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Siderophore Production by Using Free and Immobilized Cells of Two Pseudomonads Cultivated in a Medium Enriched With Fe and/or Toxic Metals (Cr, Hg, Pb) Armelle Braud, Karine Je´ze´quel, Marie-Anne Le´ger, Thierry Lebeau Laboratory G.R.E. (Risk Management and Environment, EA 2334), University of Haute-Alsace, Agency of Colmar, BP 50568, 68 008 Colmar cedex, France; telephone: 00-33-3-89-20-31-35; fax: 00-33-3-89-20-23-57; e-mail: [email protected] Received 26 October 2005; accepted 9 March 2006 Published online 3 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20937

Abstract: Pseudomonads are serious candidates for siderophore production applied to toxic metal (TM) solubilization. The bioaugmentation of contaminated soils by these TM-solubilizing bacteria combined with phytoextraction is an emerging clean-up technology. Unfortunately, siderophore synthesis may be drastically reduced by soluble iron in soils and bacteria can suffer from TM toxicity. In this study, we compared siderophore production by Pseudomonas aeruginosa and Pseudomonas fluorescens by using free and immobilized cells in Caalginate beads incubated in a medium containing Fe and/ or TM (mixture of Cr, Hg, and Pb in concentrations which represented the soluble fraction of a contaminated agricultural soil). Free cell growth was stimulated by Fe, whatever the microorganism, the inoculum size and the presence or not of TM might have been. P. aeruginosa was less sensitive to TM than P. fluorescens. By comparison with free cells, immobilization with the high inoculum size showed less sensitivity to TM most probably because of lower metal diffusion in beads. Indeed, a maximum of 99.1% of Cr, 57.4% of Hg, and 99.6% of Pb were adsorbed onto beads. The addition of iron in the culture medium reduced significantly siderophore production of free cells while it led only to a low decrease with their immobilized counterparts, in particular with P. aeruginosa. In culture medium enriched with Fe and/or TM, siderophore-specific production of immobilized cells was higher than for free cells. ß 2006 Wiley Periodicals, Inc. Keywords: cell immobilization; iron; Pseudomonas aeruginosa; Pseudomonas fluorescens; siderophore production; toxic metals

INTRODUCTION Phytoremediation is a fast-expanding technology for remediation of contaminated soils, using phytoextraction for cleaning up of soils contaminated with toxic metals (TM). Correspondence to: T. Lebeau Contract grant sponsor: French National Centre for Scientific Research Contract grant number: ACI NPD 24

ß 2006 Wiley Periodicals, Inc.

Unfortunately this technique is usually time consuming, partly because of low TM bioavailability. In order to enhance bioavailability of TM at plant disposal sites, various synthetic compounds (e.g., DTPA, EDGA, EDTA, etc.) have already been used but their biodegradability is, most of the time, low by comparison with microbial chelates (White, 2001) and they can be phytotoxic at high concentrations (Chen and Cutright, 2001). As an alternative, soil bioaugmentation coupled together with phytoextraction is an emerging technology (Glick, 2003). Several studies have shown the ability of microbial siderophores (Diels et al., 1999; Dubbin and Ander, 2003; Glick, 2003) to increase metal bioavailability in various porous matrices including soils, at a higher rate than synthetic chelates (Shenker et al., 1999; Wang et al., 1993). Among several siderophore producers, Pseudomonads are serious candidates (Cornelis and Matthijs, 2002). In a previous work (Braud et al., 2005), agricultural soils contaminated by Cr, Hg, and Pb were bioaugmented, in order to increase TM bioavailability, by either bacterial (Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, or Ralstonia metallidurans) or fungal inocula (Aspergillus niger or Penicillium simplicissimum) which synthesized either siderophores (e.g., P. aeruginosa and P. fluorescens), biosurfactants, or organic acids. We concluded that Pseudomonads were the most efficient microorganisms. Two major requirements must be fulfilled to optimize this bioaugmentation process and to guarantee a steady working whatever the physico-chemical characteristics of soil may be. On one hand, environmental conditions in soil must favor siderophore synthesis which is all the more high so as culture medium is low in Fe (Ams et al., 2002; Page, 1993; Sharma and Johri, 2003b). In spite of the high insolubility of iron (in Fe(III) form), bioavailable forms may occur in some environmental conditions, for example, anoxic soil conditions (Kabata-Pendias and Pendias, 2001). Consequently, siderophore biosynthesis can be inhibited.

