Accepted Manuscript Title: Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation Author: Matilte Halma Christine Mousty Claude Forano Martine Sancelme Pascale Besse-Hoggan Vanessa Prevot PII: DOI: Reference:
S0927-7765(14)00652-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.11.029 COLSUB 6752
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
11-9-2014 3-11-2014 19-11-2014
Please cite this article as: M. Halma, C. Mousty, C. Forano, M. Sancelme, P. Besse-Hoggan, V. Prevot, Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.11.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacteria encapsulated in Layered Double
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pollutant degradation
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Hydroxides: toward an efficient bionanohybrid for
Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale Besse-Hoggan,
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Vanessa Prevot*
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Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 CLERMONT-FERRAND, FRANCE
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CNRS, UMR 6296, ICCF, F-63171 AUBIERE, FRANCE
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* Corresponding author: Vanessa Prevot Email: [email protected] Tel 00 33 47340710 Fax 04 73 40 79 33
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Highlights
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Pseudomonas sp. strain ADP was immobilized within LDH network by a simple, quick and ecofriendly direct coprecipitation approach.
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The ratio between biological and inorganic components can be easily tuned thanks to this approach. The cell viability and the conservation of the biological activity whtin the biohybrid were evidenced.
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An enhanced atrazine degradation rate was achieved by immobilized bacteria compared to free cells
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The ADP@LDH biohybrid maintains the same biological activity after four cycles of reutilization and three weeks storage at 4°C
ABSTRACT A soft chemical process was successfully used to immobilize Pseudomonas sp. strain ADP (ADP), a well-known atrazine (herbicide) degrading bacterium, within a Mg2Allayered double hydroxide host matrix. This approach is based on a simple, quick and ecofriendly direct coprecipitation of metal salts in the presence of a colloidal suspension of bacteria in water. It must be stressed that by this process the mass ratio between inorganic and biological components was easily tuned ranging from 2 to 40. This ratio strongly influenced the biological activity of the bacteria toward atrazine degradation. The better results were obtained for ratios of
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10 or lower, leading to an enhanced atrazine degradation rate and percentage compared to free cells. Moreover the biohybrid material maintained this biodegradative activity after four cycles of reutilization and 3 weeks storage at 4°C. The ADP@MgAl-LDH bionanohybrid materials completely
characterized
by
X-ray
diffraction
(XRD),
FTIR
spectroscopy,
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thermogravimetric analysis and scanning and transmission electronic microscopy (SEM and
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TEM) evidencing the successful immobilization of ADP within the inorganic matrix. This
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synthetic approach could be readily extended to other microbial whole-cell immobilization of
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interest for new developments in biotechnological systems.
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KEYWORDS: Layered Double Hydroxides, Bacteria, Bionanohybrid, Biodegradation, Atrazine
1. INTRODUCTION
The wide bacterial enzymatic and catalytic potential is increasingly used in various fields of emerging technologies, covering applications in biocatalysis [1], bioremediation [2, 3], bioengineering [4], biosensors [5]. Immobilization of microorganisms on solid supports is often mandatory to obtain high bacteria concentrations, increased reaction rates, cells/liquid separation ease and recyclability. Thus, it is of great importance to investigate bacterial immobilization processes [6] for both academic and practical aspects. Compared to the immobilization of purified enzymes, the attachment of whole cells obviates the need for separation, isolation and
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purification steps. Moreover, in maintaining enzymes in their native state, their stability and longevity can be enhanced. It is interesting to notice that micro-organism immobilization is above all a natural phenomenon. In laboratory, different methodologies have been developed to
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mimic nature and carry out biological material immobilization [7] on inert support mainly based on adsorption, physical entrapment, cross-linking and covalent coupling. These immobilization
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techniques can be described as post-synthesis, in-situ and multistep immobilization as suggested
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by Leonard et al. in a recent review on whole-cells based hybrid materials [6]. According to the immobilization methods, a large variety of material supports has been used, ranging from
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biopolymers [8] (such as alginate, k-carragenan, cellulose and collagen) or synthetic polymers [9] (polyacrylamide, polyvinylalcohol) to inorganic compounds [10-14](silica, clays, oxides).
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Note that the nature of the matrix is of paramount importance in determining the performance of
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immobilized cells. The ideal matrix should be stable, robust, chemically inert, biocompatible and
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should not interfere with the bioreaction. Specific properties including hydrophilicity, permeability, diffusion and resistance to microbial deterioration are also crucial. Compared to
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biopolymers, inorganic host matrices are particularly interesting in terms of porosity, mechanical and physical properties. Recently, Blondeau and Coradin highlighted the interest of sol-gel chemistry to improve the preservation of living materials [15]. In the last years, among the wide variety of available supports, Layered Double Hydroxides (LDH) have proved to be highly effective as host structures for biomolecules [16, 17] such as amino acids [18-20], DNA [21, 22], proteins [23-26] mainly due to their versatile properties in terms of chemical composition, basicity, charge density, hydrophilicity and morphology. LDH are layered materials [27-30] based on the stacking of positively charged sheets [MII1III x+ xM x(OH)2] .
Anions and water molecules are located in the interlamellar domains to maintain
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the global electroneutrality. Such layered structures promote favourable electrostatic interactions with various biomolecules bearing an overall negative charge at neutral or basic pH. Recent studies showed that LDH based biohydrids can be prepared in soft conditions by direct co-
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precipitation of the inorganic matrix in the presence of biomolecules, as an elegant alternative to the adsorption or anionic-exchange process [16, 17]. The biomolecules entrapped in LDH
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matrices by such strategy are then protected. However such approach has been mainly limited to
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small biomolecules (amino acid [19], DNA strand [22]) and proteins [23-26]. To our knowledge, only adsorption process on LDH has been achieved to immobilize whole-cells. For example, Jin
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et al. [31] demonstrate the efficient use of LDH for bacteria and virus removal from waters by adsorption processes. In parallel, thanks to electrostatic interactions between a NiAlCO3 LDH
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and the bacterial Bacillus subtilis cells, biogranule-like aggregate formation and their efficiency
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for methylene blue decolourisation have been reported [32, 33]. In our previous works [34, 35],
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we investigated the Pseudomanas sp. strain ADP adsorption on different MgRAl (R = 2, 3, 4) LDH matrices evidencing that bacterial cell adhesion can improve the biodegradative activity
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towards contaminants. We choose this bacterial strain (ADP) because of its interest in bioremediation based on its ability to mineralize atrazine (2-chloro-4-ethylamino-6isopropylamino-1,3,5-s-triazine), a persistent herbicide. Only few reports described the ADP whole cell immobilization, mainly within polymeric microfibers [36], silica [37] and onto natural zeolite [38]. Interestingly, cell free extracts of strain ADP containing atrazine-degrading enzymes were entrapped in a sol-gel matrix. However, due to silica sol-gel process conditions implying methanol release, immobilization induced a significant loss of enzymatic activity[39]. In this context, the objective of this work was to investigate as an alternative to the classical adsorption process the direct LDH coprecipitation to whole-cell entrapment in order to easily
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elaborate an efficient but also, stable and recyclable Pseudomonas sp. strain ADP@LDH bionanocomposite in maintaining the biochemical activity and cell viability.
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2. MATERIALS AND METHODS 2.1 Materials
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Atrazine (AT, 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine, 98.5 % chemical purity) was supplied free of charge by Syngenta Crop Protection® (Basel). A 0.1 mM (22 mg.L) solution was prepared in mineral water (Volvic®, France) for AT biodegradation and HPLC
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1
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standard uses. For all preparations, the magnesium and aluminium nitrate Mg(NO3)2.6H2O and Al(NO3)3.9H2O salts were of analytical grade. All other solvents and reagents were of
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commercial grade (Aldrich, Acros Organics, Merck, Fluka). Mineral water (Volvic®, France) was used for all experiments.
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2.2 Cell cultures
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Pseudomonas sp. strain ADP (gift from F. Martin Laurent from INRA Dijon, France) was
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grown in 100 mL portions of Trypcase Soy broth in 500 mL Erlenmeyer flasks incubated at 27 °C and 200 rpm. Cells were harvested after 24 h of culture. Samples of 20 mL of culture were centrifuged at 12,000 x g for 15 min at 4 °C. The resulting pellets were washed first with NaCl solution (8.0 g. L-1) and then, with Volvic®. The cell concentration used was around 2x1010 colony-forming units (cfu)/mL (OD=10 at = 575 nm), corresponding to 45±5 mg of dried cells and mentioned hereafter as 1 ADP.
2.3 Bacteria immobilization The ADP@LDH bionanocomposites were prepared by the co-precipitation at constant pH method [24, 30] (pH = 8.0 ± 0.2) and room temperature. Typically, an aqueous solution
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containing Mg(NO3)2 and Al(NO3)3 ([metals]= 0.1 M, Mg/Al=2) was added (flow rate: 0.813 mL/min), simultaneously with an aqueous NaOH solution (0.2 M) to a reactor containing an aqueous suspension of ADP. The addition rate of NaOH solution was controlled in order to
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maintain the pH at a fixed value of 8.0 ± 0.2, under nitrogen atmosphere and magnetic stirring. After 5 h of aging, the suspension was centrifuged and washed three times with Volvic® and
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recovered by centrifugation (12,000 x g for 10 min). The ADP/LDH mass ratio was easily
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modulated by modifying the added salt volume in the reactor, keeping constant the ADP amount in a fixed volume (4x1011 colony-forming units (cfu) in 60 mL). Metal salt volumes of 12 ml, 23
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ml, 55 ml, 102 ml and 204 ml were used, corresponding respectively to LDH theoretical mass values of 0.1 g, 0.2 g, 0.5 g, 1 g and 2 g. Coprecipitation yields were calculated according to the
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salt volume added and theoretical LDH formulae (Mg/Al = 2). Regarding the theoretical
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LDH/ADP mass ratio involved, the bionanocomposites as-prepared are denoted hereafter
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ADP@LDH2, ADP@LDH4, ADP@LDH10, ADP@LDH20 and ADP@LDH40. The ADP@LDH bionanocomposites were dried at 30 °C for solid state analyses. For atrazine biodegradation
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experiments, the ADP@LDH biohybrids were used as suspensions.
2.4 Characterization of ADP cells and bionanocomposites Free ADP cells and ADP@LDH bionanocomposites were characterized using different analytical techniques. X-ray diffraction patterns were recorded with a Philips X’Pert automated X-ray diffractometer using CuK radiation (= 0.154051 nm), over the 2-70° (2θ). FT-IR spectra were recorded with a Nicolet 5700 spectrometer from Thermo Electron Corporation using the KBr pellet technique.
