Removal of organic load from wastewater by using Datura innoxia Mill.

N. Vaillant-Gaveau1,*, F. Monnet

2,3, H. Sallanon

4, A. Coudret

4, and A. Hitmi

5,6

1 Laboratoire de Stress, Défenses et Reproduction des Plantes, URVVC-SE EA 2069, Université de Reims Champagne-

Ardenne, UFR Sciences Exactes et Naturelles, Bâtiment 18, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2,

France 2 CEA, CNRS, Université Aix-Marseille, UMR 6191 Biologie Végétale et Microbiologie Environnementale, Laboratoire

d¹Ecophysiologie Moléculaire des Plantes, CEA Cadarache, 13108 Saint Paul lez Durance, France 3 Université d’Avignon et des Pays de Vaucluse, Faculté des Sciences, 33 Rue Louis Pasteru, 84000 Avignon, France 4 Qualité et sécurité des aliments d’origine végétale, Université d’Avignon et Pays de Vaucluse, 74 rue Louis-Pasteur,

84029 Avignon, France 5 Clermont Université, Université d'Auvergne, Laboratoire de Physiologie et Biotechnologies Végétales, BP 10448, 63000

Clermont-Ferrand, France 6 Laboratoire de Physiologie et Biotechnologies Végétales, IUT de Clermont-Ferrand, Université d’Auvergne, 100, rue de

l’Egalité, 15000 Aurillac, France.

* Corresponding author

The objectives in this work were to investigate a conceptual layout for an inexpensive and simple system that would treat

primary municipal wastewater to discharge standards. Wastewater treatment by Datura innoxia Mill. plants in horizontal

flow was investigated using the nutrient film technique, a widely used hydroponic system in commercial greenhouse

industry. A global performance evaluation of an experimental horizontal flow was performed after 6 months of

functioning. The variables measured in the influent were significantly higher than those in the effluent except for SO42-,

K+, Na+, NO2-. Forty eight hours after plant treatment, the purification efficiency was 97, 93 and 87% for SS, BOD5 and

COD, respectively. The legal discharge levels were reached. Pollutant removals (SS, BOD5 and COD) were correlated to

their respective loading and no limit has been observed. Decrease of total bacteria, total and faecal coliforms, Escherichia

coli and faecal enterococci in effluent waters were measured. SS and then indirectly BOD5 and COD were removed by

filtration and adsorption; solids, probably trapped in the root systems, were then decomposed and mineralised. D. innoxia

transforms and uses the wastewater as the only nutritive source. At the same time, D. innoxia produced alkaloids, without

affecting either the quantity or the quality of the leaves.

Keywords wastewater, phytotreatment, nutrient film technique, organic loading, Datura innoxia Mill.

1. Introduction

Conventional wastewater treatment plants involve large capital investments and operating costs. For that reason, these

systems are not suitable for small villages that cannot afford such expensive conventional treatment systems [1]. These

treatments are so expensive and produce such huge quantities of sludge that it is impossible to spread them in landfills

(European Directive 91/271/CEE of 21 May 1991). In France, more than 50% of domestic wastewater in rural areas are

not treated yet, despite the fact that all wastewater must be treated to respect environmental policy [2].

Constructed wetlands are gaining in importance as an effective alternative for the treatment of septic effluents in

small villages. Compared with conventional treatment systems, wetlands (i) could be set up where the wastewater is

produced; (ii) could be maintained by relatively untrained personnel; (iii) have relatively low energy requirements; and

(iv) are low cost systems (Ciria et al., 2005) [3]. Domestic wastewater is mainly transformed from organic matter and

contains most of the required nutrients for plant growth, generally in an appropriate ratio [4]. Duckweed (Lemna spp.)

[5], water hyacinth [Eichhornia crassipes (Mart.) Solms] [6], cattail (Typha latifolia L.) and reed [Phragmites australis

(Cav.) Trin. ex Steud. and Carex spp. are generally planted in wetlands [7]. Studies conducted on the removal of total P

and total N showed a very wide range of treatment effectiveness [8, 9]. These plants enhance biodiversity and have no

other uses for rural communities besides purification yet. Jewell (1994) has combined anaerobic treatment of primary

sewage with a specialized hydroponic (water as a growth medium) secondary or tertiary treatment system that yields

biomass [10].

