Interactions between municipal solid waste incinerator bottom ash and bacteria (Pseudomonas aeruginosa)

S CIE N CE OF T H E TOT AL E N V I RO N ME N T 3 9 3 ( 2 00 8 ) 3 8 5–3 93

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

Short communication

Interactions between municipal solid waste incinerator bottom ash and bacteria (Pseudomonas aeruginosa) G. Aouada,b,⁎, J.-L. Crovisiera , D. Damidotb , P. Stillea , E. Hutchensc , J. Muttererd , J.-M. Meyere , V.A. Geoffroye a

Ecole et Observatoire des Sciences de la Terre, Centre de Géochimie de la Surface UMR 7517, 1 rue Blessig, 67084 Strasbourg Cedex, France Civil and Environmental Engineering Department, Ecole des Mines de Douai, 941 Rue Charles Bourseul, 59500 Douai, France c School of Biological and Environmental Sciences, University College Dublin, Belfield, Dublin 4, Ireland d Institut de Biologie Moléculaire des Plantes, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France e UMR 7156 Université Louis-Pasteur/CNRS, Génétique Moléculaire, Génomique Microbiologie, Département Micro-organisme, Génomes, Environnement, 28 rue Goethe, 67083 Strasbourg Cedex, France b

AR TIC LE I N FO

ABS TR ACT

Article history:

Municipal solid waste incinerator bottom ash (MSWI BA) can be used in road construction

Received 4 July 2007

where it can become exposed to microbial attack, as it can be used as a source of

Received in revised form

oligoelements by bacteria. The extent of microbial colonization of the bottom ash and the

21 November 2007

intensity of microbial processes can impact the rate of leaching of potentially toxic

Accepted 1 January 2008

elements. As a consequence, our objective was to highlight the mutual interactions between

Available online 11 February 2008

MSWI bottom ash and Pseudomonas aeruginosa, a common bacteria found in the environment. Experiments were carried out for 133 days at 25 °C using a modified

Keywords:

soxhlet's device and a culture medium, in a closed, unstirred system with weekly renewal of

Bottom ash

the aqueous phase. The solid products of the experiments were studied using a laser

Bacteria

confocal microscopy, which showed that biofilms formed on mineral surfaces, possibly

Alteration

protecting them from leaching. Our results show that the total mass loss after 133 days is

Bioalteration

systematically higher in abiotic medium than in the biotic one in proportions going from 31

Biofilm

to 53% depending on element. Ca and Sr show that rates in biotic medium was ∼19% slower

Pseudomonas aeruginosa

than in abiotic medium during the first few weeks. However, in the longer term, both rates decreased to reach similar end values after 15 weeks. By taking into account the quantities of each tracer trapped in the layers we calculate an absolute alteration rate of MSWI BA in the biotic medium (531 μg m− 2 d− 1) and in the abiotic one (756 μg m− 2 d− 1). © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Municipal solid waste incinerator bottom ash (MSWI BA) can be used in road construction as aggregate, despite the enrichment of bottom ash in potentially toxic elements such as Cd, Zn, Cu and Pb. Since MSWI BA is incinerated at high

temperatures and then cooled fairly rapidly, the material contains some glass that is unstable under atmospheric conditions. Weathering, therefore, will change the mineralogical characteristics of the material. Three major stages in weathering have been identified (Meima and Comans, 1997), each stage having a characteristic pH: (A) unweathered bottom

⁎ Corresponding author. Tel.: +33 327 712 430; fax: +33 327 712 916. E-mail address: [email protected] (G. Aouad). 0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2008.01.017

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SC IE N CE OF T H E TOT AL E N V I RO N ME N T 3 9 3 ( 2 00 8 ) 3 8 5–3 93