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Zeta-potentials of Mg2Al-NO3, ADP cells and ADP@LDH were measured with a nano Zetasizer (Malvern instruments) apparatus, using a laser Doppler electrophoresis (LDE). Thermogravimetric analyses (TGA) were performed using a Setaram TGA92 thermogravimetric
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analyzer in the temperature range of 25-1100 °C, with a heating rate of 5 °C min-1, under air atmosphere. The bionanocomposite assemblies were studied by transmission electron
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microscopy (TEM) using a Hitachi 7650 microscope at an acceleration voltage of 80 kV. To
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perform the characterization, a drop of the suspension was deposited on a 400 mesh holey carbon-coated copper grid and dried at room temperature. FESEM characteristics of the samples
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were imaged by a Zeiss supra 55 FEG-VP operating at 3 keV combined with an energy dispersive X-ray (EDX) analyzer. Specimens were mounted on conductive carbon adhesive tabs
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and imaged after carbon sputter coating to make them conductive.
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2.5 Herbicide degradation
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ADP and ADP@LDH were both tested as biocatalysts for biodegradation of atrazine. For each duplicate, ADP or ADP@LDH were suspended in a 0.1 mM AT solution in Erlenmeyer flasks.
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In accordance with ADP/LDH ratio, various volumes of ADP@LDH gel were involved to maintain constant the cell amount for all the biodegradation tests, leading to a low dilution effect of atrazine. Then, the flasks were incubated on a platform shaker (130 rpm, 27 °C). Samples (0.6 mL) were regularly taken from each flask, centrifuged at 12,500 rpm for 3 min and then frozen until HPLC analyses. Control experiments (without atrazine or cells) were carried out under the same experimental conditions. After the first use, ADP and ADP@LDH biocatalysts were thoroughly washed by centrifugation for reuse in another biodegradation reaction, under the same experimental conditions as those employed in their first use.
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2.6 Monitoring of Atrazine concentrations by HPLC The HPLC analyses were carried out using an Agilent 1100 photodiode array detector (DAD) chromatograph on a reverse-phase Agilent Eclipse XDB-C18 (5 µm, 4.6 x 150 mm) column. The
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eluent was composed of 50 mM acetate buffer pH 4.6 and acetonitrile 60:40 (v: v), at a flow rate
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of 1 mL min-1. 10 µL of sample were injected. The UV detector was set at 225 nm.
3.1 Elaboration of ADP@LDH bionanocomposites
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3. RESULTS AND DISCUSSION
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The ADP whole cells were immobilized in LDH matrix by a direct coprecipitation method (Fig. 1) which consists of the addition of magnesium and aluminium metal salt solution in a
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colloidal suspension of ADP. In agreement with cell viability conditions, the LDH precipitation
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is conducted at room temperature in maintaining the pH constant at 8.0 ± 0.2 into the reactor.
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The coprecipitation of cells and LDH results in the formation of gelatinous precipitates. Different bionanohybrids were prepared by varying the volume of metal salt solution while keeping the
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ADP concentration constant. Various amounts of LDH were coprecipitated theoretically ranging from 0.1 g to 2.0 g. Whereas a yield of 78 % was observed for Mg2Al-NO3 formation in pure water, the solid precipitation yields in ADP@LDH biohybrid materials decrease progressively with decreasing amount of LDH, from 76% to 56% (Table 1). Obviously, it is worth noting that the cell presence in the reaction medium limits the coprecipitation efficiency probably due to the complexation of metal ions by extracellular polysaccharides.
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Figure 1. Cartoon outlining the procedure for ADP@LDH biohybrid preparation The LDH/ADP assembly is evidenced by zetametry analysis. Indeed the larger the salt volume
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involved, the higher the zeta-potential of resulting gel (Table 1). This trend evidences the increasing charge neutralization during bacteria entrapment, in good agreement with the zeta
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18 mV.
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potential values of both individual LDH nanoparticles and ADP, respectively of + 39 mV and -
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In parallel, the EDX results (Table 1) further evidence the influence of the cells not only on the yield of the LDH formation, but also on the Mg2+/Al3+ ratio. Indeed, systematically in the presence of cells, the ratio measured in the biohybrid phase is always lower to the one of initial metal salt solution, highlighting that the coprecipitation of Mg2+ and Al3+ does not occur stoichiometrically as observed usually. Such lower ratios from 1.3 to 0.58 respectively for ADP@LDH40 and ADP@LDH2 cannot be explained by the formation of a unique Al-rich LDH phase in regards with LDH structural properties [30].
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Table 1. Structural and chemical properties of ADP@LDH biohybrid materials. PXRD parameters (nm) a
c
(mV)*
25220°C
Mg2Al-NO3 LDH
78
0.3032
2.46
+39
ADP@LDH40
76
0.3032
2.33
+10
ADP@LDH20
74
0.3032
2.37
+8
ADP@LDH10
66
0.3032
2.33
ADP@LDH4
59
0.3036
-
ADP@LDH2
56
0.3032
ADP
-
-
Mg2+/Al3+
Total
16
44
1.98
18
45
1.30
19
46
1.07
+7
19
47
0.93
+3
19
53
0.63
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-9
18
60
0.58
-
-18
10
92
-
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Measured in Volvic® mineral water.
EDX
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*
TGA (weight loss, %)
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Co-precipitation yield (%)
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Samples
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It seems that despite the favourable pH value of LDH coprecipitation (pH 8), both LDH and amorphous aluminium hydroxides form in the presence of ADP cells. The side formation of
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aluminium hydroxide during the coprecipitation of MgAl LDH with high concentration of biomolecules has already been observed for the MgAl-Transketolase system [40]. The simultaneous decreases of the coprecipitation yield and the Mg/Al ratio could be explained by the decrease of the supersaturation level in the solution for the lower metal salt concentration conjointly to a local decrease of the pH value at the cell surrounding modifying the LDH coprecipitation conditions. Moreover, a specific metal cation adsorption on the microorganism surface may occur and partially hamper the precipitation reaction. To gain better insight on the immobilized cell amount into LDH materials, thermal gravimetric analyses were carried out on the different ADP@LDH biohybrids and compared to those of
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Mg2Al-NO3 and ADP cells (Fig.2). The Mg2Al-NO3 sample displays typical thermal behaviour of LDH materials with two characteristic weight loss regions corresponding to the removal of
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dehydroxylation and anion decomposition above 220°C (close to 26%).
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adsorbed and intercalated water between 20 and 220°C (18% of mass loss) and the concomitant
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Figure 2. TGA curves in the temperature range 20-1100°C for (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
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The thermogravimetric analysis of free cells shows 92 % of total weight loss with three main mass losses over the temperature ranges 25-140°C, 140°C-490°C and above 490°C. For the ADP@LDH bionanocomposites, similar behaviour to that of LDH was observed, even for the lowest amount of LDH, with an almost complete decomposition before 600°C. In all cases, the lower the LDH/ADP ratio involved in the process, the higher the experimental weight loss (Table 1). For the ADP@LDH4 and ADP@LDH2 biohybrids, the ADP and LDH proportions were computed considering total weight losses of the biohybrid and of individual components that is ADP and pristine LDH. Interestingly, results fit roughly to the theoretical LDH/ADP ratio
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of 2 and 4, evidencing that direct coprecipitation allows to easily tune the LDH/ADP ratio in a large extend. The PXRD patterns of the air-dried gels (Fig.3A) are compared to those of pure Mg2Al-NO3
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coprecipitated in the same conditions and ADP free cells. For the pure LDH material, diffraction lines are indexed according to a hexagonal lattice (R-3m space group) with cell parameters of a =
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0.3032 nm and c = 2.46 nm (Table 1). The interlayer distance (d = 0.83 nm) corresponds to
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nitrate intercalated LDH structure. For all ADP@LDH bionanocomposites, the 012 and 110 LDH characteristic diffraction lines are systematically observed, confirming the effective
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formation of the LDH layered structure. However, the presence of ADP cells in the synthetic medium induces a net decrease of the LDH platelet crystallinity. Indeed, the reflection intensity
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strongly decreases for the lowest LDH/ADP ratio. This trend is particularly clear for the 00l
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reflection lines. An enlargement of the reflections is also observed when decreasing the LDH /
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ADP ratio. These PXRD pattern modifications can be attributed to both a decrease of LDH particle size and an increase of disorder into the layer stacking. For the lowest LDH /ADP ratio,
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a complete disappearance of the 00l line is observed as for exfoliated LDH phases [41, 42] and LDH phases prepared in the presence of enzymes [24, 43]which can be attributed to an important dispersion of the layer into the biohybrid. Due to the large ADP cell dimensions (1 x 0.5 x 0.5 m), the intercalation reaction of the whole cells into the layered structure is not expected. Biohybrid formation involving both LDH matrix and bacteria was further confirmed by FTIR analyses. Fig. 3B shows the FTIR spectra of ADP cells, Mg2Al-NO3 pristine LDH and ADP@LDH bionanocomposites. The Mg2Al-NO3 compound displays the typical vibrations characterizing the LDH structure [29]. The broad intense band centred at 3741 cm-1 corresponds to the OH stretching mode of layer hydroxyl groups and interlayer water molecules whereas the
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strong vibration frequency at 1384 cm-1 corresponding to the 3 stretching vibration of NO3 confirms its presence into the layered structure. Vibration bands at 662 cm-1 and 450 cm-1 attributed respectively to (MO) and (OMO), are typical of LDH lattice. In parallel, ADP spectrum
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shows cells characteristic vibration frequencies such as bands or shoulders at 2968-2850 cm-1
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((CH2), (CH3)); 1660 cm-1 ((CO) - amide I); 1544 cm-1 ((NH) and (CN) - amide II); 1236 cm-1 ((NH), (CN)-amide III) and 1082-1053 cm-1 ((CO), (COC)-polysaccharides). On the FTIR spectra
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of ADP@LDH biohybrid materials, some vibration bands of both ADP cells and LDH matrix are identified, traducing both LDH formation and microorganism entrapment. As expected, the
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relative intensities of ADP and LDH vibration bands vary in proportion with the change in the
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relative amount of ADP and LDH. The disappearance of the 00l lines observed on PXRD patterns for the lowest amount of LDH corresponds on the FTIR spectra to the net decrease of
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the NO3 vibration.
Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
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These observations are consistent for the lower LDH/ADP ratio with both amorphous aluminium hydroxides formation and LDH exfoliation inducing layer charge compensation mainly by the cell surface rather than intercalated nitrate anions.
