Performance of Lemna gibba bioreactor for nitrogen and phosphorus retention, and biomass production in Mediterranean climate

Performance of Lemna gibba bioreactor for nitrogen and phosphorus retention, and biomass production in Mediterranean climate

Journal of Environmental Management 252 (2019) 109627 Contents lists available at ScienceDirect Journal of Environmental Management journal homepage...

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Journal of Environmental Management 252 (2019) 109627

Contents lists available at ScienceDirect

Journal of Environmental Management journal homepage: http://www.elsevier.com/locate/jenvman

Research article

Performance of Lemna gibba bioreactor for nitrogen and phosphorus retention, and biomass production in Mediterranean climate Abdeslam Ennabili a, b, *, Jamila Ezzahri b, Michel Radoux b, 1 a b

Process Engineering (GP), Superior School of Technology, Sidi Mohamed Ben Abdellah University, BP 2427, Route D’Imouzzer, 30000, Fez, Morocco MHEA® International Network, Rue de La Halte, 221, 6717, Nobressart, Belgium

A R T I C L E I N F O

A B S T R A C T

Keywords: Lemna gibba L. Wastewater Nitrogen Phosphorus Biomass Bioreactor

Lemna gibba (Lemnaceae) had been experimented in Morocco to develop macrophyte-based wastewater treatment systems adapted to the local climatic and socio-economic circumstances. This species growing on pre-treated urban wastewater, in a lagoon (Lemna bioreactor) operating in fed-batch, generates a net productivity of 28.39 t dw.ha 1.yr 1, through regular harvest of the biomass produced. In wet seasons the roots of this macrophyte generally exceed 10 cm. The Lemna lagoon clearly reduces plankton production, especially during the vegetative period, when compared to the bioreactor without macrophytes (lagoon; chlorophyll-a concen­ tration of 86.4 � 168 μg. l 1). The Lemna bioreactor also removes more particulate nitrogen (N) and phosphorus (P), and shows a highly significant total P and significant non-particulate P retention, in comparison with the lagoon. L. gibba can export daily the equivalent of 13.2% of N and 19.9% of P entering the bioreactor. The algal flora is dominated throughout the year by phytoplanktonic populations of Euglenophyceae and Chlorophyceae. Branchiopoda (Daphniidae), Insecta (Dytiscidae Chironomidae, Culicidae and Heteroptera), and Gastropoda are the main taxa of animalia developing in the Lemna bioreactor. In the Mediterranean climate, the L. gibba bioreactors would be more profitable in the tertiary wastewater treatment, especially P removal, provided regularly collect of the biomass produced.

1. Introduction Extensive wastewater treatment techniques are known for their rusticity (less energy consumption, and not requiring sophisticated mechanical equipment or skilled labor when compared with the inten­ sive ones), adapted to small communities, more convenient to high load variations, and environmentally friendly processes (Radoux and Kemp, 1988; Brix, 1993; Uysal and Zeren, 2004; Allam et al., 2014; Tatar et al., 2019). To decontaminate polluted waters, the use of duckweeds in phytor­ emediation is of increasing interest to the researchers (Uysal and Zeren, 2004; Priya et al., 2012; Verma and Suthar, 2014; Ceschin et al., 2019). The removal of nutrients, especially the absorption of nitrogen and phosphorus, is one of the most important functions of these macrophytes in wastewater treatment (e.g. Uysal and Zeren, 2004; El-Shafai et al., 2007; Liu et al., 2017).

Furthermore, duckweeds are especially used fresh or after air drying in animal feed if the transport cost and regulatory restrictions allow, given their high protein content (14–45%), low content of fat (3–7%), and fibers (7–14%) (Gumbricht, 1993; Bonomo et al., 1995; Ennabili et al., 1996; Seidel et al., 2002; Abou El-Kheir et al., 2007; El-Shafai et al., 2007). Their potential uses such as composting, renewable energy production through anaerobic digestion, pollution control of P-rich wastewater, and reduction of soluble salts in irrigation water should be developed (Reed et al., 1988; Ennabili, 1999; Abou El-Kheir et al., 2007). Lemna gibba L. and L. minor L. from Morocco indicate eutrophic waters, tolerate moderately salty or polluted ones, and occur throughout the country, except the Saharan Atlas for L. gibba (e.g. Vald�es et al., 2002; Ennabili and Gharnit, 2003a; Ozenda, 2004; Fennane et al., 2014). Duckweeds tolerate medium-polluted (L. minor) to highly polluted sites (L. gibba) thanks to their ability to eliminate carbon

* Corresponding author. Process Engineering (GP), Superior School of Technology, Sidi Mohamed Ben Abdellah University, BP 2427, Route d’Imouzzer, 30000, Fez, Morocco E-mail addresses: [email protected], [email protected] (A. Ennabili), [email protected] (J. Ezzahri), [email protected] (M. Radoux). 1 died on June 03, 2017. https://doi.org/10.1016/j.jenvman.2019.109627 Received 10 May 2019; Received in revised form 22 August 2019; Accepted 22 September 2019 Available online 3 October 2019 0301-4797/© 2019 Elsevier Ltd. All rights reserved.