On the other hand, the most important challenge in bioaugmentation consists of improving microorganism survival. It can be achieved thanks to cell immobilization into a carrier (e.g., alginate, clay . . .) which protects the microorganisms against natural competition with the soil microflora (Cassidy et al., 1996; Gentry et al., 2004; McLoughlin, 1994). Several environmental studies already tested cell immobilization for various purposes, for example, modification of the TM speciation in soils (Je´ze´quel et al., 2005), degradation of organic pollutants (Cassidy et al., 1997; Ka¨stner et al., 1998). The aim of our study was to compare siderophore production by P. aeruginosa and P. fluorescens by using free and immobilized cells in Ca-alginate beads. The bacteria were batch incubated in a liquid minimum medium containing either Fe and/or TM (mixture of Cr, Hg, and Pb) in concentrations which represented the bioavailable fraction of a TM-contaminated agricultural soil. We also studied TM adsorption onto beads and we estimated the experimental bias of the methods used for siderophore determination. MATERIALS AND METHODS Microbial Strains and Pre-Culture Conditions Pseudomonas aeruginosa ATCC 9027 and Pseudomonas fluorescens DSM 50090 were maintained at 808C in tubes containing 20% of glycerol (v/v). Bacteria were precultivated at 288C for 24 h in shaken Erlenmeyer flasks (agitation speed, 200 rpm) containing LB medium (tryptone, 10 g/L; NaCl, 5 g/L; yeast extract, 5 g/L). Cultures were centrifuged (9,400g, 10 min, 48C) and washed twice with a solution of KCl (9 g/L). Cell concentration of bacterial suspensions was determined by measuring optical density (OD) of samples at 600 nm and by relating to CFU/mL from an appropriate calibration curve.

7 (HNO3, 0.1 M). Fe-free minimal medium contained less than 3 ppb of Fe. The medium was sterilized at 1208C during 20 min. Toxic metals (Cr, 5 mg/L; Hg, 0.2 mg/L; Pb, 13 mg/L in the following forms: CrCl3 6H2O; HgCl2, Pb(NO3)2) were added or not to the medium after filtration (cellulose-acetate membrane; mean pore size, 0.45 mm). Each Erlenmeyer flask was inoculated with the same amount of free and immobilized cells which corresponded, respectively, to 6.7 104 CFU/mL of medium and 1 106 CFU/mL of alginate for the low concentration, and 6.7 107 CFU/mL of medium and 1 109 CFU/mL of alginate for the high concentration. Erlenmeyer flasks were incubated at 288C and shaked at 200 rpm during 6 days for P. fluorescens and 8 days for P. aeruginosa. Each experiment was done in triplicate.

Sorption Experiments Thirty grams of Ca-alginate beads were introduced in an Erlenmeyer flask containing 400 mL of ultra pure water along with the mixture of Cr, Hg, and Pb with or without Fe, at concentrations mentioned above. Erlenmeyer flasks were incubated at 288C and shaked at 200 rpm. qe (mg/g), metal sorbed at the equilibrium, was determined according to the equation given by Volesky (1990): qe ¼

VðCo Ce Þ M

where V (L) is the volume of solution, Co (mg/L) is the initial metal concentration, Ce (mg/L) is the metal concentration at equilibrium, and M (g) is the mass of beads.

Analytical Methods

Immobilization Procedure

Siderophore Measurement

A 100 mL sterile solution of Na-alginate (autoclaved at 1208C during 20 min) was mixed thoroughly with the cell suspension to reach a final 3% Na-alginate. Ca-alginate beads of about 3 mm diameter were obtained by dropping the Naalginate cell mixture (fall height, 7 cm) into a 750 mL stirred solution of CaCl2 (30 g CaCl2/L was used in CaCl2 2H2O form) using a peristaltic pump supplied with a calibrated needle. Beads remained 90 min in CaCl2 and were rinsed 2 h in 750 mL sterile distilled water. Five grams of Ca-alginate beads were introduced per Erlenmeyer. Alginate beads without any microorganisms were used as control.