We developed treatment of sewage using the nutrient film technique (NFT), initially developed by Cooper (1976)

[11], with a horticultural species Rosa hybrida [12] and with medicinal species such as Chrysanthemum

cinerariaefolium [13], Datura innoxia Mill. [14], and Digitalis lanata and Digitalis purpurea [15]. In previous study,

we have investigated the possibility of introducing valuable commercial species such as D. innoxia into the treatment

system [14]. D. innoxia is an herbaceous species, belonging to the Solanaceae family. Various species of Datura are

cultivated for the production of secondary metabolites. For example, the leaves of D. innoxia are an important source of

tropane alkaloids: atropine, hyoscyamine and scopolamine. Therefore, the economic importance of these molecules

relies on their medicinal applications [16].

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This paper described the growth of vegetation and the nutrient removal in an experimental wetland. The effectiveness of this system was accurately evaluated and modelized. We showed that plant grown in wastewater still, synthesize tropane alkaloids and can purify the effluent.

2. Methods

2.1 Laboratory pilot plants

Laboratory pilot plants consisted of polyvinyl chloride (PVC) tanks (4 m long, 0.15 m wide and 0.10 m deep; Fig. 1). The purification system used NFT soilless culture [11] with permanent recirculation of 30 l of wastewater, regulated by an electric pump with a flow rate of 10 l min-1. The experiment lasted six months (from July to December); this period includes three seasons and thus different qualities of wastewater. From July to November the sewage composition varied widely because the raw effluent concentration was depending on rainfall. The sewage came from a community of 980 people without industry; it was representative of a rural community (Aurillac – France, at latitude 44° North and at longitude 2°East). The raw effluent was weekly obtained and it was placed in contact with the plants without pre-processing and the 30 l of wastewater were renewed every 72 h.

Fig. 1 System of wastewater treatment: horizontal flow system with plants (D. innoxia) in the ditch (P is the pump).

D. innoxia plants were initially developed in individual pots containing a 50/50 mixture of vermiculite/compost. After six months of culture, they were transferred to a hydroponic system. These plants presented an average shoot length of 65±5 cm and a fresh mass of 75±5 g. The plants grew bare-rooted in 3 cm solution flowing by gravity. Three channels were used: one with 25 plants supplied with wastewater (planted channel), one with 25 plants supplied with a nutritive solution of Lesaint and Coïc [17] (control plant channel) and one plant-free channel supplied with wastewater (unplanted channel) to evaluate the role of plants. These experiments were performed in a green house with a controled temperature at 25±3°C/15±3°C (day/night) and with a natural photoperiod.

2.2 Measurement of water quality parameters.

During the experiment, wastewater wastewater samples were analysed after 0, 12, 24, 48 and 72 h of treatment: 5 full cycles were carried. The various samples necessary for the analysis of the physical, chemical and biological parameters were taken in compliance with international standards for water analysis (ISO 5667). Five replicates were performed for each measurement. All parameters commonly used to assess the performance and treatment capacities of such systems were examined (standard method number follows each parameter in brackets). The pH was measured using a glass electrode with a WTW pH 320 pH-meter (NF T90-008), conductivity with a LF 320/SET, WTW electrode (ISO 7888) and dissolved oxygen with a WTW OXI 320 portable oximeter (ISO 5814). Total suspended solids (SS) were determined after filtration under vacuum with 47 mm diameter glass fibre filters (Durieux, France), followed by drying to constant weight at 105°C (NF EN 872). The chemical oxygen demand (COD) was measured according to standard ISO 6060. The biochemical oxygen demand (BOD5) was determined after five days at 20°C in the dark in a thermostated incubator, by measuring the oxygen concentration, expressed in mg l-1 (ISO 5815).

Total nitrogen (TN) in water was measured by spectrophotometric assay, at 324 nm (Genesys 5, Spectronic) after potassium peroxodisulphate digestion at 120°C, 45 min, 100 KPa (ISO 7890-1).