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2. Materials and methods

pollution and to assimilate N or P. L. gibba is one of the aquatic mac­ rophytes having been experimentally used in Morocco for urban wastewater treatment (Ennabili, 1999; Radoux et al., 2000, 2003; Ezzahri, 2005, Ennabili et al., 2006; Ezzahri et al., 2001, 2006, 2010). Ambiguity is generally related to L. gibba identification because depending on the season or chemical factors (high chloride concentra­ tion or low pH), this species loses its gibbosity and becomes flat as L. minor fronds (Agences de l’Eau, 1998), but without however acquiring the other distinctive characters (trophic status, color, shape, and pres­ ence of ribs). That’s why authors mistakenly identified L. gibba as L. minor, since they worked on the same experimental pond (Radoux et al., 2000, 2003; Ezzahri et al., 2001). In addition, genetic diversity is common in vegetative reproducing species including L. gibba and L. minor, as mentioned by Ennabili and Gharnit (2003b). In this regard, the experiments carried out in Morocco since the 1980s aim to determine in situ the purification performance of based macrophyte systems, which are of great interest to developing countries. The MHEA® (Mosaic Hierarchized of Artificial Ecosystems) Experi­ mental Center in M’Diq (NW of Morocco), a project funded by the Walloon Region through the Agence de la Francophonie, has led ex­ periments on comparing and optimizing artificial ecosystems (or mac­ rophytes bioreactors) in urban wastewater treatment for several consecutive years, to develop wastewater treatment process adapted to the climatic and socio-economic context. The interest was focused on the purification efficiency of each treatment process of 3-consecutive stages according standards for treated water discharge (Radoux et al., 2000, Radoux et al., 2003; Cadelli et al., 2004; Ezzahri, 2005; Ennabili, 1999, Ennabili et al., 2008a; Ennabili et al., 2006; Ezzahri et al., 2001, 2005a, 2005b, Ezzahri et al., 2006, Ezzahri et al., 2010). Under optimal growing conditions, Lemna regenerates easily due to its large budding ability (Agences de l’Eau, 1998; Ennabili 2008b); which requires when used in wastewater treatment a frequent biomass harvest to ensure a good pu­ rification performance, as reported by Radoux and Kemp (1992), Ennabili (1999), and Ezzahri (2005), and quoted by Tabou et al. (2014). This work presents for the first time the performance of L. gibba, grown in bioreactor, for secondary treatment of urban wastewater (2nd stage of the MHEA® process), specifically in N and P retention, and biomass production, compared with an algal-bacterial bioreactor (lagoon without macrophytes).

2.1. Bioreactors Two 2 m2-basins simulating the Lemna bioreactor (Lemna lagoon) and the algal-bacterial bioreactor (lagoon), were selected (Fig. 1) among the 16 basins composing the experimental design implemented in M’Diq (NW of Morocco) according to the methodological and technological approach of the MHEA® (Ennabili, 1999; Ezzahri, 2005). The duck­ weeds settling spontaneously in the lagoon, considered as a control, are removed regularly. Pre-treated wastewater supplies semi-continuously (fed-batch) both surface water flow bioreactors, having a similar evapotranspiration (13.2 � 11.6–14.0 � 12.7 mm.day 1). By covering the water surface Lemna favors losses by evapotranspiration but at the same time limits exchanges with the atmosphere. However, the retention time of pretreated wastewater in the Lemna lagoon is distinctly lower to that in the lagoon (5.53 � 2.48 vs. 12.5 � 2.86 d). The Mediterranean climate prevails in the experimental site: rainfall of 523–683 mm (1999–2002), average daily temperatures of 14 � C–26 � C, warmest months maxima of about 35 � C (July and August), coldest months minima above 7 � C (December and January). The average rainfall, temperature, sunlight and relative humidity data per sampling cycle are compiled in Table 1. To control mosquitoes, especially in summer, about ten of Mosquito fish individuals (Gambusia affinis Baird & Girard, Poeciliidae) have been introduced, one year before the end of experiment, in each of the two Table 1 Climate data during the experimental period by sampling cycle of 13.2 � 2.80 d. Parameters

All year

Vegetative period

Vegetative rest

Rainfall (mm) Temperature (� C) Maximum temperature (� C) Minimum temperature (� C) Sunshine (w/m2) Maximum sunshine (w/m2) Relative humidity (%) Maximum relative humidity (%) Minimum relative humidity (%)

24.6 � 40.1 19.2 � 4.15 25.4 � 4.96

13.9 � 23.9 21.2 � 3.60 27.6 � 4.51

41.8 � 53.3 15.8 � 2.42 21.5 � 2.94

13.9 � 5.11 349.6 � 214.3 945.8 � 262.9 73.8 � 8.12 92.4 � 5.10

16.4 � 4.09 442.6 � 206.0 1,089 � 217.8 72.0 � 8.80 91.9 � 5.21

9.45 � 3.42 188.9 � 106.9 697.7 � 94.53 77.0 � 5.62 93.3 � 4.86

40.6 � 13.9

37.6 � 13.9

45.7 � 12.29

Sources: Ennabili (1999), Ezzahri (2005), and unpublished data.

