at 8:00 am and 8:00 pm during the eight days of the experiment. N= night, D= day. **Figure 5.** Algal chlorophyll and cyanobacteria concentration in the aquaculture wastewater measured at 8:00 am and 8:00 pm during the eight days of the experiment. N= night, D= day.

Samples of biofilm attached to the internal surface of the tanks were observed with a microscope. The genera identified are indicated in Table 1. Samples of biofilm attached to the internal surface of the tanks were observed with a microscope. The genera identified are indicated in Table 1.

**Table 1.** Genera of microalgae identified in samples of biofilm and phytoplankton built in the inter‐ nal surface of the tanks. **Table 1.** Genera of microalgae identified in samples of biofilm and phytoplankton built in the internal surface of the tanks.

#### *3.2. Nutrient Removal* and outflow water, allowed the calculation of the amount of dissolved N and P removed

*3.2. Nutrient Removal*

*Plants* **2021**, *10*, x FOR PEER REVIEW 9 of 18

The total volume of effluent treated by each tank in eight days was 1050 L. This volume, together with the cumulative differences in nutrient concentration between inflow and outflow water, allowed the calculation of the amount of dissolved N and P removed from the water. from the water. A total of 689.93 mg of nitrogen (Figure 6) and 154.44 mg of phosphorus (Figure 7) were removed from the aquaculture wastewater. These numbers were estimated from the

The total volume of effluent treated by each tank in eight days was 1050 L. This vol‐ ume, together with the cumulative differences in nutrient concentration between inflow

A total of 689.93 mg of nitrogen (Figure 6) and 154.44 mg of phosphorus (Figure 7) were removed from the aquaculture wastewater. These numbers were estimated from the difference between inflow and outflow water in unfiltered samples. The N removal rate varied between −0.16 and 2.63 mg·L −1 ·d −1 , while the P removal rate varied between 0.05 and 0.64 mg·L −1 ·d −1 . There was no significant difference between N and P removed during the day and during the night. difference between inflow and outflow water in unfiltered samples. The N removal rate varied between −0.16 and 2.63 mg∙L−1∙d−1, while the P removal rate varied between 0.05 and 0.64 mg∙L−1∙d−1. There was no significant difference between N and P removed during the day and during the night.

**Figure 6.** Cumulative Total Nitrogen removed from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Nitrogen removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Nitrogen removed between 8:00 am and 8:00 pm. The graph shows the average of three repli‐ **Figure 6.** Cumulative Total Nitrogen removed from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Nitrogen removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Nitrogen removed between 8:00 am and 8:00 pm. The graph shows the average of three replicates.

#### cates. *3.3. Nutrients Accumulated by Phytoplankton*

The phytoplankton contained in the inflow and outflow water was filtered and weighed. The filtered water samples (not containing phytoplankton) were analyzed for N and P content. The difference in nutrient concentration between filtered and unfiltered water samples represents the net nutrient removal by this biological compartment. The phytoplankton captured through filtering of the inlet and outlet water allowed the estimation that an average of 32.64 g of fresh weight phytoplankton (13.88 ± 1.22 during night hours and 18.66 ± 2.46 g during day hours) were produced, in each tank during the eight days of the experiment. The TN and TP concentrations in this biomass ranged between 14.82 and 35.2 mg N·g −1 and 5.72 and 10.09 mg P·g −1 . The sums of TN and TP accumulated by phytoplankton are in total 230.06 mg (Figure 8) and 59.08 mg, respectively (Figure 9). Table 2 shows the details of phytoplankton produced and nutrients accumulated by this compartment.

**Figure 7.** Cumulative Total Phosphorus removed from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Phosphorus removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Phosphorus removed between 8:00 am and 8:00 pm. The graph shows the average of three **Figure 7.** Cumulative Total Phosphorus removed from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Phosphorus removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Phosphorus removed between 8:00 am and 8:00 pm. The graph shows the average of three replicates.

replicates.

of three replicates.

replicates.