4 m

Circulation

Datura

innoxia

Water level

3 cm

P Tank

15 cm

_______________________________________________________________________________________

The anionic and cationic composition was determined by capillary electrophoresis (P/ACE 5000 System Series; Beckman Coulter, Fullerton, CA) equipped with a diode array detector (DAD). The capillary was 60 cm long (54 cm effective length) with a 75 mm i.d. The capillary was thermostated at 25°C and a constant voltage of 20 kV, with an initial ramp of 0.17 min, was applied during analysis. Sample injections were made with pressure mode for 30 s at 3.45 kPa. The detection of NO3

-, NO2-, SO4

2-, and Cl- was performed at 254 nm with a bandwidth of 1 nm. The carrier buffer was a chromate electrolyte solution of 4.7 mM sodium chromate (Fisher Scientific, Hampton, NH), 4 mM OFM-OH (Waters, Milford, MA), 10 mM 2-(cyclohexylamino) ethanesulfonic acid (CHES, Sigma, St. Louis, MO), and 0.1 mM calcium gluconate (Sigma) at pH 9 [18]. The detection of NH4

+, K+, Ca2+, Na+, and Mg2+ was performed at 214 nm and with a constant voltage of 25 kV. The electrolyte contained 65 mM 2-hydroxy isobutyric acid (Aldrich, St. Louis, MO), 50 mM 4-methyl benzyl amine (Fluka, Buchs, Switzerland), and 20 mM 18-crown-6-ether (Sigma, St. Louis, MO).

2.3 Enumeration of bacteria

The enumeration of total bacteria was performed by epifluorescence microscopy. Samples (2 ml of water column) were fixed with 2 ml of 4% formaldehyde for 30 min or more at room temperature; 1 ml of an appropriate dilution of the mixture was stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma) at a final concentration of 1 µg/ml for 20–30 min in the dark and filtered on a polycarbonate filter (Millipore, pore size: 0.2 µm, type GTBP). The filter was mounted on a slide with immersion oil (Olympus) and the bacteria counted with an Olympus BH 2 epifluorescence microscope [19]. Total and faecal coliforms were performed in accord with ISO 9308-2. Enumeration of Escherichia coli (ISO 9308-3) and faecal enterococci (ISO 7899-1) in effluent waters were determined by miniaturized microtiter plate method for MPN determination: MUG/EC for Escherichia coli and MUD/SF for faecal enterococci (Biokar Diagnostics).

2.4 Modelling of the process - Kinetics of SS, BOD5, COD

The removal of biodegradable constituents such as SS, BOD5 and COD in wetland systems can be described by the following first-order kinetic model:

C(t) = C1 . e-k.t + C0

where C(t) is the effluent concentration (mg l-1) at time t, (C1 + C0) is the initial influent concentration (mg l-1), C0 is the final influent concentration at infinite time (mg l-1), k is the first order reaction rate constant (h-1) and t is the treatment time (h). This model has been used previously to calculate rate constants for organic removal in urban wastewater treatment [13].

2.5 Tropane alkaloid extraction and analyses (hyoscyamine and scopolamine)

Freeze-dried tissues (3 leaves and 3 seeds) were ground, and tropane alkaloids were extracted twice from samples weighing 150 mg, with 50 ml of CHCl3 – 30% NH4OH (49/1, v/v) under reflux. Combined extracts were dried over Na2SO4, filtered and evaporated under reduced pressure in a thermostatic bath (45°C). Three extractions were carried out for treatment. The residue was redissolved in the HPLC solvent and analysed in replicate sets [20]. Alkaloids were assayed by quantitative HPLC using a Waters HPLC system [21]. The stationary phase was a C18 Nucleosil (250 x 4.6 mm) column. The elution solvent was composed of water – acetonitrile – phosphoric acid – triethylamine (83.4/16.5/0.1/0.02, v/v/v/v, isocratic mode). A spectrophotometric UV detection was used (λ = 210 nm). The flow rate was 1 ml min-1. The calibration was made with standard scopolamine hydrochloride (Rt = 6.2±0.5 min) and L-hyoscyamine (Rt = 8.3±0.5 min). Every sample was assayed twice. Alkaloids were quantified as mg g-1 dry weight.