Fig. 1. Experimental design of bioreactors. Legend: BOD, biological oxygen demand; COD, chemical oxygen demand; Ec, electrical conductivity; TN, total N; O2, dissolved oxygen; pH, potential hydrogen; PT, total P; Q, flow; T, temperature; V, wet volume. 2

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400 nm for the medium range (0–5 mg N.l 1). For the low range (0–0.5 mg N.l 1), the diazonium reagent reacts with chromotropic acid and develops a yellow color measured at 507 nm. Total phosphorus (TP) and total non-particulate phosphorus (TPf) are oxidized as orthophosphate ions in acidic medium in the presence of persulfate. Two ranges are used: (i) the low range (0–3.5 mg P.l 1) for which orthophosphate reacts with molybdate in an acid medium to produce a phosphomolybdic complex which, in turn, is reduced by ascorbic acid giving an intense molybdenum blue coloration determined at 890 nm; (ii) the high range (0–100 mg P.l 1) for which the phos­ phomolybdic complex forms in the presence of vanadium the yellow vanadomolybdophosphoric complex measured at 420 nm. Orthophosphate ions (P-PO34 ) react, in acid medium, with the ammonium molybdate producing the molybdophosphoric acid. This complex is then reduced by the amino-acid reagent to form the bluemolybdenum intensely colored to determine at 530 nm. The method range is 0–9.8 mg P.l 1.

bioreactors in narrow collaboration with the National Center of Hy­ drobiology and Fish Farming of Azrou (Region of Fez-Meknes). This endemic species of the SE of the United States was introduced in Algeria in 1926, and cited in the South Atlas of Morocco (Le Berre and Chevalier, 1989). Water samples are taken on each bioreactor inlet and outlet approximately every two weeks, during the “December 1999–June 2003” period, for analyzes of nitrogen compounds (total nitrogen, ammonium, nitrates and nitrites) and phosphorus ones (total phos­ phorus and orthophosphates). In the period from June 1999 to September 2000, water samples were also collected to quantify phyto­ plankton. L. gibba growth parameters were measure, from May 1998 to October 1999. 2.2. Biomass and mineral content assessment To estimate the net productivity of L. gibba, the biomass completely covering the water surface is collected at 50%, or only 25% during the warmer season, i.e. every 8 � 3 d (Ennabili, 1999). The collected biomass samples are washed abundantly to remove the excess impurity and intruders (flocs, biofilm, and fauna), drained, dried to constant mass before weighing. To assess N and P contents of L. gibba biomass, 50 g of dry matter were powdered using a 0.5 mm trapezoidal-perforation grinder. 0.25 gplant powder, put into a 100 ml flask, was added with 4 ml of H2SO4 (99–100%) and after 2–3 min, placed on a Hach-Digestdahl apparatus at 440 � C. After 3.3 min, 5 ml of hydrogen peroxide (H2O2) were added. The final mixture (mineralized solution) was obtained after chilling and completing to 100 ml with demineralized water. 0.4 ml of the mineralized solution and 1 ml of Nessler reagent were placed in a 25 ml-flask, and completed with polyvinyl alcohol (0.1 g. l 1). By 420 nm spectrophotometric-reading, the N content was deduced from a calibration curve, in dry weight percent. An atomic absorption spectrophotometer with a PK DDL program was used for the P content-estimate from the mineralized solution. As for phytoplankton, an adequate volume is taken depending on the density of algae at a 10 cm-depth for qualitative examination. The algal biomass was evaluated by the chlorophyll-a determination according to the spectrophotometric method described by Standard Methods (1995).

3. Results 3.1. Lemna biomass The daily net productivity of L. gibba (containing negligible biomass of L. minor, especially during the wet season) in culture on pre-treated wastewater shows a primary peak (0.25 ton dry weight.ha 1.day 1) in the middle of the vegetative period (VP), and a secondary one (0.16 t dw.ha 1.d 1) at the beginning of the vegetative rest (VR). This pro­ ductivity exceeds the average (0.08 � 0.06 t dw.ha 1.d 1, N ¼ 34) dur­ ing the first part of VP, but remains generally low during VR and the second part of VP (Fig. 2). By carrying out a regular harvest of biomass, ranging from 0.296 to 1.709 t dw.ha 1, the net productivity of L. gibba could average 28.39 t dw.ha 1.year 1, i.e. 18.95 t dw.ha 1 during VP and 9.43 t dw.ha 1 throughout VR. The L. gibba biomass collected is partially charged with sludge and plankton at the root level, especially during the cooler season, expressly VR and the first half of VP (Table 2). At the end of VP, biomass harvest contributes to the mechanical disintegration of flocs in the upper part of the water column, and those adhered to the Lemna roots, promoting their subsequent settling at the bioreactor bottom. We also noted that in addition to the conditions prevailing in the bioreactor, the windy weather indirectly favors the production of Lemna biomass, by increasing the free water surface. In wet season either during VP (March–April) or throughout VR (November), corresponding to moderate temperatures (Table 1), the L. gibba roots grow longer, generally exceeding 10 cm (Fig. 3), and reach for several individuals 20 cm, that is about 19% of the water column, apparently due to low nutrient concentrations. Conditions of organic load dilution favor the appearance of L. minor fronds in the Lemna lagoon, and filamentous algae also occur in May. By stopping the bioreactor supply during four days, the L. gibba fronds generally lose their gibbosity and are easily confused with those of L. minor. The above-average biomass production in L. gibba corresponds to the following pre-treated wastewater: COD (310.5 � 51.17 mg O2.l 1), TN (41.75 � 6.292 mg N.l 1), and TP (7.15 � 1.31 mg P.l 1), of which approximately 58%, 43.3% and 56.3% are particulate form, in the same order. It’s the same for the N–NO-2 and N–NO-3 inlet-content (1.163 � 0.469 mg N.l 1), the hydraulic retention time (8.45 � 0.56 d), the air and water temperatures (22.6 � 1.28 and 22.2 � 3.83 � C), and sunshine (686.8 � 105.1 w.m 2). These values are slightly higher than the averages recorded throughout the experimentation period (Fig. 1; Table 1).