Table 1 – Chemical composition of the MSWI BA %

SiO2

Al2O3

MgO

CaO

FeO

Fe2O3

MnO

TiO2

Na2O

K2O

P2O5

S

C

MSWI BA

51.30

7.00

1.98

13.60

4.12

3.32

0.09

0.46

5.96

1.20

1.03

0.39

4.84

ppm

Sr

Zr

Mo

Ni

Co

Cr

Zn

Cu

Pb

V

Th

U

MSW BA

269.0

142.0

220.0

25.1

514.0

1626.0

988.0

768.6

263.0

9.6

ash, with pH N12 (unquenched samples); (B) quenched/noncarbonated bottom ash, with pH 10–10.5 (freshly quenched and 6-weeks-old samples); and (C) carbonated bottom ash, with pH 8–8.5 (1.5- and 12-years-old samples). It has generally been observed that the leaching of contaminants from bottom ash is reduced upon natural ageing, probably due to the combined effect of the pH-decrease and the neo-formation of clay-like minerals from glasses (Zevenbergen et al., 1994) that can bind contaminants (Chandler et al., 1997). In addition, sorption is potentially an important mechanism in controlling the leaching of Cd, Zn, Cu and Pb from bottom ash, because reactive sorbent minerals such as Fe/Al-hydroxides are present in weathered bottom ash (Meima et al., 2002; Piantone et al., 2004). Micro-organisms such as bacteria, are likely to be present in the weathered MSWI bottom ash used as an aggregate in road construction. Microbial activity may impact the weathering process and thus the rate of leaching of potentially toxic elements. Indeed, the deterioration of minerals, and materials of anthropogenic origin (stained glasses, cements, blastfurnace slags) depends partly on organic compounds and micro-organisms (Ehrlich, 1981; Crundwell 2003). However, the exact role of micro-organisms remains poorly understood. Some studies on bioalteration have been undertaken, but were limited to a physical description of materials altered by bacteria (Krumbein et al., 1991; Thorseth et al., 1992, 1995; Staudigel et al., 1995; Torsvik et al., 1998; Alt and Mata, 2000). Banerjee and Muehlenbachs (2003) are of the opinion that bacteria accelerate the deterioration of marine basaltic glasses due to the observation of “hair channels” that could represent bacterial activity. In studies where the solution compositions were determined, aluminium was used as a tracer (Maurice et al., 2001). The reliability of this tracer is subject, however, to caution since it can be part of secondary mineralisation products (Mevel, 1980; Crovisier at al., 1983, 1985), and also can be complexed by siderophores (iron chelating compound) (Rosenberg and Maurice, 2003). In such case, the apparent solubility of aluminium could be increased without leading to the conclusion of an increased alteration rate. In our opinion, apart for some well-defined materials, such as the degradation of books by fungi, metal drains or cement by bacteria such as Acidithiobacillus (Sand 1987; Hiernaux, 2003; Idachaba et al., 2003; Beech, 2004), the exact role of micro-organisms remains to be demonstrated. The principal difficulty, in such complex media (bacteria/material interactions), is to measure the alteration rate with reliable tracers. It is questionable whether the degradations observed under the layers rich in living micro-organisms are due to microbial action (acidification, complexation), or is simply the fact that these layers retain more easily water. Micro-organisms may only benefit from the availability of water and nutrients produced by reactions between minerals and aqueous solutions (dissolution, pre-

4.7

2.3

cipitation, sorption reactions) without actively accelerating deterioration processes. Another aspect of microbial activity is the possibility that potentially toxic metals may be sequestered by bacteria and the exopolysaccharides they produce (Kinzler et al., 2003; Aouad et al., 2006). Studies have been conducted on the role of bacterial cells (Abdelouas et al., 1998; Anderson and Pedersen, 2003), but there is a lack of data that compare the degradation due to bacterial biofilms (cells–exopolymers-solution) and the degradation that occurs in abiotic (sterile) systems. In order to clarify the role of micro-organisms, our aim was to investigate the effect of Pseudomonas aeruginosa (common bacteria found in the environment) on the weathering of MSWI bottom ash under well-defined conditions, similar to the natural environment (pH 6.7 and 25 °C). We characterized the rates of elemental release during bacteria-bottom ash interactions under laboratory scale conditions. We especially focused on major elements involved in the carbonate–silicate geochemical cycle (e.g., Si and Ca) as well as life-supporting nutrients (e.g., Fe and Mg) and trace elements (Sr, Pb, Ni, Cu, Cr, Zn and Co). Our findings ultimately demonstrate that actively metabolizing bacteria can substantially decrease elemental release rates from bottom ash compared to abiotic conditions.