2.6 Statistical analysis

All data were analysed using the ANOVA test at the 0.05 probability level.

3. Results and Discussion

3.1 Pollutant removal versus loadings

When the pollutant removal capacity is plot versus the pollutant loading, we observed a close relationship (Fig. 2). The effluent composition varied widely: it was composed of SS between 35 and 170 mg l-1, BOD5 between 32 and 307 mg l-

1, and COD between 142 and 637 mg l-1. The pollutant removal was highly correlated to the effluent pollutant concentration, in the plant-free channel and in the planted channel (Fig. 2). In the case of SS (Fig. 2a), the linear regression coefficient (r2) is 0.99 in the planted channel and 0.89 in the plant-free channel. Similar results were

_______________________________________________________________________________________

observed with BOD5 and COD (Fig 2b, 2c). The slope of the curve indicated the means of removal efficiencies observed after 48 h. The removal means of SS, BOD5 and COD were 97, 93 and 87 %, respectively in the planted channel. However, these values were only 25, 65 and 46 % in the plant-free channel (Fig. 2a, 2b, 2c). This suggerats that the system purified the wastewater correctly whatever the influent organic load. No saturation sign was detected, even at the highest load. The removal of biodegradable constituents in the system could be described using a first-order kinetic model (Vaillant et al., 2003). SS, BOD5 and COD decreased exponentially and the C1, C0, k and r2 values are represented in Table 1. The values of r2 indicated that the kinetic modelling was correct and that the model fits reality. C1 (quantity of removed pollution) varied with the sewage load, but C0 (minimal value of pollution after one infinite treatment time) remained constant. The k values change. However, no correlation could be demonstrated between k and the influent load or the experiment duration. The k variation may have been due to the sewage composition. The C0 values obtained after the modelisation of the system purification capacity were identical to those obtained experimentally after 48 h of plant treatment (Table 1). Thus, we can assume that the system has reached maximal efficiency after 48h. Total bacteria, total and faecal coliforms, Escherichia coli and faecal enterococci decreased more quickly in the presence of D. innoxia than in the control ditch (Fig. 4). The NFT system with D. innoxia can strongly reduce the total organic load without root system saturation and without sedimentation of the hydroponic channels. SS and thus indirectly BOD5 and COD were decreased by filtration and adsorption; the solids trapped in the root system were then decomposed and mineralised by bacteria [14]. Research on the function of wetland plants revealed that macrophytes principally create appropriate conditions for microbial activity through increasing the substrate surface area in the water and oxygenating the environment around root hairs for aerobic reaction. They also facilitate filtration and sedimentation by encouraging quiescent conditions. Moreover, plants also assimilate nutrients via their growth metabolism [3, 16, 22]. D. innoxia preferably removed Mg2+. The average reduction in BOD5 concentrations was 93 % in the planted system and 65 % in the plant free system. Nonetheless, in comparison with the control system, the treatment with plants frequently provided better performance. This agreed with Tanner et al. (1995) who reported that BOD5 removal in planted units was greater than that in unplanted units, especially at high loading rates [23]. As has been reported elsewhere [16, 22, 24], it is well known that macrophytes accelerate performance of constructed wetlands by several mechanisms. Nevertheless, Lim et al. (2001) reported no significant difference for BOD5 removal between planted and unplanted wetlands due to microbes playing a prominent role for BOD5 removal whereas vegetation provide more oxygen and a support medium for microbial degradation to occur [25]. Table 1. Modelisation of wastewater quality evolution in the planted channel.