**Figure 8.** Cumulative Total Nitrogen removed by phytoplankton from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Nitrogen removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Nitrogen removed between 8:00 am and 8:00 pm. The graph shows the average **Figure 8.** Cumulative Total Nitrogen removed by phytoplankton from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Nitrogen removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Nitrogen removed between 8:00 am and 8:00 pm. The graph shows the average of three replicates.

**Figure 9.** Cumulative total phosphorus removed from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Phosphorus removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Phosphorus removed between 8:00 am and 8:00 pm. The graph shows the average of three

**Figure 8.** Cumulative Total Nitrogen removed by phytoplankton from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Nitrogen removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Nitrogen removed between 8:00 am and 8:00 pm. The graph shows the average

**Figure 9.** Cumulative total phosphorus removed from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Phosphorus removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Phosphorus removed between 8:00 am and 8:00 pm. The graph shows the average of three **Figure 9.** Cumulative total phosphorus removed from 70 L of aquaculture effluent every 12 h. N (Night) indicates the Total Phosphorus removed between 8:00 pm and 8:00 am; D (Day) indicates the Total Phosphorus removed between 8:00 am and 8:00 pm. The graph shows the average of three replicates.

replicates. **Table 2.** Biomass gained and nutrients accumulated by phytoplankton during the experiment. The total biomass produced was estimated from the phytoplankton filtered from the water samples collected twice a day from the inlet and outlet water.

#### *3.4. Duckweed Biomass*

of three replicates.

The area of the duckweed mat increased from 831.81 ± 23.03 cm2 to 1936.47 ± 455.54 cm2 . The RGR calculated for the duration of the experiment was 0.11 ± 0.003 d−1 . Figure 10 shows the daily increase in duckweed surface. The plants grew slowly during the first days, while the absolute growth rate increased over the last two days.

**Table 2.** Biomass gained and nutrients accumulated by phytoplankton during the experiment. The total biomass produced was estimated from the phytoplankton filtered from the water samples col‐

**Phytoplankton Biomass**

**N accumulated by phytoplankton (mg) 230.06** P in tot. initial biomass (mg) 199.3 P in tot. final biomass (mg) 258.38

**P accumulated by phytoplankton (mg) 59.08**

days, while the absolute growth rate increased over the last two days.

The area of the duckweed mat increased from 831.81 ± 23.03 cm2 to 1936.47 ± 455.54 cm2. The RGR calculated for the duration of the experiment was 0.11 ± 0.003 d−1. Figure 10 shows the daily increase in duckweed surface. The plants grew slowly during the first

Total final biomass 142.75 N in tot. initial biomass (mg) 775.83 N in tot. final biomass (mg) 1005.89

Total biomass that entered the system (g of fresh weight) 110.11

lected twice a day from the inlet and outlet water.

*3.4. Duckweed Biomass*

**Figure 10.** Increase in surface area covered by duckweed mat during the eight days of the experi‐ ment. Error bars are standard deviations. **Figure 10.** Increase in surface area covered by duckweed mat during the eight days of the experiment. Error bars are standard deviations.

The relationship between the increase in the area of the duckweed mat and the re‐ moval of nutrients was not linear (Supplementary Material); the non‐parametric Kendall Tau test failed to identify a significant correlation between the duckweed area and the amount of N and P removed from the water. The relationship between the increase in the area of the duckweed mat and the removal of nutrients was not linear (Supplementary Material); the non-parametric Kendall Tau test failed to identify a significant correlation between the duckweed area and the amount of N and P removed from the water.

The dry weight of duckweed, during the experiment, increased by 5.81 g. The content of N and P in duckweed biomass is indicated in Table 3. The aquatic plants removed, in 8 days, 210.1 mg of N and 44.5 mg of P from the entire water volume treated. The dry weight of duckweed, during the experiment, increased by 5.81 g. The content of N and P in duckweed biomass is indicated in Table 3. The aquatic plants removed, in 8 days, 210.1 mg of N and 44.5 mg of P from the entire water volume treated.