2.3. Nitrogen and phosphorus determination Total nitrogen (TN) and total non-particulate nitrogen (TNf) are determined after digestion of the sample in an alkaline medium (NaOH) in the presence of persulfate whereby the nitrogen compounds convert to nitrates. Undigested persulfate is reduced by sodium metabisulfite. Nitrates react with chromotropic acid in a strong acid medium (H2SO4) to form a yellow complex with maximum absorbance at 410 nm. Two ranges are proposed by the firm Hach: the low range (0.2–25 mg N.l 1) and the high range (7–150 mg N.l 1). Ammonium ions (N–NHþ 4 ) are determined according to the Nessler method on filtered samples. The hardness of the sample is complexed by a mineral stabilizer. The polyvinyl alcohol, a dispersing agent, helps to form a yellow color with the Nessler reagent in the presence of the ammonium ions. The coloring developed is proportional to the con­ centration of ammonia. The reading is at 425 nm for a concentration range 0.05–2.5 mg N.l 1. Nitrite ions (N–NO-2) are determined according to the sulfanilamide method whose principle is the formation of a diazonium compound by the reaction of nitrites with sulfanilamide. This compound gives, by coupling with N- (1-naphthyl) -ethylenediamine dichloride, a redmauve color measured at 540 nm. The range used is 0.003–0.3 mg N. l 1. Nitrate ions (N–NO-3) are reduced by cadmium to nitrite ones which react in acidic medium with sulphanilic acid to form an intermediate diazonium salt. The latter reacts with gentisic acid and forms an amber color determined at 500 nm for the high range (0–30 mg N.l 1) and at

3.2. Chlorophyll-a The Lemna Lagoon distinctly reduces plankton production, 3

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Fig. 2. Daily production of biomass by L. gibba grown on pre-treated urban wastewater (May 98-October 99). M, Mean. Table 2 Nitrogen and phosphorus contents (mg.l 1) of bioreactors inlet and outlet, and one way analysis of variance (significant difference at p < 0.0500, N ¼ 98). Bioreactors inlet Lagoon outlet Lemna lagoon outlet F-ratio F-prob. Effects

TN

TNf

NHþ 4

NO2

NO2 þ NO3

TP

TPf

PO34

37.73 � 9.545 26.33 � 9.534 27.65 � 8.536 0.527 0.470 NS

27.67 � 8.887 19.61 � 6.773 20.87 � 7.008 0.821 0.367 NS

22.23 � 7.446 14.89 � 5.558 17.46 � 6.057 4.806 0.031 *

0.027 � 0.049 0.110 � 0.155 0.059 � 0.118 3.331 0.071 NS

0.981 � 1.553 0.912 � 0.863 0.708 � 0.929 1.263 0.264 NS

6.365 � 2.303 6.237 � 2.190 4.852 � 1.739 12.014 0.001 **

3.801 � 1.615 3.781 � 1.794 3.071 � 1.266 5.123 0.026 *

2.475 � 1.634 2.312 � 1.939 2.082 � 1.374 0.459 0.500 NS

TN, Total N; TNf, Non-particulate TN; PT, Total P; TPf, Non-particulate TP.

During the VP, heat waves induce a superabundant development of plankton in the inter-Lemna fronds space, contributing to a suffocating superficial heterogeneous biofilm, sometimes accompanied by sludge rising, gas release, and lack of oxygen in the bioreactor bottom. This was accompanied by slowdown in Lemna multiplication, and sometimes calls for lagoon supplying with additional fresh biomass of Lemna, decreasing the rate of regular biomass removal from 50% to 25% of growing area, or increasing the bioreactor wet volume. Dissolved oxygen concentrations are higher in the lagoon during the warm season (June–September), and decrease significantly in autumn related to the temperature and lighting decline, which in turn affect photosynthesis. In the Lemna Lagoon, the oxygen concentration never passed 3.4 mg O2.l 1 because of shading function of duckweeds, inhib­ iting plankton photosynthesis and restraining gaseous exchanges be­ tween water surface and atmosphere. In the autumn, oxygen content reaches the lowest minimum of 0.1 mg O2.l 1 when compared with the lagoon, and generates some odors the months of November and December. The pH variation is low in the presence of duckweed due to the in­ hibition of the photosynthetic activity of the plankton by shading. Maximum values are associated with heat waves, causing algal blooms and slow multiplication of L. gibba: pH ¼ 9.6 and 660 μg. l 1 of chlorophyll-a in June–August for the lagoon, and pH ¼ 8.7 in July–September for the Lemna lagoon. Moreover, the phytoplankton populations of Euglenophyceae and Chlorophyceae dominate throughout the year. In spring and summer, Euglenales [Euglena texta (Dujard.) K. Hübner and E. splendens Dan­ geard], Chlamydomonadales [Chlamydomonas spp., Eudorina spp. and Pandorina morum (O.F.Müller) Bory.)], Chlorellales (Chlorella sorokiniana Shihira & R.W.Krauss, Micractinium pusillum Fresenius and Oocystis lacustris Chodat), Desmidiales (Closterium ralfsii Br�ebisson),

Fig. 3. Upper part of the water column 52.7 � 2.50 cm deep - Lemna Lagoon.

apparently thanks to the screen effect provided by the floating biomass in comparison with the lagoon (Fig. 4); the latter generates more newly formed organic matter from algae, sometimes exceeding that of the bioreactor inlet in warm season. The chlorophyll-a content at the Lemna lagoon outlet is significantly lower compared to the lagoon one (Fratio ¼ 6.0808, F-prob. ¼ 0.0169, p < 0.0500). 4

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Fig. 4. Chlorophyll-a content of bioreactors inlet and outlets (June 99-September 00).