BOD5 k r2

C1

(mg l-1) C0 calculated

(mg l-1) C0 experimental

(mg l-1) 1 0.1633±0.0094 0.999 145±2 1.9±1.2 1.6±1.0 2 0.1310±0.0127 0.992 256±8 18.8±2.1 20.3±4.5 3 0.4693±0.0203 0.998 121±2 20.8±0.8 18.5±3.6 4 0.4972±0.0601 0.988 151±6 11.4±2.8 6.7±0.5 5 0.1827±0.0198 0.987 150±6 4.1±3.4 4.4±0.8

SS

k r2 C1

(mg l-1) C0 calculated

(mg l-1) C0 experimental

(mg l-1)

1 0.2891±0.0161 0.9996 181±1 1.2±0.5 1.2±0.4 2 0.5781±0.0364 0.9965 156±3 6.7±1.3 3.5±1.1 3 0.4805±0.0326 0.9947 164±4 9.8±1.7 6.6±1.4 4 0.7743±0.0794 0.9958 89±2 3.8±0.9 2.2±0.4 5 0.6532±0.0620 0.9947 104±2 4.3±1.2 2.9±0.7

COD k r2 C1

(mg l-1) C0 calculated

(mg l-1) C0 experimental

(mg l-1) 1 0.2370±0.0163 0.998 328±4 78.8±1.9 81.1±3.2 2 0.1298±0.0144 0.982 267±12 64.6±7.2 51.4±6.1 3 0.2576±0.0141 0.995 527±12 104.7±6.1 95.6±5.6 4 0.6199±0.1045 0.998 205±10 116.9±4.6 104.7±2.4 5 0.7198±0.0862 0.993 196±6 30.4±2.6 23.1±0.4

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Fig. 2 Removal from SS (a), BOD5 (b), COD (c) in the plant system

with D. innoxia and with the control versus the effluent load after 48 h.

n = 9.

Fig. 3 Concentration of N-Total, N-NH4+, N-

NO2-, N-NO3

- in the plant system with D.

innoxia (a) and in the control (b). Values are

means ± standard error; n = 5.

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3.2 Nitrogen removal

Total nitrogen sums the nitrogen in ammonium (NH4+), oxidised forms (Nox = NO2

- + NO3-) and aggregate and soluble

organic forms (Fig. 3). The TN decreased more quickly in the presence of D. innoxia than in the control ditch. Initially, the wastewater had a high concentration of TN mainly composed of NH4

+ with a very low content of NO2- and NO3

-. After 48 h treatment, the ammonium concentrations in the effluent were strongly reduced, by approximately 93±12 % of D. innoxia and were no longer measurable after 72 h processing with the plants. The decrease was slower in the control ditch. Thus, 72±19 % of NH4

+ still present in the wastewater at 48 h. In contrast, NO2- concentration values

remained very low (Fig. 3). The highest content was observed after 48 h of processing. A significant proportion of the NH4

+ removed from the wastewater was found to be converted by nitrifying bacteria into NO2

- and NO3-. In presence of plants, the removed NH4

+ was mainly transformed into NO2- and NO3

-. NH4+ was

either reduced by other processes such as absorption by the plants, or by the combined nitrification-denitrification process which has been reported to transform the NO2

- and NO3- into gaseous N2 in anoxic regions [26]. This process

also seems to be responsible for NH4+ removal in the control. Three major nitrogen removal mechanisms have been

identified: microbial denitrification [27] and plant nitrogen uptake [28, 29] and volatilisation [30]. The conversion of ammonium to nitrite and subsequent oxidation of nitrite to nitrate was more marked in the plant system. The removal of nitrogen is based on the nitrification/denitrification activity of root-associated bacteria [31]. Plant roots with organic matter provide a large surface area for microbial growth and allow the biofilm formation [32].