2.

Materials and methods

2.1.

Characterization and preparation of MSWI BA samples

A matured bottom ash from a French municipal solid waste incinerator was used (chemical composition is given in Table 1). The bottom ash was ground, sieved between 100 and 125 μm then sterilized by autoclaving for 2 h at 120 °C. The specific surface area of the bottom ash (44,400 cm2/g) was measured using a Coulter SA3100 device (multipoint N2, BET data and Langmuir surface area). CO2 and S contents were determined by combustion under oxygen at 1400 °C (LECO SC 144DRPC, Centre de Recherches Pétrographiques et Géochimiques, Nancy). Organic carbon was determined by the same method after dissolution of carbonates by HCl. X-ray diffraction analysis was realised on 9 representative samples using a BRUCKER AXS D8 diffractometer.

2.2.

Growth media

A primary objective of this study was to examine how bacteria influence the release of major and trace elements from MSWI BA. However, most formulae yield media with very high base cation concentrations that could potentially mask the effects of rock dissolution. To circumvent this problem, we elaborated a defined medium allowing both the growth of P. aeruginosa

387

1 Osmium oxide method: Bottom ash grains were rinsed with deionised water (suprapure) and were then treated with osmium tetroxide according to the method of Jones et al. (2003) but using a reduced quantity of osmium tetroxide. Samples were fixed in glutaraldehyde to maintain cell structure and to secure attached cells to glass surfaces. Samples were immersed overnight in 2% glutaraldehyde in a 0.1 mol/L sodium cacodylate (CAC), rinsed in a CAC 0.1 mol/L, and postfixed for 1 h with 0.5% OsO4 in 0.1 mol CAC at 4 °C. Samples were then dehydrated sequentially in graded ethanol solutions, (50, 70, 85, 95, and 100% EtOH). Finally, samples were dried for one week at 45 °C and then stored in a desiccator until examination by scanning electron micro-

− 0.44 0.25 0.70 − 69.47 14.55 84.01

+ 23.54 52.49 76.04

− 61.22 44.57 105.79

+ 0.18 0.35 0.53

Co Zn

− 30.06 35.90 65.96

+ 59.23 1.19 60.42

Cr − 25.76 2.57 28.32

+ 23.31 19.70 43.01

Cu − 12.45 6.93 19.38

+ 16.43 4.52 20.95

Ni − 12.65 13.61 26.26

+ 5.03 9.73 14.76

Pb − 70 711 781

+ 9.80 8.63 18.43

Sr − 415 131 546

+ 367 162 529

Fe + 388 11 399 − 8004 1580 9584 + 6230 470 6700

Microscope analysis

At the end of experiments (133 days), grains from both conditions were characterized by microscopy. Duplicate samples were analysed. For grains corroded in biotic conditions: three methods were used:

− 16,574 4920 21,494

2.4.1.

+ 14,759 435 15,194

Grains treatments at the end of experiments

Bacteria Solutions Layers Total (Q)

2.4.

Mg

Experiments were carried out at 25 °C for 133 days. Two tests were performed in parallel (experiments were both carried out on previously sterilized samples); the first one in a sterile medium (abiotic) and the second one in the presence of P. aeruginosa (biotic). Conventional plate counting techniques showed that the initial cell density in the biotic experiment was 2.106 CFU/mL. Bottom ash grains were placed in polypropylene containers with 50 mL of PS medium. The ratio between the surface area of the solid and the volume of solution was 200 cm− 1. Containers were incubated in airconditioned rooms with constant temperature, and were not agitated. Solutions were renewed each week to allow bacteria to start a new growth cycle and maintain their activity for a long period of time (Aouad et al., 2006). At the end of each cycle, solutions were analysed using ICP/AES (Jobin-Yvon JY 124 spectrometer) and ICP/MS (Fisons VG PQ2+ spectrometer). The pH was measured using a “WTW pH 340i” pH-meter. It is important to specify that the number of planktonic bacteria measured for biotic experiments by plating did not exceed 108 CFU/mL with each renewal, which means that our conditions were close to a natural environment. The sterility of the abiotic experiments was also verified by plating.