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The morphology of ADP based biohybrid materials was further investigated by SEM and TEM. On FESEM image (Fig. 4A), ADP@LDH2 displays a rather porous network of aggregated
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nanoparticles. Clearly, there is no evidence of ADP, a rod shaped bacteria (see supporting
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information Fig. SI 1) at the surface of the LDH materials. Consequently, TEM analyses (Fig. 4B and Fig. SI 2) were required to get further information about the dispersion of ADP cells in the
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LDH material. Interestingly, analysis of the ADP@LDH2 suspension deposited on a grid allows an unmistakable visualization of cells surrounding by aggregated plate-like LDH nanoparticles
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with size in the range of 50-100 nm. Such observations indicate that the microorganisms are
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really embedded within the LDH gel, thanks to direct coprecipitation synthetic approach.
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The ADP encapsulation within LDH material was also evidenced by the change of coloration of solid. Whereas pristine LDH display a white colour, a primrose yellow solid is obtained in the
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presence of ADP. Moreover, the resulted ADP@LDH aggregates were more compact and larger than the spread LDH material.
Figure 4. A) FESEM and B) TEM images of ADP@LDH2.
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3.2 Viability of cells after immobilization in LDH and Biodegradation of atrazine To check the viability of ADP once immobilized in Mg2Al- LDH matrix aliquots (100 µL) from ADP@LDH2 suspensions were spread on TSA plates and incubated at 27 °C. After 24h, it
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was observed that ADP remains viable when anchored in LDH support. Nevertheless it is difficult to estimate adequately the bacterial number cfu due to the aggregated nature of the
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biohybrid but ADP obviously maintain its ability to grow evidencing that LDH formation
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process is compatible with ADP cell viability.
As ADP entrapped within LDH was shown to be viable, the biohybrids were tested as catalysts
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for the biodegradation of atrazine. Indeed since immobilisation method and support properties can affect the bacterial properties in a positive or negative way, the effect of entrapment on the
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ADP metabolic activity was evaluated. Systematically, biodegradation of atrazine (0.1 mM, in
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Volvic® mineral water) was carried out using ADP@LDH biohybrids and compared to ADP
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free cells stored under the same conditions (See supporting data and Fig. SI3). As an exemple, the Fig. 5 presents the kinetic curves of atrazine biodegradation for the two biohybrids
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ADP@LDH2 and ADP@LDH10. Strikingly, the biodegradation activity of ADP cells was strongly improved after immobilization into LDH for the lower mass ratios LDH/ADP (Table 2 and supporting data, Fig. SI 3) in terms of initial kinetics but also of biodegradation percentages. It seems that the flexible bidimensional structure of the LDH support combined with specific interactions between ADP and the mineral surface provides a favourable environment and boosts the biodegradation activity of ADP cells. It is worth noting that no sorption of atrazine occurred on LDH. Similar behaviour was previously observed for adsorbed ADP onto LDH [34, 35], evidencing that the ADP bacterial metabolic activity was systematically promoted, regardless of the immobilization method and the interactions between ADP and LDH. Moreover, the direct
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LDH formation involving acidic metal salt and basic agent in presence of ADP does not induce denaturation of proteins or cell death as observed in using sol-gel procedure [37]. However, the increase of the biodegradation rate depends on the ADP@LDH ratio (Table 2
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and supporting data, Fig. S1B). The higher biodegradation rates and percentages are only
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observed for LDH / ADP mass ratios lower than 20.
Figure 5. Kinetics of atrazine biodegradation by ADP free cells and ADP-LDH bionanocomposites: (■) ADP free cells; () ADP@LDH10; (▲) ADP-LDH2 and (■) LDH.
For the biohybrids ADP@LDH40 and ADP@LDH20, an opposite effect was observed. In these cases, high amounts of LDH lead to lower biodegradation activities compared with ADP free cells. This trend could be explained by a high entrapment of cells in a large quantity of LDH gel. Since no atrazine adsorption occurred on LDH surface (Fig. 5) [34], such high amount of inorganic matter may limit to some extend the solution diffusion to the cell and consequently
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reduces the biodegradation performance. Moreover when increasing the coprecipitated LDH amount, the viscosity of the medium increases since the cell quantity is maintained constant affecting then the atrazine diffusion and mobility in the medium.
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To gain better insight on the cell stability which is of major importance for bionanocomposite applications, biocatalysts were thoroughly washed by centrifugation after the first biodegradation
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experiment and reuse to biodegrade atrazine under the same conditions. Fig. 6 shows that ADP
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free cells present progressive loss of metabolic activity after each cycle of atrazine biodegradation. On the contrary, entrapped ADP into LDH maintains its full bio-catalytic
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activity over 4 cycles, highlighting the positive effect inorganic matrix has on the ADP viability. This behaviour should be correlated to the ability of LDH to limit the protein denaturation thanks
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to their physico-chemical surface properties, as previously underlined in the literature [17].
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These results confirm the great positive influence of direct coprecipitation approach to entrap
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ADP into LDH on the bacteria stability and biodegradation activity. Indeed even if a slightly higher boosting effect was observed in our previous work using adsorption method, direct
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coprecipitation leads to an efficient ADP entrapment limiting the direct exposure to the surrounding medium and the decrease of activity and lysis of bacteria even after several regeneration cycles. On the contrary, in the case of bacteria adsorption process, a progressive decrease in biocatalytic activity was reported with the increase of the reuse cycles [33]. Moreover, ADP@LDH2 stored at 4°C in suspension for three weeks maintains a similar bioactivity confirming the robustness of such bionanocomposite and its excellent stability over time.
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Table 2. Kinetic parameters of 0.1 mM atrazine biodegradation by Pseudomonas sp. ADP free cells and ADP@LDH, considering kinetics to be of first order. Degradation rate (h-1)
Degradation after 8h (%)
ADP
0.164 (R2 = 0.998)
71
ADP@LDH40
0.053 (R2 = 0.998)
ADP@LDH20
0.071 (R2 = 0.995)
ADP@LDH10
0.371 (R2 = 0.999)
94
ADP@LDH4
0.207 (R2 = 0.998)
82
ADP@LDH2
0.341 (R2 = 0.999)
94
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Samples
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100 80
1st cycle 2nd cycle 3rd cycle 4th cycle
40 20 0
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60
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Atrazine Biodegradation(% )
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ADP@LDH2
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ADP free cells
Figure 6. Atrazine biodegradation percentage after 8h of incubation with free and immobilized ADP.
4. CONCLUSIONS
The ADP@LDH bionanocomposites were successfully prepared by direct LDH formation in the presence of Pseudomonas sp. ADP suspensions. Such one step process allows to easily tune the LDH/ADP ratio of the bionanocomposites. Cells entrapped within LDH gel can maintain their cell viability and the inorganic part does not hamper the microorganism growth. Moreover ADP immobilization leads to a great enhancement of the metabolic activity toward atrazine
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degradation for LDH /ADP mass ratio lower than 20. Such immobilization method leading to LDH based bionanocomposites also provides interesting advantages in terms of cell stability and reusability of the living materials with no loss of bioactivity. This approach based on whole cell
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immobilization within LDH can be easily extended to other microorganisms and applied to various pollutant bioremediation. Moreover since a gel easy to handle and to cast is elaborated, it
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opens also the way for applications in other fields such as biosensor or biofuel cell development.
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Author Contributions
The manuscript was written through contributions of all authors. All authors have given
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approval to the final version of the manuscript and contributed equally.
ACKNOWLEDGMENT
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The authors would like to thank the Conseil Régional Auvergne and Fonds Européen de
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REFERENCES
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Développement Régional (FEDER) for the financial support.
[1] R. Eibl, S. Kaiser, R. Lombriser, D. Eibl, Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology, Appl. Microbiol. Biotechnol., 86 (2010) 41-49. [2] P. Kanmani, J. Aravind, D. Preston, Remediation of chromium contaminants using bacteria, Int. J. Environ. Sci. Techn., 9 (2012) 183-193. [3] L. Wessels Perelo, In situ and bioremediation of organic pollutants in aquatic sediments, J. Hazard. Mater., 177 (2010) 81-89.
[4] Y. Tomoko, Yoshiaki; Maeda, M. Tadashi, Bioengineering of bacterial magnetic particles and their applications in biotechnology, Recent Pat Biotechnol., 4 (2010) 214-225. [5] E. Eltzov, R. Marks, Whole-cell aquatic biosensors, Anal. Bioanal. Chem., 400 (2011) 895913.
20
Page 20 of 34
[6] A. Leonard, P. Dandoy, E. Danloy, G. Leroux, C.F. Meunier, J.C. Rooke, B.-L. Su, Wholecell based hybrid materials for green energy production, environmental remediation and smart cell-therapy, Chem. Soc. Rev., 40 (2011) 860-885. [7] L. Betancor, H.R. Luckarift, Bioinspired enzyme encapsulation for biocatalysis, Trends
ip t
Biotechnol., 26 (2008) 566-572.
[8] J.K. Park, H.N. Chang, Microencapsulation of microbial cells, Biotechnol. Adv., 18 (2000)
cr
303-319.
[9] M. Qi, Y. Gu, N. Sakata, Y. Kim, Y. Shirouzu, C. Yamamoto, A. Hiura, S. Sumi, K. Inoue,
us
PVA hydrogel sheet macroencapsulation for the bioartificial pancreas., Biomaterials, 25 (2004) 5885-5892.
[10] A.T. Aikaterini, E. Kalogeris, E. Apostolos, A.T. Ali, G. Dimitrios, S. Haralambos,
an
Effective Immobilization of Candida Antarctica lipase B in organic modified clays: Application for the epoxidation of Terpenes, Mater. Sci. Eng., A, 165 (2009) 173-177.
M
[11] M. Amoura, R. Brayner, M. Perullini, C. Sicard, C. Roux, J. Livage, T. Coradin, Bacteria encapsulation in a magnetic sol-gel matrix, J. Mater. Chem., 19 (2008) 1241-1244.
d
[12] M. Amoura, N. Nassif, C. Roux, J. Livage, T. Coradin, Sol-gel encapsulation of cells is not 4015-4016.
te
limited to silica: bacteria long-term viability in alumina matrices, Chem. Commun., (2007) [13] A. Coiffier, T. Coradin, C. Roux, O.M.M. Bouvet, J. Livage, Sol-gel encapsulation of 2044.