3.3 pH, conductivity, O2 concentration and ionic composition of wastewater

Cl- and Ca2+ concentrations remain stable throughout the experiment and no significant difference could be observed between the planted system and the free-planted system (Fig.5). In contrast, SO4

2-, K+ and Na+ concentrations increased in the planted system and remained stable in the control. Mg2+ concentration decreased in the planted system and remained stable in the control. The pH of the water quickly increased in both systems between 0 and 12 h (Fig. 6). After 12 h, it remained constant in the control, but, decreased with D. innoxia after 24 h. The conductivity of the water is constant and identical in both systems between 0 and 24 h and afterwards, it decreased in the planted system while it remained stable in the control. The O2 concentration in the effluent was 2 mg l-1. During the treatment, O2 concentration increased after 48 h in both systems. The oxygenation is less important in the control. In nitrogen removal, the pH is a significant parameter. Princic et al., (1998) have shown that the optimal pH range for NH4

+ conversion to nitrite is between 5.8 and 8.5 and between 6.5 and 8.5 for nitrification [33]. In wetlands, pH values were between 6 and 7 [34, 35]. In our planted system, the pH was between 7 and 8; hence the nitrification was active. Moreover, Sanchez-Monedero (2001) reported the nitrogen may be volatilised and be given off at these pH values [30]. The system of wastewater recirculation led to water oxygenation. The dissolved oxygen determined in the inflow was generally 2 mg l-1. Dissolved oxygen concentrations were approximately 5.8 mg l-1 after 48 h, and provided the oxygen necessary for the reduction of BOD5 then for the conversion of ammonium to nitrite and to nitrate. In comparison, other studies have indicated a low efficiency of ammonium conversion to nitrite in wetland systems owing to limited oxygen transfer capability [36].

Fig 4 Total bacteria, total and faecal coliforms, Escherichia coli and faecal enterococci in waters in the plant system with D. innoxia (a) and in the control (b). Values are means ± standard error; n = 5.

_______________________________________________________________________________________

3.4. Alkaloid production

The alkaloid production in leaf and seed extracts is given in Table 2. The analysis of the total alkaloid content in leaves

no significant differences between of the seedlings cultivated on wastewater or nutrient solution. The total alkaloid

concentration content in seeds generated from seedling cultivated on the nutrient solution was higher than these

cultivated on the wastewater. Additionally, the hyoscyamine and scopolamine production in leaves showed no

difference between the two growth types, while significant differences could be detected in seeds.

The leaves of Datura are an important source of tropane alkaloids: atropine, hyoscyamine and scopolamine. These

alkaloids are used as parasympathicolitics because of their ability to suppress the activity of the parasympathic nerve

system. The sole source for these compounds are plants and they are therefore of commercial interest to the

pharmaceutical industry [37]. This plant is commonly found in the tailings of abandoned mines, in high dry places and

it develops naturally in some French regions. In this study we demonstrated that it is possible to purify wastewater,

while at the same time to produce alkaloids, without affecting either the quantity or the quality of the leaves extracts.

Fig. 5 Concentration of Cl- (a), SO42- (b), Ca2+ (c), K+ (d),

Mg2+ (e), Na+ (f) in the plant system with D. innoxia and in the

control during the 1st month. Values are means ± standard

error; n = 5.

Fig. 6 pH (a), conductivity (b), O2

concentration (c) in the plant system with D.

innoxia and in the control. Values are means

± standard error; n = 5.

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Table 2 Total plant biomass, shoot/root ratio and alkaloids productions in leaves and in seed derived from plant cultivated with nutrient solution and with wastewater. Values are means ± standard deviation (n = 6).

Nutrient solution Wastewater Plant dry weight (g.plant-1) 51.7 ± 3.1 53.5 ± 5.7

Shoot dry weight/root dry weight

3.61 ± 0.39

3.49 ± 0.32

Alkaloids (mg.g-1 DW leave)

Hyoscyamine 0.14 ± 0.04 0.12 ± 0.03 Scopolamine 0.38 ± 0.08 0.46 ± 0.10 Total alkaloids 0.52 ± 0.13 0.58 ± 0.13

Alkaloids (mg.g-1 DW seed) Hyoscyamine 0.72 ± 0.07 0.42 ± 0.05 Scopolamine 3.50 ± 0.31 2.47 ± 0.39 Total alkaloids 4.22 ± 0.38 2.79 ± 0.43

5. Conclusion

The system of wastewater purification with NFT using D. innoxia allowed to reach the permitted levels for discharge as defined by the European directive 21/05/1991 after 48 h of processing. Moreover, it was demonstrated previously that the wastewater provided the necessary elements for plant growth.

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