Sphaeropleales [Ankistrodesmus convolutus Corda, Scenedesmus quad­ ricauda (Turpin) Br� ebisson and S. apiculatus Corda], and Chlorococcales (Pediastrum spp.) predominate. In autumn (November–December), Euglenophyceae and Cyanophyceae, covering Oscillatoriales (Oscillatoria spp. and Phormidium spp.), Synechococcales (Synechococcus spp.) and Chroococcales (Synechocystis spp.), co-dominate with Chlorophyceae (Chlamydomonas spp.), with appearance also of a pink coloring of water. During the winter, Chlorophyceae and Cyanophyceae generally show less

development, probably related to the decrease of temperature and lighting. 3.3. Nitrogen and phosphorus removal The Lemna lagoon generally exports more particulate N and P with respect to the lagoon (Figs. 5 and 6), mainly due to Lemna role as a screen preventing overdevelopment of plankton, and to its roots fixing

Fig. 5. Variation of particulate-Total nitrogen of bioreactors inlet and outlets versus time. 5

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Fig. 6. Variation of particulate-Total phosphorus of bioreactors inlet and outlets versus time.

suspended flocs. In addition, the ANOVA analysis (significant difference at p < 0.0500, N ¼ 98) shows that the Lemna lagoon reveals a highly sig­ nificant total P retention and significant non-particulate P removal with regard to the lagoon, in turn exhibiting a significant retention of N–NHþ 4 (Table 2). By comparing the N and P concentrations of inlet and outlet of each bioreactor (Table 2), the total N retention by the Lemna lagoon is analogous to the one observed in the lagoon, averaging 28.2% and 28.9% respectively, while these respective rates are 23.1% and 0.99% for total P removal. The L. gibba biomass collection allows a reduction of 13.2% of N and 19.9% of P entering daily in the Lemna lagoon, vs. 34.1% of N and 57.8% of P removed by this bioreactor. The N and P retention as a result of regular harvest of the L. gibba biomass achieves 1.749 t N. ha 1.yr 1 and 0.457 t P.ha 1.yr 1, equivalent to a variable miner­ alomass throughout the year from 18.23 to 105.3 kg N.ha 1 and from 4.766 to 27.51 kg P.ha 1.

the year, mostly at the water level. 4. Discussion 4.1. Biomass production The specific growth rate of Lemna ranges from 0.10 to 0.35 d 1, and each frond is able to produce at least 10 to 20 more during its lifetime, a doubling time of 2.3–7.3 d related by Reed et al. (1988) and Edeline (1997) or cited by Bonomo et al. (1995), which converges with the period between two successive collections of Lemna biomass. This plant can thrive under anoxic conditions, but the biological activity rate in water will be lower than in an aerobic environment (Reed et al., 1988). The Lemna specific growth rate would increase with the water and at­ mosphere temperatures, the wastewater nutrient content, the collection frequency of biomass, and the windy conditions (Reed et al., 1988; Radoux and Kemp, 1992). The optimal growth of Lemnaceae is observed under pH (4.5–8.0), temperature (13.5–22 � C), electrical conductivity (900–3,000 μS/cm), and COD (300–500 mg O2.l 1), as underlined by Agences de l’Eau (1998), El-Shafai et al. (2007), Sanchez Morales and Coral Caldas (2009) and Priya et al. (2012), and mentioned by Bonomo et al. (1995), which are similar characteristics of the pre-treated waters supplying the bio­ reactors in this study. Duckweeds can also grow under conditions of pH superior to 11 (Rodríguez-Miranda et al., 2010). Lemnaceae can tolerate minimum temperatures of 4–7 � C (water) and 1–3 � C (atmosphere); underneath the plant survives in a dormant layer in the bioreactor bottom until restoration of the favorable conditions to repopulate the water surface again, as evidenced by Reed et al. (1988) or discussed by these authors, Brix (1993) and Bonomo et al. (1995). Lemna can also grow under high organic loads of COD (700 mg O2.l 1), and 1 1 and P-PO3N–NHþ 4 contents of 16 mg N.l 4 of 6 mg P.l , as emphasized by Tabou et al. (2014) or mentioned by Bonomo et al. (1995). However the duckweeds growth is inhibited when pH, and N–NHþ 4 1 and P-PO3and 8 mg P.l 1 in the 4 concentration exceed 10, 32 mg N.l same order, like highlighted by Tabou et al. (2014) and Seidel et al.