**P removed by duckweed (mg) 44.5** ± **6.2**

**Table 3.** Biomass gained and nutrients removed by duckweed during the experiment.

#### *3.5. Biofilm*

Most of the biofilm on the internal surface of the tanks constituted inorganic sediments. The organic matter represented only 15.6% of the biofilm at the start of the experiment and only 3.4% at the end of the experiment. The biofilm did not contribute substantially to the removal of nutrients from the wastewater. On the contrary, 11.2 mg of N and 4.9 mg of P were released from this compartment over the eight-day experimental period (Table 4).

**Table 4.** Biofilm weight, % of organic matter, and nutrient content at the start and at the end of the experiment.

#### *3.6. Nutrient Removal from Different Compartments*

The calculations illustrated in Section 2.5. were used to estimate the relative nutrient uptake by the different biological compartments. Duckweed is responsible for the uptake from the wastewater of 31% of the total nitrogen removed, while phytoplankton is responsible for another 33% of nitrogen removal. Some 29% of total phosphorus removal can be attributed to duckweed, while 38% is removed by phytoplankton. The biofilm did not contribute to the removal of nutrients. The remaining 36% and 33% of N and P, respectively, were removed from the aquaculture effluent by other means, such as denitrification and volatilization (Figure 11). *Plants* **2021**, *10*, x FOR PEER REVIEW 14 of 18

**Figure 11.** Percentages of total nitrogen and total phosphorus removed by the different compart‐ **Figure 11.** Percentages of total nitrogen and total phosphorus removed by the different compartments during the experiment.

#### ments during the experiment. **4. Discussion**

mainly on the fish density.

cumulate in the water.

**4. Discussion**  The present study assessed the amount of N and P removed from aquaculture wastewater by duckweed species, biofilm, and phytoplankton. The respective proportion of nutrients removed by these three compartments will inform system management and The present study assessed the amount of N and P removed from aquaculture wastewater by duckweed species, biofilm, and phytoplankton. The respective proportion of nutrients removed by these three compartments will inform system management and predict the portion of the nutrient load that can effectively be converted into valuable biomass as part of a circular economy approach.

predict the portion of the nutrient load that can effectively be converted into valuable bi‐

The water quality parameters observed during the experiment are consistent with the parameters observed the year before by O'Neill et al. [28] during the same season, and at the same aquaculture farm. The nutrient concentration observed are also in the range previously identified by Paolacci et al. [29] The latter authors reviewed the characteristics of wastewater generated by rainbow trout and perch farms. They found that TN ranges between 0.5 and 70 mg∙L−1, while TP ranges between 0.42 and 15 mg∙L−1, depending

The chlorophyll concentration is an indicator of the phytoplankton density in the wa‐ ter. Phytoplankton plays an important role in recirculating systems as it removes the NH4+‐N from the effluents, contributing to the waterrestoration process [12]. The average chlorophyll concentration measured by [30] in a rainbow trout farm was 5 μg∙L−1, consid‐ erably lower than the concentrations observed in the effluents used for this experiment (between 251.5 and 342.2 μg∙L−1). However, Sen and Sonmez' data [30] refer to a flow through system. In an outdoor recirculating system the phytoplankton is never released into natural waters and it seems plausible that higher chlorophyll concentrations can ac‐

Water turbidity depends mainly on the concentration and the characteristics of the suspended particles which depend, amongst others, on the geological substrate [31]. The experimental site is located in a cutaway peatland and the values of turbidity measured are consistent with those measured by [32] in lakes present in the same area (Offaly, IE), and generated by flooding cutaway bogs without removing the residual peat (as it was done in the IMTA system of the present study). Turbidity affects light diffraction in the water column and can limit phytoplankton growth. Despite the high turbidity, the chlo‐ rophyll concentration measured suggests that this parameter was not suppressing phyto‐ plankton. However, the turbidity can explain the low number of taxa observed in the bio‐

omass as part of a circular economy approach.

film and in the phytoplankton community [33].

#### *4.1. Characteristics of the Effluent*

The water quality parameters observed during the experiment are consistent with the parameters observed the year before by O'Neill et al. [28] during the same season, and at the same aquaculture farm. The nutrient concentration observed are also in the range previously identified by Paolacci et al. [29] The latter authors reviewed the characteristics of wastewater generated by rainbow trout and perch farms. They found that TN ranges between 0.5 and 70 mg·L −1 , while TP ranges between 0.42 and 15 mg·L −1 , depending mainly on the fish density.