Ca

Batch experiments and chemical analyses

Si

2.3.

μg

and a precise measurement of the elements solubilized from the minerals. The culture medium which we called “PS” is made up of (g/L): Na2HPO4 (9), (NH4)2SO4 (1), succinic acid (4), NaOH (1.52). This medium was buffered at pH 6.7 and does not contain iron and magnesium to allow the growth of bacteria. These elements are provided by the dissolution of the bottom ash (Aouad et al., 2005). The PS culture medium was especially developed to detect the highest number of the elements dissolved during deterioration. As a consequence, this medium contains high concentrations of sodium and phosphorus that are required by bacteria and it allows a precise analysis of all the other elements after treatment on chromatographic columns (Aouad et al., 2005).

Table 2 – Mass balance of each element in solutions and layers. In biotic “+” medium layer represent the biofilm, by contrast in abiotic “−” experiment it represent a mineral alteration layer

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388

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goal of these methods is to dissolve, at the end of the experiments, only phases formed during alteration. Bottom ash grains corroded in biotic conditions were treated with 5 mL 30% hydrogen peroxide for 10 min. Grains were then washed 5 times in 5 mL of ultrapure water and the wash solution was added to hydrogen peroxide, dried, redissolved in 1 N HNO3 and analysed using ICP/AES and ICP/MS. A non-altered material was tested in parallel with exact the same extraction procedures and was used as a blank for the values presented in Table 2 (layers line). Bottom ash grains corroded in abiotic condition were reacted for 10 min with 5 mL of a mixture of 0.01 N CH3COOH and 0.45 N HCl. Grains were then washed and solutions were mixed, dried and analysed as described previously. This method was also validated by a control.

Fig. 1 – Scanning electron micrographs showing the biofilm cementing two bottom ash grains after: (a) Freeze–dry technique treatment and (b) Osmium oxide treatment.

scopy (SEM) (JEOL JSM 840 equipped with an energy dispersive spectrometer, Tracor TN 5500). The osmium oxide treatment dissolved the exopolysaccharide matrix of the biofilm allowing a better observation of bacterial cells colonizing the grains (Aouad et al., 2006). 2 Freeze–dry technique method was used to preserve the integrity of the whole biofilm, including exopolysaccharides for SEM analysis (Bozola and Russell, 1992). 3 Some grains were observed under a confocal microscopy (Zeiss LSM 510 Model Axiovert 100).

3.

Results and discussion

3.1.

Mineralogical and chemical composition of MSWI BA

Chemical composition of the MSWI BA is given in Table 1. Because MSWI BA is a source of oligoelements (e.g. Fe, Mg) for bacteria, their growth and activity may impact the rate of leaching of potentially toxic elements (Cr, Co, Ni, Cu, Zn and Pb) present in the MSWI BA. X-ray diffraction analysis indicates that the most abundant minerals in the crystalline phase are quartz (SiO2), calcite (CaCO3), magnetite (Fe2.96 (Cr0.03, Ni0.01)O4), gehlenite (Ca2(Al(AlSi)O7), wollastonite (CaSiO3) and gypsum (Ca(SO4)(H2O)2). The presence of wustite and hydroxylapatite is probable. Calcite, gypsum and hydroxylapatite are likely to be formed during maturation. The other main component of the bottom ash is glass.

3.2. Observation of the surface of MSWI BA at the end of experiments The first remarkable observation is that grains leached in the biotic medium are agglomerated and form a compact cluster, whereas the grains resulting from deterioration in the abiotic medium remain free. This result is explained by the following

Grains corroded in abiotic conditions were observed by SEM. Ultramicrotomic thin sections were visualised on a Transmission Electron Microscopy (TEM) (Philips CM12 equipped with EDS detector: PV9900 EDAX) according to the method of Ehret et al. (1986).

2.4.2.

Layers dissolution

One of the critical points is to precisely evaluate the mass balance of dissolved elements during alteration. Elements can be mobilized to the solution and/or immobilized at the surface of grains. Taking into account immobilized quantity of every element is of primary importance for a precise evaluation of alteration rate in biotic and abiotic conditions. Thus, methods for selective dissolution of biofilm (for biotic condition) and alteration layer (for abiotic condition) were developed. The

Fig. 2 – Thickness of the biofilm observed by confocal microscopy.