3.4. Animalia Cladocera (Daphnia spp.) and Culicidae (mosquito larvae) progres­ sively develop in the two bioreactors at the beginning of VP, favoring Gambusia. Red waters show up in May, followed by a partial death of the mosquito fishes, owing to heat waves in June, especially in the Lemna lagoon. In addition, macro-zooplankton larvae of Chironomidae develop intensively, especially in the Lemna lagoon in July, decline in August (warmest month), and return in large numbers the end of VP as well as the mosquito larvae. Heteroptera and Coleoptera (Dytiscidae) are also observed in summer. Several factors could be responsible for death of Gambusia individuals in the Lemna lagoon, including high density, large plankton growth, decrease of dissolved oxygen content, and invasion by Dytiscidae larvae; the lack of regular harvest of Lemna biomass would also have a com­ parable effect. A simultaneous influx of young Gambusia and mosquito larvae was observed during the VR, also associated with the death of adults of this fish. Gastropoda fixed to the edge are observed throughout 6

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(2002) or indicated by Bonomo et al. (1995). The average concentra­ tions obtained in this study for these elements are 22.9 � 6.98 mg N.l 1 and 2.66 � 1.95 mg P.l 1 respectively. It was also shown that greater than 75 mM salinity causes salt stress in L. minor and therefore inhibits retention of N and P by this macrophyte (Liu et al., 2017). The extensive length of L. gibba roots in the wet season would be an adaptation mechanism to the lowering of nutriment concentrations, as related by Bonomo et al. (1995). The Lemna biomass fluctuates from 0.296 to 1.709 t dw.ha 1 and consequently ranks among the biomass values reported in Lemnaceae under different conditions of experimentation or management (climate, supplying conditions, and harvesting frequency); these are from 0.24 to 3.5 t dw.ha 1, as reported by Agences de l’Eau (1998), Ennabili et al. (1998), Greenway and Woolley (1999), and Ennabili (1999, 2008b) or evoked by Gumbricht (1993) and Bonomo et al. (1995), despite its high water content from 82 to 97.2% (Reed et al., 1988; Ennabili et al., 1998; Ennabili and Radoux, 2006; Verma and Suthar, 2014; Sudiarto et al., 2019). The average biomass production by L. gibba of 0.08 t dw.ha 1.d 1 is comprised in the production range highlighted by previous studies (0.01–0.29 t dw.ha 1.d 1) under different conditions of experimenta­ tion and management (Ennabili 1999, 2008b; El-Shafai et al., 2007). The biomass production of 18.95 t dw.ha 1 during the VP remains similar to that reported previously in semi-continuous wastewater treatment with a regular biomass collection (16.5 t dw.ha 1), but far exceeds that achieved in batch wastewater culture under greenhouse (3.3 t dw.ha 1), such as Ennabili(1999, 2008b) or mentioned by Bonomo et al. (1995). The maximum net productivity reached under our experimental condi­ tions (0.25 t dw.ha 1.d 1) is one of those reported in the laboratory by Verma and Suthar (2014) in the same species grown on 75% and 50% diluted wastewater (0.239 and 0.268 t dw.ha 1.d 1 respectively). The net productivity of L. gibba recorded (28.39 t dw.ha 1.yr 1) slightly surpasses the values reported by other authors (6–26 t dw.ha 1. yr 1), and factors such as temperature, electrical conductivity, nutrient availability, collection frequency of biomass, and impurities presence influence the surface density of duckweeds, both underlined by Reed et al. (1988), Gumbricht (1993) and Framework Programme (2004) or recalled by Bonomo et al. (1995). In the VP, the concentration of chlorophyll-a is greater and more variable in the lagoon (86.4 � 168 μg l 1) comparatively with the Lemna lagoon (21.3 � 19.4 μg l 1), and far from reaching the levels recorded in stagnant water (300–500 μg l 1), but cross in summer the high risk limit of algae blooming, eventually associated to a massive mortality of Cyanobacteria colonies, as reported by Agences de l’Eau (1998). In wastewater treatment ponds, duckweeds result a decrease of chlorophyll-a concentration as well as algal diversity, pushing the tro­ phic status of the bioreactor from eutrophication to mesotrophication (Abou El-Kheir et al., 2007). Moreover, light and temperature are considered as the best indicator explaining the variation of the maximum chlorophyll-a biomass. The dense duckweed cover on water surface causes a weak light penetration, and consequently a low photosynthetic oxygen production by the plankton. The water column then becomes largely anaerobic, favoring denitrification, which was obtained by Reed et al. (1988), Bonomo et al. (1995), Agences de l’Eau (1998) and Abou El-Kheir et al. (2007), or discussed by Brix (1993) and Framework Programme (2004). Other factors may be involved in the reduction of algae in the bioreactor, such as competition for nutrients and the secretion of organic substances by the duckweed rhizosphere, as highlighted and discussed by Abou El-Kheir et al. (2007). This float layer of biomass on water surface also reduces the aeration of the water by direct diffusion and evaporation (Bonomo et al., 1995).