The chlorophyll concentration is an indicator of the phytoplankton density in the water. Phytoplankton plays an important role in recirculating systems as it removes the NH4+-N from the effluents, contributing to the water restoration process [12]. The average chlorophyll concentration measured by [30] in a rainbow trout farm was 5 µg·L −1 , considerably lower than the concentrations observed in the effluents used for this experiment (between 251.5 and 342.2 µg·L −1 ). However, Sen and Sonmez' data [30] refer to a flow through system. In an outdoor recirculating system the phytoplankton is never released into natural waters and it seems plausible that higher chlorophyll concentrations can accumulate in the water.

Water turbidity depends mainly on the concentration and the characteristics of the suspended particles which depend, amongst others, on the geological substrate [31]. The experimental site is located in a cutaway peatland and the values of turbidity measured are consistent with those measured by [32] in lakes present in the same area (Offaly, IE), and generated by flooding cutaway bogs without removing the residual peat (as it was done in the IMTA system of the present study). Turbidity affects light diffraction in the water column and can limit phytoplankton growth. Despite the high turbidity, the chlorophyll concentration measured suggests that this parameter was not suppressing phytoplankton. However, the turbidity can explain the low number of taxa observed in the biofilm and in the phytoplankton community [33].

#### *4.2. Nutrient Removal from Water*

The results show that the total amount of N removed from the water was nearly five times higher than the amount of P removed (684.9 g vs. 154.4 g). This proportion is consistent with the observations of other authors. For example, [34] reported that, in a duckweedcovered sewage lagoon, the removal rates for N and P were, respectively, 0.26 g·m−2 ·d −1 and 0.05 g·m−2 ·d −1 . This proportion reflects the N:P ratio in duckweed biomass [34] and also the N:P ratio observed in the biomass of other aquatic plants [35]. Similar N and P uptake data were also previously reported by [36] using dairy processing waste.

No significant difference was observed in nutrient removal during the night and the day. This can be explained by considering that the samples were taken at 8 am and 8 pm, in the summer, in Ireland. Between July and August (when the experiment was performed) this latitude experiences around 16 h of daylight. This means the 'night' uptake includes four hours of light during which duckweed and phytoplankton can perform photosynthesis and remove nutrients. Moreover, during night hours, sedimentation of nutrients is active and can contribute to N and P removal from the water column.

During the experiment, duckweed increased their surface density from 50% to >90%. The growth rate was consistent with the growth rates observed earlier for duckweed during the same time of the year in Ireland [37]. Around 30% of both N and P in the water was removed by duckweed, presumably to support growth, although some luxury uptake cannot be excluded. This percentage is consistent with the results of a small-scale experiment (1 L batches) performed by [38]. The latter authors determined the uptake rates of N and P in domestic wastewater by duckweed, algae and bacteria, at different initial densities of duckweed. They observed that duckweed was responsible for removing between 30% and 47% of N and up to 54% of P, depending on the initial plant density. The results of the present study are also in accordance with the observations of [23] who performed a competition experiment between phytoplankton and duckweed. When the

duckweed mat was dense (like in the present study), the authors observed an increase in N and P content in the plant biomass, while at lower duckweed density the phytoplankton was competing with duckweed for nutrients, and this resulted in a decreased content of N and P in the biomass. The values reported by [23] for N and P content at high duckweed density are 36.65 and 10.95 mg·g −1 respectively, while the values observed in the present study are 36.8 (N) and 7.99 (P) mg·g −1 of biomass. The slightly lower value for P content is probably due to a lower concentration of this element in effluents used in the present study.

It is important to highlight that the removal of nutrients by duckweed measured in this study was determined by analysing N and P content in the initial and in the final biomass harvested at the end of the experiment. As a consequence, the values include the indirect contribution of algae and bacteria attached to, or incorporated into, the duckweed.