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389

The confocal micrograph in Fig. 2, obtained by transmitted light, shows that the thickness of the biofilm covering the grain is close to 40 μm. Living bacteria (stained green using orange acridine) inside the biofilm were also observed by confocal microscopy. The fluorochrome fixed on the ADN of bacteria, is excited at 488 nm and emits a light at 545 nm which enables the bacteria to be imaged (Fig. 3).

3.3.

Fig. 3 – Biofilm and the bacteria in it observed by confocal microscopy.

microscopic observations: SEM analyses of the grains corroded in abiotic conditions showed no detectable alteration layer while a very thin non-crystalline layer was identified by TEM; conversely, the grains deteriorated in biotic experiments are systematically covered with biofilm which explains their agglomeration. Fig. 1a, obtained by freeze–dry technique, shows the biofilm (including exopolysaccharides) cementing bottom ash grains together, while Fig. 1b, show bacteria colonizing grains after osmium oxide treatment leading to dissolution of exopolysaccharide matrix of the biofilm.

Solution analysis

Cell yield reached during growth was constant within each incubation period representing an average value of 2.5 108 CFU/mL. A pH increase from 6.7 to 7.8 was observed in parallel with the bacterial growth due to the consumption of succinic acid. In abiotic medium pH slightly increased (the PS medium is buffered) each week from 6.7 to 6.9 due to proton uptake during inorganic MSWI BA dissolution. Leaching of elements is represented as cumulated concentration curves in Figs. 4 and 5. The amount of dissolved Si, Ca and Mg decreased in the presence of bacteria. By contrast with other elements, iron concentrations are smaller in abiotic than in biotic solutions. This is normal since iron, not very soluble in sterile conditions at neutral pH, is complexed by organic ligands in the biotic medium. In addition, the phenomenon is probably accentuated by the fact that the bacterial metabolism imposes a reducing condition. The reduced iron concentration in sterile solutions determines the high iron concentration in the sterile layers (further support is given by analyzing of alteration layers discussed in Section 3.4). Iron is probably amorphous hydroxides as indicated by TEM analysis.

Fig. 4 – Major elements leaching represented as cumulated concentration curves as function of time.

390

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Fig. 5 – Trace elements leaching represented as cumulated concentration curves as function of time.

Fig. 5 shows that solubilization of trace elements decreases in the presence of bacteria. This result can have beneficial consequences on environmental pollution with regard to the mobilization of potentially toxic elements.

3.4.

Mass balance

One of the critical points in experiments such as ours is to precisely evaluate the mass balance of elements between

solutions and layers (biofilm in biotic and alteration layer in abiotic conditions). Another point is to evaluate the role of bacteria on the alteration rate of materials. The last point of each curve of Figs. 4 and 5 represents the total loss (to the solution) of each element during 133 days. Knowing that experiments were performed in 50 mL, Table 2 shows the total mass of each element (in μg) mobilized to the solutions. The quantities found in the layers are also represented in μg, as well as the total solubilized mass (Q).

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391

Fig. 6 – Apparent alteration rate evolution of bottom ash expressed in masses standardized to the reactive surface area calculated using Si, Ca and Sr as tracers.

Pb, Ni and Zn are immobilized in greater amount in the biofilm than in the sterile layers. Iron concentration in the abiotic layer is 4.3 times greater than that in the biofilm, which can explain the tendency of iron concentrations in solution (Fig. 4). Table 2 shows the results of mass balance analyses. The total solubilized mass (Q) is systematically higher in abiotic medium than in the biotic experiments with values ranging from 31 to 53% depending on the element. One explanation could be that the alteration rate of bottom ash in the biotic medium is weaker than in the abiotic one. This hypothesis will be tested in the next section.

3.5.

Alteration rate

The analysis of the reaction solution constitutes, in particular cases, the simplest way to evaluate the alteration rate.