mineralomasses. Each frond of the Lemnaceae absorbs its nutrients directly through the whole plant and not only through the root system, as mentioned by Bonomo et al. (1995). N–NHþ 4 and N–NO3 are the two N forms preferentially assimilated by aquatic plants (Reddy and Debusk, 1984), while the direct assimilation of P by these plants mainly concerns P-PO34 (Richardson et al., 1995). Furthermore, the contribution of L. gibba lagoon in the significant export of particulate N and P, total P (also underlined by Sudiarto et al., 2019) and non-particulate P, high­ lighted in this study, assumes an auto-heterotrophic status of this plant species; nitrification and denitrification would be low apparently because of the short hydraulic retention time. However, the increase in hydraulic retention time could adversely affect the yield of Lemna gibba biomass as reported by Sanchez Morales and Coral Caldas (2009). The average export of about 1.749 t N.ha 1.yr 1 and 0.457 t P.ha 1. yr 1 by L. gibba, with regular biomass collection, largely exceeds cor­ responding values in Lemnaceae for N (0.35–1.20 t N.ha 1.yr 1), and is similar to those for P (0.116–0.400 t P.ha 1.yr 1), as discussed by Bonomo et al. (1995). Compared with the inlet load, the N equivalent (13.2%) exported by regular biomass collection is lower than that underlined by Reed et al. (1988) (25%), but the equivalent in P (19.9%) exceeds that reported by the same authors (16%). Leaching of dead duckweeds between successive biomass exports can restore significant amounts of N in the bioreactor (Reed et al., 1988; Radoux and Kemp, 1992; Ceschin et al., 2019; Sudiarto et al., 2019). In this study, the lagoon exhibits a significant retention of N–NH4þ when compared with the Lemna lagoon, finding confirmed by Ozimek et al. (2015) in L. minor. 4.3. Bio-components interaction The zooplankton population identified in the studied bioreactors partially overlaps the taxa quoted by Kone (2002) and Ezzahri (2005), in particular Rotifera (Brachionus spp.), Crustacea-Ostracoda, Crustacea-­ Clodocera (Moina spp.), Ciliophora (Paramecium spp. and Vorticella spp.), Zooflagellates, Amoebae, Hemiptera (Notonectidae), and larvae of Diptera (Syrphidae and Culicidae). In addition to the climate conditions, the proliferation of algae is also influenced by the filter-feeders such as Rotifera or Cladocera (Kone, 2002); although Dawidowicz and Ozimek (2013) confirmed a negligible contribution of Moina branchiata (Cladocera) in the removal of organic particles from Lemna based wastewater-treatment plant. Also, there is a seasonal and cyclical evolution of P concentrations in lagoon ponds, in close correlation with phytoplankton and zooplankton populations (Ouazzani et al., 1997). The high algal charge in the bioreactor outlets may be also related to the low zooplankton biomass when compared to the phytoplankton one (Kone, 2002). The zooplankton importantly acts on purification processes; pro­ tozoa eliminate bacteria, especially the free ones. Rotifera and Cladocera remove algae and particulate organic substances by filtration, contrib­ uting to the medium clarification. Daphnia (Cladocera) directly con­ tributes to the wastewater treatment by removing suspended solids, including algae, and indirectly reduces the P concentration in feces (Reed et al., 1988; Moss, 1995; Ezzahri, 2005). But the variability of biotic and abiotic parameters in sewage treatment ponds makes survival of zooplankton random, which is directly reflected in the reduction of algae and bacteria (Reed et al., 1988; Moss, 1995; Kone, 2002). The zooplankton consumes little or no Cyanobacteria, given their characteristics of mucilaginous envelope, potential toxicity, and cell size, instead promoting their growth by eliminating competitor algae (Agences de l’Eau, 1998). The massive development of planktonic Cyanobacteria generally generates a coloring of water (Moss, 1995; Agences de l’Eau, 1998), a phenomenon observed in this study. The mosquito fishes consume mainly the mosquito larvae, the benthic invertebrates (either in the bottom sediments or at their appearance as adult insects), crustaceans, young fishes and detritus, and probably take rotifers as other food sources become scarce (Le Berre and Chevalier, 1989; Moss, 1995).

4.2. Nitrogen and phosphorus retention The most productive macrophytes generally have the largest N and P 7

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The temperature and pH recorded in the studied bioreactors are among the values tolerated by the mosquito fish “Gambusia affinis” (Reed et al., 1988; Le Berre and Chevalier, 1989), but the dissolved oxygen concentration (2.62 mg O2.l 1) in the Lemna lagoon is ranging the lower threshold (2 mg O2.l 1) limiting its growth and that of 5 mg O2.l 1 allowing slow growth (Reed et al., 1988); on the contrary, the lagoon is more oxygenated (14.27 mg O2.l 1). Although Gambusia can survive under low dissolved oxygen concentrations (Ezzahri et al., 2005a), their long-term effectiveness in eliminating mosquito larvae would to be confirmed, and the injection of oxygen into the Lemna lagoon would be relevantly applicable. Duckweeds greatly reduce the open water surface, and as a result, lessen the laying area for mosquitoes and the respiration of existing larvae (Reed et al., 1988; Bonomo et al., 1995; Tariq et al., 2009). But this indirect effect of duckweeds on the limitation of mosquito larvae on the water surface is inversely related to the wastewater organic load and associated microorganisms (Tennessen, 1993).