Ac ce p
bacteria: a comparison betweeen alkoxide and aqueous routes., J. Mater. Chem., 11 (2001) 2039[14] M. Perullini, M. Jobbagy, N. Mouso, F. Forchiassin, S.A. Bilmes, Silica-Alginate-fungi bionanocomposites for remediation of polluted water, J. Mater. Chem., 20 (2010) 6479-6483. [15] M. Blondeau, T. Coradin, Living materials from sol–gel chemistry: current challenges and perspectives J. Mater. Chem., 22 (2012) 22335-22343. [16] C. Mousty, V. Prevot, Hybrid and biohybrid Layered Double Hydroxides for electrochemical analysis Anal. Bioanal. Chem., 405 (2013) 3513-3523. [17] V. Prevot , C. Mousty, C. Forano, State of the art in Biomolecules and LDH association, Adv. Chem. Res., 17 (2013) 35-83.
21
Page 21 of 34
[18] S. Aisawa, S. Takahashi, W. Ogasawara, Y. Umetsu, E. Narita, Direct intercalation of amino acids into layered double hydroxides by coprecipitation, J. Solid State Chem., 162 (2001) 52-62. [19] Á. Fudala, I. Pálinkó, I. Kiricsi, Preparation and characterization of hybrid Organic-
ip t
Inorganic composite materials using the Amphoteric property of amino acids : amino acid intercalated layered double hydroxide and montmorillonite., Inorg. Chem., 38 (1999) 4653-4658.
cr
[20] M. Wei, Z. Shi, D.G. Evans, X. Duan, Study on the intercalation and interlayer oxidation transformation of L-cysteine in a confined region of layered double hydroxides, J. Mater. Chem.,
us
16 (2006) 2102-2109.
[21] J.-H. Choy, S.-Y. Kwak, J.-S. Park, Y.-J. Jeong, J. Portier, Intercalative Nanohybrids of (1999) 1399-1400.
an
Nucleoside Monophosphates and DNA in Layered Metal Hydroxide, J. Am. Chem. Soc, 121 [22] L. Desigaux, M.B. Belkacem, P. Richard, J. Cellier, P. Léone, L. Cario, F. Leroux, C. B.
Pitard,
Self-Assembly
and
M
Taviot-Guého,
Characterization
of
Layered
Double
Hydroxide/DNA Hybrids, Nano Lett., 6 (2006) 199-204.
d
[23] K. Benaissi, V. Helaine, V. Prevot, C. Forano, L. Hecquet, Efficient Immobilization of Yeast Transketolase on Layered Double Hydroxides and Application for Ketose Synthesis, Adv.
te
Synth. Catal., 353 (2011) 1497-1509.
[24] K. Charradi, C. Forano, V. Prevot, D. Madern, A. Ben Haj Amara, C. Mousty,
Ac ce p
Characterization of Hemoglobin Immobilized in MgAl-Layered Double Hydroxides by the Coprecipitation Method, Langmuir, 26 (2010) 9997-10004. [25] E. Geraud, V. Prevot, C. Forano, C. Mousty, Spongy gel-like layered double hydroxidealkaline phosphatase nanohybrid as a biosensing material, Chem. Commun., (2008) 1554-1556. [26] C. Guérard-Hélaine, B. Légeret, C. Fernandes, V. Prévot, C. Forano, M. Lemaire, Efficient immobilization of fructose-6-phophate aldolase in layered double hydroxide: improved stereoselective synthesis of sugar analogues, New J. Chem., 35 (2011) 779-779. [27] X. Duan, D.G. Evans, Structure and bonding: Layered Double Hydroxides, Springer, Berlin/Heidelberg, 2006. [28] D.G. Evans, X. Duan, Preparation of layered double hydroxides and their applications as additives in polymers, as precursors to magnetic materials and in biology and medicine, Chem. Commun., (2006) 485-486.
22
Page 22 of 34
[29] V. Rives, Layered Double Hydroxides: Present and Future, Nova Science, New York, 2001. [30] C. Taviot-Gueho, V. Prevot, C. Forano, Layered Double Hydroxides in: F. Bergaya, L. G. (Eds.) Handbook of Clays Science 2nd Edition, Elsevier 2013. [31] S. Jin, P.H. Fallgren, J.M. Morris, Q. Chen, Removal of bacteria and viruses from waters
ip t
using layered double hydroxide nanocomposites Original Research Article, Sci. Tech. Adv. Mater., 8 (2007) 67-70.
cr
[32] J. Liu, C. Duan, J. Zhou, X. Li, G. Qian, Z.P. Xu, Adsorption of bacteria onto layered double hydroxide particles to form biogranule-like aggregates, Appl. Clay Sci., 75 (2013) 39-45.
us
[33] J. Liu, X. Li, J. Luo, C. Duan, H. Hu, G. Qian, Enhanced decolourisation of methylene blue by LDH-bacteria aggregates wit bioregeneration, Chem. Eng. J., 242 (2014) 187-194. [34] T. Alekseeva, V. Prevot, M. Sancelme, C. Forano, P. Besse-Hoggan, Enhancing atrazine
an
biodegradation by Pseudomonas sp. strain ADP adsorption to Layered Double Hydroxide bionanocomposites, J. Hazard. Mater., 191 (2011) 126-135.
M
[35] P. Besse-Hoggan, T. Alekseeva, M. Sancelme, A.-M. Delort, C. Forano, Atrazine biodegradation modulated by clays and clays/humic acid complexes, Environ. Pollut., 157
d
(2009) 2837-2844.
[36] S. Klein, R. Avrahami, E. Zussman, M. Beliavski, S. Tarre, M. Green, Encapsulation of
te
Pseudomonas sp. ADP cells in electrospun microtubes for atrazine bioremediation, J. Ind. Microbiol Biotechnol, 39 (2012) 1605-1613.
Ac ce p
[37] M. Rietti-Shati, D. Ronen, R.T. Mandelbaum, Atrazine degradation by Pseudomonas Strain ADP Entrapped in Sol-Gel Glass, J. Sol-Gel Sci. Technol., 7 (1996) 77-79. [38] S. Stelting, R.G. Burns, A. Sunna, G. Visnovsky, C.R. Bunt, Immobilization of Pseudomonas sp. strain ADP: A stable inoculant for the bioremediation of atrazine, Appl. Clay Sci., 64 (2012) 90-93.
[39] C.G. Kauffmann, R.T. Mandelbaum, Entrapment of atrazine-degrading enzymes in sol-gel glass, J. Biotechnol., 51 (1996) 219-225. [40] N. Touisni, F. Charmantray, V. Helaine, C. Forano, L. Hecquet, C. Mousty, Optimized immobilization of transketolase from E. coli in MgAl-layered double hydroxides, Colloids Surf. B Biointerfaces, 112 (2013) 452-459. [41] M. Adachi-Pagano, C. Forano, J.-P. Besse, Delamination of layered double hydroxides by use of surfactants, Chem. Commun., (2000) 91-92.
23
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[42] Q. Wang, D. O'Hare, Recent advances in the synthesis and application of layered double hydroxide (LDH) nanosheets Chem. Rev., 112 (2012) 4124-4155. [43] Z. An, S. Lu, J. He, Y. Wang, Colloidal Assembly of Proteins with Delaminated Lamellas
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te
d
M
an
us
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of Layered Metal Hydroxide, Langmuir, 25 (2009) 10704-10710.
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Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation
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Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale BesseHoggan,
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Vanessa Prevot*
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Figure Captions
Figure 1. Cartoon outlining the procedure for ADP@LDH biohybrid preparation
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Figure 2. TGA curves in the temperature range 20-1100°C for (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
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Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells
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Figure 4. A) FESEM and B) TEM images of ADP@LDH2. Figure 5. Kinetics of atrazine biodegradation by ADP free cells and ADP-LDH bionanocomposites: (■) ADP free cells; () ADP@LDH10; (▲) ADP-LDH2 and (■) LDH Figure 6. Atrazine biodegradation percentage after 8h of incubation with free and immobilized ADP.
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Figure 1. Cartoon outlining the procedure for ADP@LDH biohybrid preparation
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Figure 2. TGA curves in the temperature range 20-1100°C for (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
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Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells
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Figure 4. A) FESEM and B) TEM images of ADP@LDH2.
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Figure 5. Kinetics of atrazine biodegradation by ADP free cells and ADP-LDH bionanocomposites: (■) ADP free cells; () ADP@LDH10; (▲) ADP-LDH2 and (■) LDH
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80
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60 40 20 0
ADP free cells
1st cycle 2nd cycle 3rd cycle 4th cycle
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Atrazine Biodegradation(% )
100
ADP@LDH2
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Figure 6. Atrazine biodegradation percentage after 8h of incubation with free and immobilized ADP
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Graphical Abstract
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Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation
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Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale Besse-Hoggan, Vanessa Prévot
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Bacteria@LDH biohybrid materials were elaborated by one pot direct coprecipitation, leading to efficient, stable and reusable assembly in atrazine biodegradation
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Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation
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Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale Besse-Hoggan,
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Vanessa Prevot*
Table 1. Textural properties of ADP@LDH biohybrid materials.
c
0.3032
ADP@LDH40
76
0.3032
ADP@LDH20
74
0.3032
ADP@LDH10
66
ADP@LDH4
59
ADP@LDH2
56
ADP
-
Mg2+/Al3+
2.46
+39
44
1.98
2.33
+10
45
1.30
2.37
+8
46
1.07
0.3032
2.33
+7
47
0.93
0.3036
-
+3
53
0.63
0.3032
-
-9
60
0.58
-
-
-18
92
-
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*
EDX
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Mg2Al-NO3 LDH
TGA (Total weight loss, %)
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a
(mV)*
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PXRD parameters (nm)
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Coprecipitation yield (%)
Samples
Measured in Volvic® mineral water.
Table 2. Kinetic parameters of 0.1 mM atrazine biodegradation by Pseudomonas sp. ADP free cells and ADP@LDH, considering kinetics to be of first order. Samples
Degradation rate (h-1)
Degradation after 8h (%)
ADP
0.164 (R2 = 0.998)
71
ADP@LDH40
0.053 (R2 = 0.998)
33
ADP@LDH20
0.071 (R2 = 0.995)
45
ADP@LDH10
0.371 (R2 = 0.999)
94
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0.207 (R2 = 0.998)
82
ADP@LDH2
0.341 (R2 = 0.999)
94
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ADP@LDH4
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S0927-7765(14)00652-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.11.029 COLSUB 6752
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
11-9-2014 3-11-2014 19-11-2014
Please cite this article as: M. Halma, C. Mousty, C. Forano, M. Sancelme, P. Besse-Hoggan, V. Prevot, Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.11.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacteria encapsulated in Layered Double
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pollutant degradation
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Hydroxides: toward an efficient bionanohybrid for
Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale Besse-Hoggan,
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Vanessa Prevot*
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Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 CLERMONT-FERRAND, FRANCE
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CNRS, UMR 6296, ICCF, F-63171 AUBIERE, FRANCE
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* Corresponding author: Vanessa Prevot Email: [email protected] Tel 00 33 47340710 Fax 04 73 40 79 33
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Highlights
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Pseudomonas sp. strain ADP was immobilized within LDH network by a simple, quick and ecofriendly direct coprecipitation approach.