Knowing the concentration of an element E in solution after the duration (T) of deterioration as well as the concentration of E in the initial rock, the rate of degradation can be calculated. This rate is considered “absolute” only if the totality of solubilized element E remains in solution and does not take part in the formation of alteration layers, in which case the calculated rate is only “apparent”.

3.5.1.

Rn ¼ Rn n [Xi] V T S %Xi

Fig. 7 – Absolute alteration rate of bottom ash expressed in masses standardized to the reactive surface area calculated using Si, Ca and Sr as tracers.

Apparent alteration rate

From the weekly measured concentrations of Ca, Sr and Si (potential tracers), the apparent rates (Rn) of dissolution, expressed in masses standardized to the reactive surface area (g m− 2 d− 1) were calculated with Eq. (1). ½Xi n  V 100  TS kXi

ð1Þ

Apparent alteration rate of bottom ash (g m− 2 d− 1) Week number Concentration of element i considered as tracer (g/L) Volume (L) Time (days (d)) Surface area of bottom ash (m2) Percentage of tracer i in the bottom ash

The rates calculated using concentrations of Si, Ca and Sr are not identical (Fig. 6). We can notice that the three tracers show rates decreasing with function of time. Ca and Sr show that rates in biotic medium was ∼ 19% slower than in abiotic medium during the first few weeks. However, in the longer term, both rates decreased to reach similar end values after 15 weeks.

392 3.5.2.

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Absolute alteration rate

By taking into account the quantities trapped in the layers of each tracer, we can calculate the absolute alteration rate with Eq. (2). R¼

R Qi T S %Xi

Q 100  T  S kXi

ð2Þ

Absolute alteration rate of bottom ash (μg m− 2 d− 1) Total solubilized mass (Q) of element i considered as tracer (μg) Time (days (d)) = 133 d Surface area of bottom ash (m2) Percentage of tracer i in the bottom ash

The values of Q are given in Table 2. These values make possible to calculate an absolute alteration rate over the time of the experiments (133 days). The three tracers give very similar rates in both biotic and sterile conditions (Fig. 7). These rates are 503(Si), 547(Ca) and 544(Sr) μg m− 2 d− 1 in biotic condition and 711(Si), 783(Ca) and 775(Sr) in abiotic medium. Finally, the mean alteration rate of MSWI BA is 531 μg m− 2 d− 1 in biotic and 756 μg m− 2 d− 1 in abiotic media.

4.

Conclusions

The interaction of MSWI bottom ash with a growth medium depleted of iron and magnesium (at 25 °C) leads to the fast growth of P. aeruginosa and the formation of a biofilm on the surface of the grains. We have already shown in a previous study (Aouad et al., 2004) that bacteria can colonize various other type of glass when the chemical elements necessary for their development become available. The alteration rate of bottom ash decreases quickly in the biotic medium despite of the renewal of the growth medium. One explanation could be that the biofilms acts as a protective barrier, thus preventing dissolution. The rate also decreases, although it is less rapid in the absence of P. aeruginosa. This is probably due to the formation of precipitated minerals at the surface that have also protective properties. Our results show that the total mass loss after 133 days is systematically higher in abiotic medium than in the biotic conditions in proportions ranging from 31 to 53% depending on element. The role of bacterial exopolymers versus bacterial cells in the sorption of potentially toxic metals is yet to be established. The composition of the biofilm clearly indicates the diversity of affinity for various elements with respect to the occupation of the complexation sites in these poly-anionic substances. A better comprehension of these complex mechanisms undoubtedly can be obtained by experiments in simplified mediums, but the results of our studies already show that alteration rate of MSWI BA is 42% smaller in a biotic system. Taking into account the chemical resistance of biofilms, it is very possible that the biosorption of various chemical elements leads, in the long-term, to an intense passivebiomineralisation. The results presented in this study are applicable for P. aeruginosa which grows at neutral pH, as a perspective of this result we have begun to study, under the same conditions,

the influence of the biofilm formed by bacteria which impose an acid pH like Acidithiobacillus on the alteration rate of silicates.

Acknowledgements This work was supported by grants from the Réseau Alsace de Laboratoires en Ingénierie et Sciences pour l'Environnement (RÉALISE).

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