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5. Conclusion The L. gibba bioreactor would be more profitable in wastewater treatment, only on condition of biomass harvesting and valorization, to improve the purification efficiency and to amortize the high manage­ ment costs. This bioreactor concerns, inter alia, the tertiary treatment of wastewater, especially the P removal, and the rehabilitation of existing wetlands by favoring the installation of the trophic chain, chiefly the duckweed grazers like fish, birds, rodents, etc. The use of L. gibba in wastewater treatment for the cold-dominated localities (e.g. high alti­ tudes for the Mediterranean climate) could restraint the macrophyte growth and engenders odors. Direct N and P assimilation by duckweeds remains low, and the contribution of other processes such as sedimentation and biodegrada­ tion of organic matter, microbial nitrification and denitrification, adsorption, precipitation … should be quantified. The artificial oxygenation of the lower part of the L. gibba lagoon could favor nitrifi­ cation, activate more the other bio-components (algae, bacteria, zooplankton, fish …), limit occasional odors related to the conditions of anoxia at the bottom, and adequately ensure mosquitoes control. Acknowledgments This study was carried out as part of the MHEA® project, achieved in M’Diq (NW of Morocco) via the Agence de la Francophonie (Walloon Region-Morocco). References Abou El-Kheir, W., Ismail, G., Abou El-Nour, F., Tawfik, T., Hammad, D., 2007. Assessment of the efficiency of duckweed (L. Gibba) in wastewater treatment. Int. J. Agric. Biol. 9 (5), 681–687. Agences de l’Eau, 1998. Biologie et �ecologie des esp� eces v�eg�etales prolif�erant en France, Synth�ese bibliographique, N� 68. Agences de l’Eau, Paris. Allam, A., Tawfik, A., El-Saadi, A., Negm, A., 2014. Potentials of using duckweed (Lemna gibba) for treatment of drainage water for reuse in irrigation purposes. Desalin. Water Treat. 57, 1–9. https://doi.org/10.1080/19443994.2014.966760. Bonomo, L., Pastorelli, G., Zambon, N., 1995. The use of duckweed for wastewater treatment: a review and practical experience in Northern Italy. In: Ramadori, R., Cingolani, L., Cameroni, L. (Eds.), Natural and Constructed Wetlands for Wastewater Treatment and Reuse: Experiences, Goals and Limits, pp. 47–58. Perugia. Brix, H., 1993. Wastewater treatment in constructed wetlands: system design, removal processes, and treatment performance. In: Moshiri, G.A. (Ed.), Constructed Wetlands for Water Quality Improvement. CRC Press, Inc., pp. 9–21 Cadelli, C., Nemcova, M., Ezzahri, J., Ennabili, A., Ater, M., Radoux, M., 2004. Influence of evapotranspiration on the design of extensive wastewater treatment systems under Mediterranean conditions at the MHEA® Experimental Centre of M’Diq (Tetouan, Morocco). In: 6th International Conference on Waste Stabilization Ponds & 9th International Conference on Wetland Systems. Avignon, France, pp. 95–103. Ceschin, S., Sgambato, V., William Ellwood, N.T., Zuccarello, V., 2019. Phytoremediation performance of Lemna communities in a constructed wetland system for wastewater treatment. Environ. Exp. Bot. 162, 67–71. https://doi.org/10 .1016/j.envexpbot.2019.02.007.

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Radoux, M., Kemp, D., 1992. R^ ole de la fr�equence des pr�el�evements de la biomasse produite sur les capacit� es �epuratrices de L. minor L. Rev. Sci. Eau 1, 55–68. https:// doi.org/10.7202/705120ar. Radoux, M., Cadelli, D., Nemcova, M., Ennabili, A., Ezzahri, J., Ater, M., 2000. Optimisation of Natural Wastewater Treatment Technologies in the Mhea® Experimental Centre in M’Diq, Mediterranean Coast of Morocco, 7th International Conference on Wetland Systems for Water Pollution Control. Lake Buena Vista, Florida, pp. 1145–1152. Radoux, M., Cadelli, D., Nemcova, M., Ennabili, A., Ezzahri, J., 2003. Optimisation of extensive wastewater treatment systems under Mediterranean conditions (Morocco): compared purification efficiency of artificial ecosystems. In: Vymazal, J. (Ed.), Wetlands: Nutrients, Metals and Mass Cycling. Backhuys Publishers, Leiden, The Netherlands, pp. 143–168. Reddy, K.R., Debusk, W.F., 1984. Growth characteristics of aquatic macrophytes cultured in nutrient-enriched water: I. Water hyacinth, water lettuce, and pennywort. Econ. Bot. 2, 229–239. https://doi.org/10.1007/BF02858838. Reed, S.C., Crites, R.W., Middlebrooks, E.J., 1988. Natural Systems for Waste Management and Treatment. McGraw-Hill, Inc., New York. Richardson, C.J., Quin, S., Craft, C.B., 1995. Predictive model for phosphorus retention in wetlands. In: Proceedings of the International Workshop, Nutrient Cycling and Retention in Wetlands and Their Use for Wastewater Treatment. Trebon, Czech Republic, September 6-9, pp. 125–148. Rodríguez-Miranda, J.P., G� omes, E., Garavito, L., L� opez, F., 2010. Estudio de comparaci� on del tratamiento de aguas residuales dom�esticas utilizando lentijas y buch� on de agua en humedales artificiales. Technol. Cienc. Agua 1 (1), 59–68. Sanchez Morales, R., Coral Caldas, Y.M., 2009. Evaluaci� on del tratamiento de aguas residuales con Lemna gibba en estanques con r� egimen de flujo de pist� on. Afinidad 541, 238–242. Seidel, M., Laouali, S., Idder, T., Mouchel, J.M., 2002. Lentille d’eau et Tilapia : une solution � ecologique pour le traitement des eaux us� ees dans les pays en voie de

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