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The ratio between biological and inorganic components can be easily tuned thanks to this approach. The cell viability and the conservation of the biological activity whtin the biohybrid were evidenced.
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An enhanced atrazine degradation rate was achieved by immobilized bacteria compared to free cells
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The ADP@LDH biohybrid maintains the same biological activity after four cycles of reutilization and three weeks storage at 4°C
ABSTRACT A soft chemical process was successfully used to immobilize Pseudomonas sp. strain ADP (ADP), a well-known atrazine (herbicide) degrading bacterium, within a Mg2Allayered double hydroxide host matrix. This approach is based on a simple, quick and ecofriendly direct coprecipitation of metal salts in the presence of a colloidal suspension of bacteria in water. It must be stressed that by this process the mass ratio between inorganic and biological components was easily tuned ranging from 2 to 40. This ratio strongly influenced the biological activity of the bacteria toward atrazine degradation. The better results were obtained for ratios of
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10 or lower, leading to an enhanced atrazine degradation rate and percentage compared to free cells. Moreover the biohybrid material maintained this biodegradative activity after four cycles of reutilization and 3 weeks storage at 4°C. The ADP@MgAl-LDH bionanohybrid materials completely
characterized
by
X-ray
diffraction
(XRD),
FTIR
spectroscopy,
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were
thermogravimetric analysis and scanning and transmission electronic microscopy (SEM and
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TEM) evidencing the successful immobilization of ADP within the inorganic matrix. This
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synthetic approach could be readily extended to other microbial whole-cell immobilization of
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interest for new developments in biotechnological systems.
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KEYWORDS: Layered Double Hydroxides, Bacteria, Bionanohybrid, Biodegradation, Atrazine
1. INTRODUCTION
The wide bacterial enzymatic and catalytic potential is increasingly used in various fields of emerging technologies, covering applications in biocatalysis [1], bioremediation [2, 3], bioengineering [4], biosensors [5]. Immobilization of microorganisms on solid supports is often mandatory to obtain high bacteria concentrations, increased reaction rates, cells/liquid separation ease and recyclability. Thus, it is of great importance to investigate bacterial immobilization processes [6] for both academic and practical aspects. Compared to the immobilization of purified enzymes, the attachment of whole cells obviates the need for separation, isolation and
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purification steps. Moreover, in maintaining enzymes in their native state, their stability and longevity can be enhanced. It is interesting to notice that micro-organism immobilization is above all a natural phenomenon. In laboratory, different methodologies have been developed to
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mimic nature and carry out biological material immobilization [7] on inert support mainly based on adsorption, physical entrapment, cross-linking and covalent coupling. These immobilization
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techniques can be described as post-synthesis, in-situ and multistep immobilization as suggested
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by Leonard et al. in a recent review on whole-cells based hybrid materials [6]. According to the immobilization methods, a large variety of material supports has been used, ranging from
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biopolymers [8] (such as alginate, k-carragenan, cellulose and collagen) or synthetic polymers [9] (polyacrylamide, polyvinylalcohol) to inorganic compounds [10-14](silica, clays, oxides).
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Note that the nature of the matrix is of paramount importance in determining the performance of
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immobilized cells. The ideal matrix should be stable, robust, chemically inert, biocompatible and
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should not interfere with the bioreaction. Specific properties including hydrophilicity, permeability, diffusion and resistance to microbial deterioration are also crucial. Compared to
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biopolymers, inorganic host matrices are particularly interesting in terms of porosity, mechanical and physical properties. Recently, Blondeau and Coradin highlighted the interest of sol-gel chemistry to improve the preservation of living materials [15]. In the last years, among the wide variety of available supports, Layered Double Hydroxides (LDH) have proved to be highly effective as host structures for biomolecules [16, 17] such as amino acids [18-20], DNA [21, 22], proteins [23-26] mainly due to their versatile properties in terms of chemical composition, basicity, charge density, hydrophilicity and morphology. LDH are layered materials [27-30] based on the stacking of positively charged sheets [MII1III x+ xM x(OH)2] .
Anions and water molecules are located in the interlamellar domains to maintain
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the global electroneutrality. Such layered structures promote favourable electrostatic interactions with various biomolecules bearing an overall negative charge at neutral or basic pH. Recent studies showed that LDH based biohydrids can be prepared in soft conditions by direct co-
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precipitation of the inorganic matrix in the presence of biomolecules, as an elegant alternative to the adsorption or anionic-exchange process [16, 17]. The biomolecules entrapped in LDH
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matrices by such strategy are then protected. However such approach has been mainly limited to
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small biomolecules (amino acid [19], DNA strand [22]) and proteins [23-26]. To our knowledge, only adsorption process on LDH has been achieved to immobilize whole-cells. For example, Jin
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et al. [31] demonstrate the efficient use of LDH for bacteria and virus removal from waters by adsorption processes. In parallel, thanks to electrostatic interactions between a NiAlCO3 LDH
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and the bacterial Bacillus subtilis cells, biogranule-like aggregate formation and their efficiency
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for methylene blue decolourisation have been reported [32, 33]. In our previous works [34, 35],
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we investigated the Pseudomanas sp. strain ADP adsorption on different MgRAl (R = 2, 3, 4) LDH matrices evidencing that bacterial cell adhesion can improve the biodegradative activity
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towards contaminants. We choose this bacterial strain (ADP) because of its interest in bioremediation based on its ability to mineralize atrazine (2-chloro-4-ethylamino-6isopropylamino-1,3,5-s-triazine), a persistent herbicide. Only few reports described the ADP whole cell immobilization, mainly within polymeric microfibers [36], silica [37] and onto natural zeolite [38]. Interestingly, cell free extracts of strain ADP containing atrazine-degrading enzymes were entrapped in a sol-gel matrix. However, due to silica sol-gel process conditions implying methanol release, immobilization induced a significant loss of enzymatic activity[39]. In this context, the objective of this work was to investigate as an alternative to the classical adsorption process the direct LDH coprecipitation to whole-cell entrapment in order to easily
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elaborate an efficient but also, stable and recyclable Pseudomonas sp. strain ADP@LDH bionanocomposite in maintaining the biochemical activity and cell viability.
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2. MATERIALS AND METHODS 2.1 Materials
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Atrazine (AT, 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine, 98.5 % chemical purity) was supplied free of charge by Syngenta Crop Protection® (Basel). A 0.1 mM (22 mg.L) solution was prepared in mineral water (Volvic®, France) for AT biodegradation and HPLC
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1
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standard uses. For all preparations, the magnesium and aluminium nitrate Mg(NO3)2.6H2O and Al(NO3)3.9H2O salts were of analytical grade. All other solvents and reagents were of
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commercial grade (Aldrich, Acros Organics, Merck, Fluka). Mineral water (Volvic®, France) was used for all experiments.
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2.2 Cell cultures
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Pseudomonas sp. strain ADP (gift from F. Martin Laurent from INRA Dijon, France) was
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grown in 100 mL portions of Trypcase Soy broth in 500 mL Erlenmeyer flasks incubated at 27 °C and 200 rpm. Cells were harvested after 24 h of culture. Samples of 20 mL of culture were centrifuged at 12,000 x g for 15 min at 4 °C. The resulting pellets were washed first with NaCl solution (8.0 g. L-1) and then, with Volvic®. The cell concentration used was around 2x1010 colony-forming units (cfu)/mL (OD=10 at = 575 nm), corresponding to 45±5 mg of dried cells and mentioned hereafter as 1 ADP.
2.3 Bacteria immobilization The ADP@LDH bionanocomposites were prepared by the co-precipitation at constant pH method [24, 30] (pH = 8.0 ± 0.2) and room temperature. Typically, an aqueous solution
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containing Mg(NO3)2 and Al(NO3)3 ([metals]= 0.1 M, Mg/Al=2) was added (flow rate: 0.813 mL/min), simultaneously with an aqueous NaOH solution (0.2 M) to a reactor containing an aqueous suspension of ADP. The addition rate of NaOH solution was controlled in order to
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maintain the pH at a fixed value of 8.0 ± 0.2, under nitrogen atmosphere and magnetic stirring. After 5 h of aging, the suspension was centrifuged and washed three times with Volvic® and
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recovered by centrifugation (12,000 x g for 10 min). The ADP/LDH mass ratio was easily
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modulated by modifying the added salt volume in the reactor, keeping constant the ADP amount in a fixed volume (4x1011 colony-forming units (cfu) in 60 mL). Metal salt volumes of 12 ml, 23
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ml, 55 ml, 102 ml and 204 ml were used, corresponding respectively to LDH theoretical mass values of 0.1 g, 0.2 g, 0.5 g, 1 g and 2 g. Coprecipitation yields were calculated according to the
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salt volume added and theoretical LDH formulae (Mg/Al = 2). Regarding the theoretical
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LDH/ADP mass ratio involved, the bionanocomposites as-prepared are denoted hereafter
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ADP@LDH2, ADP@LDH4, ADP@LDH10, ADP@LDH20 and ADP@LDH40. The ADP@LDH bionanocomposites were dried at 30 °C for solid state analyses. For atrazine biodegradation
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experiments, the ADP@LDH biohybrids were used as suspensions.
2.4 Characterization of ADP cells and bionanocomposites Free ADP cells and ADP@LDH bionanocomposites were characterized using different analytical techniques. X-ray diffraction patterns were recorded with a Philips X’Pert automated X-ray diffractometer using CuK radiation (= 0.154051 nm), over the 2-70° (2θ). FT-IR spectra were recorded with a Nicolet 5700 spectrometer from Thermo Electron Corporation using the KBr pellet technique.
7
Page 7 of 34
Zeta-potentials of Mg2Al-NO3, ADP cells and ADP@LDH were measured with a nano Zetasizer (Malvern instruments) apparatus, using a laser Doppler electrophoresis (LDE). Thermogravimetric analyses (TGA) were performed using a Setaram TGA92 thermogravimetric
ip t
analyzer in the temperature range of 25-1100 °C, with a heating rate of 5 °C min-1, under air atmosphere. The bionanocomposite assemblies were studied by transmission electron
cr
microscopy (TEM) using a Hitachi 7650 microscope at an acceleration voltage of 80 kV. To
us
perform the characterization, a drop of the suspension was deposited on a 400 mesh holey carbon-coated copper grid and dried at room temperature. FESEM characteristics of the samples
an
were imaged by a Zeiss supra 55 FEG-VP operating at 3 keV combined with an energy dispersive X-ray (EDX) analyzer. Specimens were mounted on conductive carbon adhesive tabs
M
and imaged after carbon sputter coating to make them conductive.
d
2.5 Herbicide degradation
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ADP and ADP@LDH were both tested as biocatalysts for biodegradation of atrazine. For each duplicate, ADP or ADP@LDH were suspended in a 0.1 mM AT solution in Erlenmeyer flasks.
Ac ce p
In accordance with ADP/LDH ratio, various volumes of ADP@LDH gel were involved to maintain constant the cell amount for all the biodegradation tests, leading to a low dilution effect of atrazine. Then, the flasks were incubated on a platform shaker (130 rpm, 27 °C). Samples (0.6 mL) were regularly taken from each flask, centrifuged at 12,500 rpm for 3 min and then frozen until HPLC analyses. Control experiments (without atrazine or cells) were carried out under the same experimental conditions. After the first use, ADP and ADP@LDH biocatalysts were thoroughly washed by centrifugation for reuse in another biodegradation reaction, under the same experimental conditions as those employed in their first use.
8
Page 8 of 34
2.6 Monitoring of Atrazine concentrations by HPLC The HPLC analyses were carried out using an Agilent 1100 photodiode array detector (DAD) chromatograph on a reverse-phase Agilent Eclipse XDB-C18 (5 µm, 4.6 x 150 mm) column. The
ip t
eluent was composed of 50 mM acetate buffer pH 4.6 and acetonitrile 60:40 (v: v), at a flow rate
cr
of 1 mL min-1. 10 µL of sample were injected. The UV detector was set at 225 nm.
3.1 Elaboration of ADP@LDH bionanocomposites
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3. RESULTS AND DISCUSSION
an
The ADP whole cells were immobilized in LDH matrix by a direct coprecipitation method (Fig. 1) which consists of the addition of magnesium and aluminium metal salt solution in a
M
colloidal suspension of ADP. In agreement with cell viability conditions, the LDH precipitation
d
is conducted at room temperature in maintaining the pH constant at 8.0 ± 0.2 into the reactor.
te
The coprecipitation of cells and LDH results in the formation of gelatinous precipitates. Different bionanohybrids were prepared by varying the volume of metal salt solution while keeping the
Ac ce p
ADP concentration constant. Various amounts of LDH were coprecipitated theoretically ranging from 0.1 g to 2.0 g. Whereas a yield of 78 % was observed for Mg2Al-NO3 formation in pure water, the solid precipitation yields in ADP@LDH biohybrid materials decrease progressively with decreasing amount of LDH, from 76% to 56% (Table 1). Obviously, it is worth noting that the cell presence in the reaction medium limits the coprecipitation efficiency probably due to the complexation of metal ions by extracellular polysaccharides.
9
Page 9 of 34
ip t cr us
an
Figure 1. Cartoon outlining the procedure for ADP@LDH biohybrid preparation The LDH/ADP assembly is evidenced by zetametry analysis. Indeed the larger the salt volume
M
involved, the higher the zeta-potential of resulting gel (Table 1). This trend evidences the increasing charge neutralization during bacteria entrapment, in good agreement with the zeta
te
18 mV.
d
potential values of both individual LDH nanoparticles and ADP, respectively of + 39 mV and -
Ac ce p
In parallel, the EDX results (Table 1) further evidence the influence of the cells not only on the yield of the LDH formation, but also on the Mg2+/Al3+ ratio. Indeed, systematically in the presence of cells, the ratio measured in the biohybrid phase is always lower to the one of initial metal salt solution, highlighting that the coprecipitation of Mg2+ and Al3+ does not occur stoichiometrically as observed usually. Such lower ratios from 1.3 to 0.58 respectively for ADP@LDH40 and ADP@LDH2 cannot be explained by the formation of a unique Al-rich LDH phase in regards with LDH structural properties [30].
10
Page 10 of 34
Table 1. Structural and chemical properties of ADP@LDH biohybrid materials. PXRD parameters (nm) a
c
(mV)*
25220°C
Mg2Al-NO3 LDH
78
0.3032
2.46
+39
ADP@LDH40
76
0.3032
2.33
+10
ADP@LDH20
74
0.3032
2.37
+8
ADP@LDH10
66
0.3032
2.33
ADP@LDH4
59
0.3036
-
ADP@LDH2
56
0.3032
ADP
-
-
Mg2+/Al3+
Total
16
44
1.98
18
45
1.30
19
46
1.07
+7
19
47
0.93
+3
19
53
0.63
us
an -
-9
18
60
0.58
-
-18
10
92
-
M
Measured in Volvic® mineral water.
EDX
d
*
TGA (weight loss, %)
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Co-precipitation yield (%)
cr
Samples
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It seems that despite the favourable pH value of LDH coprecipitation (pH 8), both LDH and amorphous aluminium hydroxides form in the presence of ADP cells. The side formation of
Ac ce p
aluminium hydroxide during the coprecipitation of MgAl LDH with high concentration of biomolecules has already been observed for the MgAl-Transketolase system [40]. The simultaneous decreases of the coprecipitation yield and the Mg/Al ratio could be explained by the decrease of the supersaturation level in the solution for the lower metal salt concentration conjointly to a local decrease of the pH value at the cell surrounding modifying the LDH coprecipitation conditions. Moreover, a specific metal cation adsorption on the microorganism surface may occur and partially hamper the precipitation reaction. To gain better insight on the immobilized cell amount into LDH materials, thermal gravimetric analyses were carried out on the different ADP@LDH biohybrids and compared to those of
11
Page 11 of 34
Mg2Al-NO3 and ADP cells (Fig.2). The Mg2Al-NO3 sample displays typical thermal behaviour of LDH materials with two characteristic weight loss regions corresponding to the removal of
d
M
an
us
cr
dehydroxylation and anion decomposition above 220°C (close to 26%).
ip t
adsorbed and intercalated water between 20 and 220°C (18% of mass loss) and the concomitant
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Figure 2. TGA curves in the temperature range 20-1100°C for (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
Ac ce p
The thermogravimetric analysis of free cells shows 92 % of total weight loss with three main mass losses over the temperature ranges 25-140°C, 140°C-490°C and above 490°C. For the ADP@LDH bionanocomposites, similar behaviour to that of LDH was observed, even for the lowest amount of LDH, with an almost complete decomposition before 600°C. In all cases, the lower the LDH/ADP ratio involved in the process, the higher the experimental weight loss (Table 1). For the ADP@LDH4 and ADP@LDH2 biohybrids, the ADP and LDH proportions were computed considering total weight losses of the biohybrid and of individual components that is ADP and pristine LDH. Interestingly, results fit roughly to the theoretical LDH/ADP ratio
12
Page 12 of 34
of 2 and 4, evidencing that direct coprecipitation allows to easily tune the LDH/ADP ratio in a large extend. The PXRD patterns of the air-dried gels (Fig.3A) are compared to those of pure Mg2Al-NO3
ip t
coprecipitated in the same conditions and ADP free cells. For the pure LDH material, diffraction lines are indexed according to a hexagonal lattice (R-3m space group) with cell parameters of a =
cr
0.3032 nm and c = 2.46 nm (Table 1). The interlayer distance (d = 0.83 nm) corresponds to
us
nitrate intercalated LDH structure. For all ADP@LDH bionanocomposites, the 012 and 110 LDH characteristic diffraction lines are systematically observed, confirming the effective
an
formation of the LDH layered structure. However, the presence of ADP cells in the synthetic medium induces a net decrease of the LDH platelet crystallinity. Indeed, the reflection intensity
M
strongly decreases for the lowest LDH/ADP ratio. This trend is particularly clear for the 00l
d
reflection lines. An enlargement of the reflections is also observed when decreasing the LDH /
te
ADP ratio. These PXRD pattern modifications can be attributed to both a decrease of LDH particle size and an increase of disorder into the layer stacking. For the lowest LDH /ADP ratio,
Ac ce p
a complete disappearance of the 00l line is observed as for exfoliated LDH phases [41, 42] and LDH phases prepared in the presence of enzymes [24, 43]which can be attributed to an important dispersion of the layer into the biohybrid. Due to the large ADP cell dimensions (1 x 0.5 x 0.5 m), the intercalation reaction of the whole cells into the layered structure is not expected. Biohybrid formation involving both LDH matrix and bacteria was further confirmed by FTIR analyses. Fig. 3B shows the FTIR spectra of ADP cells, Mg2Al-NO3 pristine LDH and ADP@LDH bionanocomposites. The Mg2Al-NO3 compound displays the typical vibrations characterizing the LDH structure [29]. The broad intense band centred at 3741 cm-1 corresponds to the OH stretching mode of layer hydroxyl groups and interlayer water molecules whereas the
13
Page 13 of 34
strong vibration frequency at 1384 cm-1 corresponding to the 3 stretching vibration of NO3 confirms its presence into the layered structure. Vibration bands at 662 cm-1 and 450 cm-1 attributed respectively to (MO) and (OMO), are typical of LDH lattice. In parallel, ADP spectrum
ip t
shows cells characteristic vibration frequencies such as bands or shoulders at 2968-2850 cm-1
cr
((CH2), (CH3)); 1660 cm-1 ((CO) - amide I); 1544 cm-1 ((NH) and (CN) - amide II); 1236 cm-1 ((NH), (CN)-amide III) and 1082-1053 cm-1 ((CO), (COC)-polysaccharides). On the FTIR spectra
us
of ADP@LDH biohybrid materials, some vibration bands of both ADP cells and LDH matrix are identified, traducing both LDH formation and microorganism entrapment. As expected, the
an
relative intensities of ADP and LDH vibration bands vary in proportion with the change in the
M
relative amount of ADP and LDH. The disappearance of the 00l lines observed on PXRD patterns for the lowest amount of LDH corresponds on the FTIR spectra to the net decrease of
Ac ce p
te
d
the NO3 vibration.
Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
14
Page 14 of 34
These observations are consistent for the lower LDH/ADP ratio with both amorphous aluminium hydroxides formation and LDH exfoliation inducing layer charge compensation mainly by the cell surface rather than intercalated nitrate anions.
ip t
The morphology of ADP based biohybrid materials was further investigated by SEM and TEM. On FESEM image (Fig. 4A), ADP@LDH2 displays a rather porous network of aggregated
cr
nanoparticles. Clearly, there is no evidence of ADP, a rod shaped bacteria (see supporting
us
information Fig. SI 1) at the surface of the LDH materials. Consequently, TEM analyses (Fig. 4B and Fig. SI 2) were required to get further information about the dispersion of ADP cells in the
an
LDH material. Interestingly, analysis of the ADP@LDH2 suspension deposited on a grid allows an unmistakable visualization of cells surrounding by aggregated plate-like LDH nanoparticles
M
with size in the range of 50-100 nm. Such observations indicate that the microorganisms are
d
really embedded within the LDH gel, thanks to direct coprecipitation synthetic approach.
te
The ADP encapsulation within LDH material was also evidenced by the change of coloration of solid. Whereas pristine LDH display a white colour, a primrose yellow solid is obtained in the
Ac ce p
presence of ADP. Moreover, the resulted ADP@LDH aggregates were more compact and larger than the spread LDH material.
Figure 4. A) FESEM and B) TEM images of ADP@LDH2.
A
B 15
Page 15 of 34
3.2 Viability of cells after immobilization in LDH and Biodegradation of atrazine To check the viability of ADP once immobilized in Mg2Al- LDH matrix aliquots (100 µL) from ADP@LDH2 suspensions were spread on TSA plates and incubated at 27 °C. After 24h, it
ip t
was observed that ADP remains viable when anchored in LDH support. Nevertheless it is difficult to estimate adequately the bacterial number cfu due to the aggregated nature of the
cr
biohybrid but ADP obviously maintain its ability to grow evidencing that LDH formation
us
process is compatible with ADP cell viability.
As ADP entrapped within LDH was shown to be viable, the biohybrids were tested as catalysts
an
for the biodegradation of atrazine. Indeed since immobilisation method and support properties can affect the bacterial properties in a positive or negative way, the effect of entrapment on the
M
ADP metabolic activity was evaluated. Systematically, biodegradation of atrazine (0.1 mM, in
d
Volvic® mineral water) was carried out using ADP@LDH biohybrids and compared to ADP
te
free cells stored under the same conditions (See supporting data and Fig. SI3). As an exemple, the Fig. 5 presents the kinetic curves of atrazine biodegradation for the two biohybrids
Ac ce p
ADP@LDH2 and ADP@LDH10. Strikingly, the biodegradation activity of ADP cells was strongly improved after immobilization into LDH for the lower mass ratios LDH/ADP (Table 2 and supporting data, Fig. SI 3) in terms of initial kinetics but also of biodegradation percentages. It seems that the flexible bidimensional structure of the LDH support combined with specific interactions between ADP and the mineral surface provides a favourable environment and boosts the biodegradation activity of ADP cells. It is worth noting that no sorption of atrazine occurred on LDH. Similar behaviour was previously observed for adsorbed ADP onto LDH [34, 35], evidencing that the ADP bacterial metabolic activity was systematically promoted, regardless of the immobilization method and the interactions between ADP and LDH. Moreover, the direct
16
Page 16 of 34
LDH formation involving acidic metal salt and basic agent in presence of ADP does not induce denaturation of proteins or cell death as observed in using sol-gel procedure [37]. However, the increase of the biodegradation rate depends on the ADP@LDH ratio (Table 2
ip t
and supporting data, Fig. S1B). The higher biodegradation rates and percentages are only
Ac ce p
te
d
M
an
us
cr
observed for LDH / ADP mass ratios lower than 20.
Figure 5. Kinetics of atrazine biodegradation by ADP free cells and ADP-LDH bionanocomposites: (■) ADP free cells; () ADP@LDH10; (▲) ADP-LDH2 and (■) LDH.
For the biohybrids ADP@LDH40 and ADP@LDH20, an opposite effect was observed. In these cases, high amounts of LDH lead to lower biodegradation activities compared with ADP free cells. This trend could be explained by a high entrapment of cells in a large quantity of LDH gel. Since no atrazine adsorption occurred on LDH surface (Fig. 5) [34], such high amount of inorganic matter may limit to some extend the solution diffusion to the cell and consequently
17
Page 17 of 34
reduces the biodegradation performance. Moreover when increasing the coprecipitated LDH amount, the viscosity of the medium increases since the cell quantity is maintained constant affecting then the atrazine diffusion and mobility in the medium.
ip t
To gain better insight on the cell stability which is of major importance for bionanocomposite applications, biocatalysts were thoroughly washed by centrifugation after the first biodegradation
cr
experiment and reuse to biodegrade atrazine under the same conditions. Fig. 6 shows that ADP
us
free cells present progressive loss of metabolic activity after each cycle of atrazine biodegradation. On the contrary, entrapped ADP into LDH maintains its full bio-catalytic
an
activity over 4 cycles, highlighting the positive effect inorganic matrix has on the ADP viability. This behaviour should be correlated to the ability of LDH to limit the protein denaturation thanks
M
to their physico-chemical surface properties, as previously underlined in the literature [17].
d
These results confirm the great positive influence of direct coprecipitation approach to entrap
te
ADP into LDH on the bacteria stability and biodegradation activity. Indeed even if a slightly higher boosting effect was observed in our previous work using adsorption method, direct
Ac ce p
coprecipitation leads to an efficient ADP entrapment limiting the direct exposure to the surrounding medium and the decrease of activity and lysis of bacteria even after several regeneration cycles. On the contrary, in the case of bacteria adsorption process, a progressive decrease in biocatalytic activity was reported with the increase of the reuse cycles [33]. Moreover, ADP@LDH2 stored at 4°C in suspension for three weeks maintains a similar bioactivity confirming the robustness of such bionanocomposite and its excellent stability over time.
18
Page 18 of 34
Table 2. Kinetic parameters of 0.1 mM atrazine biodegradation by Pseudomonas sp. ADP free cells and ADP@LDH, considering kinetics to be of first order. Degradation rate (h-1)
Degradation after 8h (%)
ADP
0.164 (R2 = 0.998)
71
ADP@LDH40
0.053 (R2 = 0.998)
ADP@LDH20
0.071 (R2 = 0.995)
ADP@LDH10
0.371 (R2 = 0.999)
94
ADP@LDH4
0.207 (R2 = 0.998)
82
ADP@LDH2
0.341 (R2 = 0.999)
94
ip t
Samples
33
M
100 80
1st cycle 2nd cycle 3rd cycle 4th cycle
40 20 0
d
60
te
Atrazine Biodegradation(% )
an
us
cr
45
ADP@LDH2
Ac ce p
ADP free cells
Figure 6. Atrazine biodegradation percentage after 8h of incubation with free and immobilized ADP.
4. CONCLUSIONS
The ADP@LDH bionanocomposites were successfully prepared by direct LDH formation in the presence of Pseudomonas sp. ADP suspensions. Such one step process allows to easily tune the LDH/ADP ratio of the bionanocomposites. Cells entrapped within LDH gel can maintain their cell viability and the inorganic part does not hamper the microorganism growth. Moreover ADP immobilization leads to a great enhancement of the metabolic activity toward atrazine
19
Page 19 of 34
degradation for LDH /ADP mass ratio lower than 20. Such immobilization method leading to LDH based bionanocomposites also provides interesting advantages in terms of cell stability and reusability of the living materials with no loss of bioactivity. This approach based on whole cell
ip t
immobilization within LDH can be easily extended to other microorganisms and applied to various pollutant bioremediation. Moreover since a gel easy to handle and to cast is elaborated, it
cr
opens also the way for applications in other fields such as biosensor or biofuel cell development.
us
Author Contributions
The manuscript was written through contributions of all authors. All authors have given
M
an
approval to the final version of the manuscript and contributed equally.
ACKNOWLEDGMENT
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The authors would like to thank the Conseil Régional Auvergne and Fonds Européen de
Ac ce p
REFERENCES
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Développement Régional (FEDER) for the financial support.
[1] R. Eibl, S. Kaiser, R. Lombriser, D. Eibl, Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology, Appl. Microbiol. Biotechnol., 86 (2010) 41-49. [2] P. Kanmani, J. Aravind, D. Preston, Remediation of chromium contaminants using bacteria, Int. J. Environ. Sci. Techn., 9 (2012) 183-193. [3] L. Wessels Perelo, In situ and bioremediation of organic pollutants in aquatic sediments, J. Hazard. Mater., 177 (2010) 81-89.
[4] Y. Tomoko, Yoshiaki; Maeda, M. Tadashi, Bioengineering of bacterial magnetic particles and their applications in biotechnology, Recent Pat Biotechnol., 4 (2010) 214-225. [5] E. Eltzov, R. Marks, Whole-cell aquatic biosensors, Anal. Bioanal. Chem., 400 (2011) 895913.
20
Page 20 of 34
[6] A. Leonard, P. Dandoy, E. Danloy, G. Leroux, C.F. Meunier, J.C. Rooke, B.-L. Su, Wholecell based hybrid materials for green energy production, environmental remediation and smart cell-therapy, Chem. Soc. Rev., 40 (2011) 860-885. [7] L. Betancor, H.R. Luckarift, Bioinspired enzyme encapsulation for biocatalysis, Trends
ip t
Biotechnol., 26 (2008) 566-572.
[8] J.K. Park, H.N. Chang, Microencapsulation of microbial cells, Biotechnol. Adv., 18 (2000)
cr
303-319.
[9] M. Qi, Y. Gu, N. Sakata, Y. Kim, Y. Shirouzu, C. Yamamoto, A. Hiura, S. Sumi, K. Inoue,
us
PVA hydrogel sheet macroencapsulation for the bioartificial pancreas., Biomaterials, 25 (2004) 5885-5892.
[10] A.T. Aikaterini, E. Kalogeris, E. Apostolos, A.T. Ali, G. Dimitrios, S. Haralambos,
an
Effective Immobilization of Candida Antarctica lipase B in organic modified clays: Application for the epoxidation of Terpenes, Mater. Sci. Eng., A, 165 (2009) 173-177.
M
[11] M. Amoura, R. Brayner, M. Perullini, C. Sicard, C. Roux, J. Livage, T. Coradin, Bacteria encapsulation in a magnetic sol-gel matrix, J. Mater. Chem., 19 (2008) 1241-1244.
d
[12] M. Amoura, N. Nassif, C. Roux, J. Livage, T. Coradin, Sol-gel encapsulation of cells is not 4015-4016.
te
limited to silica: bacteria long-term viability in alumina matrices, Chem. Commun., (2007) [13] A. Coiffier, T. Coradin, C. Roux, O.M.M. Bouvet, J. Livage, Sol-gel encapsulation of 2044.
Ac ce p
bacteria: a comparison betweeen alkoxide and aqueous routes., J. Mater. Chem., 11 (2001) 2039[14] M. Perullini, M. Jobbagy, N. Mouso, F. Forchiassin, S.A. Bilmes, Silica-Alginate-fungi bionanocomposites for remediation of polluted water, J. Mater. Chem., 20 (2010) 6479-6483. [15] M. Blondeau, T. Coradin, Living materials from sol–gel chemistry: current challenges and perspectives J. Mater. Chem., 22 (2012) 22335-22343. [16] C. Mousty, V. Prevot, Hybrid and biohybrid Layered Double Hydroxides for electrochemical analysis Anal. Bioanal. Chem., 405 (2013) 3513-3523. [17] V. Prevot , C. Mousty, C. Forano, State of the art in Biomolecules and LDH association, Adv. Chem. Res., 17 (2013) 35-83.
21
Page 21 of 34
[18] S. Aisawa, S. Takahashi, W. Ogasawara, Y. Umetsu, E. Narita, Direct intercalation of amino acids into layered double hydroxides by coprecipitation, J. Solid State Chem., 162 (2001) 52-62. [19] Á. Fudala, I. Pálinkó, I. Kiricsi, Preparation and characterization of hybrid Organic-
ip t
Inorganic composite materials using the Amphoteric property of amino acids : amino acid intercalated layered double hydroxide and montmorillonite., Inorg. Chem., 38 (1999) 4653-4658.
cr
[20] M. Wei, Z. Shi, D.G. Evans, X. Duan, Study on the intercalation and interlayer oxidation transformation of L-cysteine in a confined region of layered double hydroxides, J. Mater. Chem.,
us
16 (2006) 2102-2109.
[21] J.-H. Choy, S.-Y. Kwak, J.-S. Park, Y.-J. Jeong, J. Portier, Intercalative Nanohybrids of (1999) 1399-1400.
an
Nucleoside Monophosphates and DNA in Layered Metal Hydroxide, J. Am. Chem. Soc, 121 [22] L. Desigaux, M.B. Belkacem, P. Richard, J. Cellier, P. Léone, L. Cario, F. Leroux, C. B.
Pitard,
Self-Assembly
and
M
Taviot-Guého,
Characterization
of
Layered
Double
Hydroxide/DNA Hybrids, Nano Lett., 6 (2006) 199-204.
d
[23] K. Benaissi, V. Helaine, V. Prevot, C. Forano, L. Hecquet, Efficient Immobilization of Yeast Transketolase on Layered Double Hydroxides and Application for Ketose Synthesis, Adv.
te
Synth. Catal., 353 (2011) 1497-1509.
[24] K. Charradi, C. Forano, V. Prevot, D. Madern, A. Ben Haj Amara, C. Mousty,
Ac ce p
Characterization of Hemoglobin Immobilized in MgAl-Layered Double Hydroxides by the Coprecipitation Method, Langmuir, 26 (2010) 9997-10004. [25] E. Geraud, V. Prevot, C. Forano, C. Mousty, Spongy gel-like layered double hydroxidealkaline phosphatase nanohybrid as a biosensing material, Chem. Commun., (2008) 1554-1556. [26] C. Guérard-Hélaine, B. Légeret, C. Fernandes, V. Prévot, C. Forano, M. Lemaire, Efficient immobilization of fructose-6-phophate aldolase in layered double hydroxide: improved stereoselective synthesis of sugar analogues, New J. Chem., 35 (2011) 779-779. [27] X. Duan, D.G. Evans, Structure and bonding: Layered Double Hydroxides, Springer, Berlin/Heidelberg, 2006. [28] D.G. Evans, X. Duan, Preparation of layered double hydroxides and their applications as additives in polymers, as precursors to magnetic materials and in biology and medicine, Chem. Commun., (2006) 485-486.
22
Page 22 of 34
[29] V. Rives, Layered Double Hydroxides: Present and Future, Nova Science, New York, 2001. [30] C. Taviot-Gueho, V. Prevot, C. Forano, Layered Double Hydroxides in: F. Bergaya, L. G. (Eds.) Handbook of Clays Science 2nd Edition, Elsevier 2013. [31] S. Jin, P.H. Fallgren, J.M. Morris, Q. Chen, Removal of bacteria and viruses from waters
ip t
using layered double hydroxide nanocomposites Original Research Article, Sci. Tech. Adv. Mater., 8 (2007) 67-70.
cr
[32] J. Liu, C. Duan, J. Zhou, X. Li, G. Qian, Z.P. Xu, Adsorption of bacteria onto layered double hydroxide particles to form biogranule-like aggregates, Appl. Clay Sci., 75 (2013) 39-45.
us
[33] J. Liu, X. Li, J. Luo, C. Duan, H. Hu, G. Qian, Enhanced decolourisation of methylene blue by LDH-bacteria aggregates wit bioregeneration, Chem. Eng. J., 242 (2014) 187-194. [34] T. Alekseeva, V. Prevot, M. Sancelme, C. Forano, P. Besse-Hoggan, Enhancing atrazine
an
biodegradation by Pseudomonas sp. strain ADP adsorption to Layered Double Hydroxide bionanocomposites, J. Hazard. Mater., 191 (2011) 126-135.
M
[35] P. Besse-Hoggan, T. Alekseeva, M. Sancelme, A.-M. Delort, C. Forano, Atrazine biodegradation modulated by clays and clays/humic acid complexes, Environ. Pollut., 157
d
(2009) 2837-2844.
[36] S. Klein, R. Avrahami, E. Zussman, M. Beliavski, S. Tarre, M. Green, Encapsulation of
te
Pseudomonas sp. ADP cells in electrospun microtubes for atrazine bioremediation, J. Ind. Microbiol Biotechnol, 39 (2012) 1605-1613.
Ac ce p
[37] M. Rietti-Shati, D. Ronen, R.T. Mandelbaum, Atrazine degradation by Pseudomonas Strain ADP Entrapped in Sol-Gel Glass, J. Sol-Gel Sci. Technol., 7 (1996) 77-79. [38] S. Stelting, R.G. Burns, A. Sunna, G. Visnovsky, C.R. Bunt, Immobilization of Pseudomonas sp. strain ADP: A stable inoculant for the bioremediation of atrazine, Appl. Clay Sci., 64 (2012) 90-93.
[39] C.G. Kauffmann, R.T. Mandelbaum, Entrapment of atrazine-degrading enzymes in sol-gel glass, J. Biotechnol., 51 (1996) 219-225. [40] N. Touisni, F. Charmantray, V. Helaine, C. Forano, L. Hecquet, C. Mousty, Optimized immobilization of transketolase from E. coli in MgAl-layered double hydroxides, Colloids Surf. B Biointerfaces, 112 (2013) 452-459. [41] M. Adachi-Pagano, C. Forano, J.-P. Besse, Delamination of layered double hydroxides by use of surfactants, Chem. Commun., (2000) 91-92.
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[42] Q. Wang, D. O'Hare, Recent advances in the synthesis and application of layered double hydroxide (LDH) nanosheets Chem. Rev., 112 (2012) 4124-4155. [43] Z. An, S. Lu, J. He, Y. Wang, Colloidal Assembly of Proteins with Delaminated Lamellas
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of Layered Metal Hydroxide, Langmuir, 25 (2009) 10704-10710.
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Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation
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Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale BesseHoggan,
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Vanessa Prevot*
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Figure Captions
Figure 1. Cartoon outlining the procedure for ADP@LDH biohybrid preparation
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Figure 2. TGA curves in the temperature range 20-1100°C for (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
te
d
Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells
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Figure 4. A) FESEM and B) TEM images of ADP@LDH2. Figure 5. Kinetics of atrazine biodegradation by ADP free cells and ADP-LDH bionanocomposites: (■) ADP free cells; () ADP@LDH10; (▲) ADP-LDH2 and (■) LDH Figure 6. Atrazine biodegradation percentage after 8h of incubation with free and immobilized ADP.
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Figure 1. Cartoon outlining the procedure for ADP@LDH biohybrid preparation
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Figure 2. TGA curves in the temperature range 20-1100°C for (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
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Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells.
Figure 3. Powder X-ray diffraction patterns (A) and FTIR spectra (B) of (a) Mg2Al-NO3 LDH; (b) ADP@LDH40; (c) ADP@LDH20; (d) ADP@LDH10; (e) ADP@LDH4; (f) ADP@LDH2 and (g) ADP free cells
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Figure 4. A) FESEM and B) TEM images of ADP@LDH2.
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Figure 5. Kinetics of atrazine biodegradation by ADP free cells and ADP-LDH bionanocomposites: (■) ADP free cells; () ADP@LDH10; (▲) ADP-LDH2 and (■) LDH
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60 40 20 0
ADP free cells
1st cycle 2nd cycle 3rd cycle 4th cycle
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Atrazine Biodegradation(% )
100
ADP@LDH2
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Figure 6. Atrazine biodegradation percentage after 8h of incubation with free and immobilized ADP
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Graphical Abstract
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Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation
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Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale Besse-Hoggan, Vanessa Prévot
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Bacteria@LDH biohybrid materials were elaborated by one pot direct coprecipitation, leading to efficient, stable and reusable assembly in atrazine biodegradation
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Bacteria encapsulated in Layered Double Hydroxides: toward an efficient bionanohybrid for pollutant degradation
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Matilte Halma, Christine Mousty, Claude Forano, Martine Sancelme, Pascale Besse-Hoggan,
cr
Vanessa Prevot*
Table 1. Textural properties of ADP@LDH biohybrid materials.
c
0.3032
ADP@LDH40
76
0.3032
ADP@LDH20
74
0.3032
ADP@LDH10
66
ADP@LDH4
59
ADP@LDH2
56
ADP
-
Mg2+/Al3+
2.46
+39
44
1.98
2.33
+10
45
1.30
2.37
+8
46
1.07
0.3032
2.33
+7
47
0.93
0.3036
-
+3
53
0.63
0.3032
-
-9
60
0.58
-
-
-18
92
-
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EDX
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Mg2Al-NO3 LDH
TGA (Total weight loss, %)
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(mV)*
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PXRD parameters (nm)
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Coprecipitation yield (%)
Samples
Measured in Volvic® mineral water.
Table 2. Kinetic parameters of 0.1 mM atrazine biodegradation by Pseudomonas sp. ADP free cells and ADP@LDH, considering kinetics to be of first order. Samples
Degradation rate (h-1)
Degradation after 8h (%)
ADP
0.164 (R2 = 0.998)
71
ADP@LDH40
0.053 (R2 = 0.998)
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ADP@LDH20
0.071 (R2 = 0.995)
45
ADP@LDH10
0.371 (R2 = 0.999)
94
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0.207 (R2 = 0.998)
82
ADP@LDH2
0.341 (R2 = 0.999)
94
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ADP@LDH4
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