All siderophores were detected, according to the method of Schwyn and Neilands (1987), by using 100 mL of blue CAS reagent (chromeazurol S). Siderophore concentrations were determined by measuring OD at 630 nm of centrifuged samples (7,155g for 10 min). The affinity of microbial siderophores for both Fe derived from CAS reagent and metals in the liquid medium was estimated. Two experiments were performed in which OD of samples at 630 nm was measured: (i) siderophore-containing supernatant derived from a microbial cell culture with either P. aeruginosa or P. fluorescens was concentrated and added to the minimal medium with Fe and/or TM (multiples of the following concentrations: Cr, 5 mg/L; Fe, 4 mg/L; Hg, 0.2 mg/L; Pb, 13 mg/L), and (ii) serial dilutions of the concentrated supernatant (1/1, 1/2, 1/4, 1/8, 1/16, 1/32) were added to both metal-free minimal medium and to the medium containing Fe (4 mg/L) and/or a mixture of Cr (5 mg/L), Hg (0.2 mg/L), Pb (13 mg/L).

Microbial Cell Cultures Erlenmeyer flasks (100 mL volume) were filled with 75 mL of modified minimal medium (L-asparagin, 0.5 g/L; K2HPO4, 0.5 g/L; MgSO4 7H2O, 0.2 g/L) with or without 4 mg Fe/L (in FeCl3 6H2O form). pH was adjusted to

Braud et al.: Siderophore Production by Using Free and Immobilized Cells of Pseudomonads Biotechnology and Bioengineering. DOI 10.1002/bit

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Microbial Biomass Determination Cell growth in Erlenmeyer flasks was determined from both liquid media at 600 nm, as mentioned above, and from the beads at the same wavelength after dissolution with 50 mM of trisodium citrate. Cell concentrations were determined by measuring OD of samples and by relating to CFU/mL from an appropriate calibration curve. TM Measurement The liquid medium samples were filtered through a filter (same characteristics as above mentioned) and Cr, Fe, Hg, and Pb concentrations were measured, using ICP-AES (Inductively Coupled Plasma, Atomic Emission Spectrometers, JY ULTIMA, Jobin Yvon HORIBA Group, Kyoto, Japan) at the wavelength of 267.72, 259.94, 194.23, and 220.35 nm, respectively. Statistical Analysis The experiments were done in triplicate. Statistical analysis of data included Student’s t-test and analysis of variance (ANOVA) from a randomized multifactorial design with a mean comparison according to the Newman–Keuls test, by using Statbox Pro software (GrimmerSoft, version 5, Paris, France). Statistical significance was determined at P ¼ 0.05. Homogeneous groups were designated by a, b, c, etc. RESULTS AND DISCUSSION Bias in the Siderophore Measurement The method of Schwyn and Neilands (1987), which allows to determine siderophore production, is based on the higher affinity of siderophores than CAS reagent for Fe. Thus siderophores remove iron from the dye CAS reagent whose color turns from blue to orange (detection at 630 nm). In that case, the amount of cleaved Fe-CAS chelates is proportional to the amount of siderophores. This method is valid only for studies concerning the metal-free culture media, otherwise the siderophore affinity may be higher for metals added in the medium than for Fe chelated to CAS reagent. In that case, the amount of siderophore may be underestimated since the Fe-CAS chelates are not cleaved or not in proportion to the amount of siderophores in the medium. The respective affinity of siderophores produced by P. aeruginosa and P. fluorescens for Cr, Fe, Hg, and Pb was unknown (a priori) in our experimental conditions. To compare the affinity of microbial siderophores for the above-mentioned metals, siderophore-containing supernatant resulting from a microbial cell culture with either P. aeruginosa or P. fluorescens was concentrated and added to the minimal medium with TM and/or Fe. The results showed that microbial siderophores produced by either P. aeruginosa (Fig. 1a) or P. fluorescens (Fig. 1b) had higher affinity—as shown by the decrease of OD at 630 nm—for metals in the

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Figure 1. Comparison of siderophore affinity of P. aeruginosa and P. fluorescens for Cr, Fe, Hg, and Pb, added individually or in mixture to the medium, by measuring OD at 630 nm in relation to metal concentration in the medium. The values in abscissa are multiples of the following concentrations: Cr, 5 mg/L; Fe, 4 mg/L; Hg, 0.2 mg/L; Pb, 13 mg/L. Cr (&), Fe (^), Hg (~) or Pb (*), mixture of Cr, Hg, and Pb with (~) or without Fe (&). a: P. aeruginosa, (b) P. fluorescens. Results show mean confidence interval.

medium, that is, Cr, Fe, Hg, and Pb than for Fe chelated to the CAS reagent. The affinity for Fe was the highest by comparison with other metals (added alone or as a mixture), whatever the bacterium might have been. Cr, Hg, and Pb mixture led to a significant decrease of OD, by comparison with each of these three metals, which was reinforced with the addition of Fe to TM. As we could expect it, siderophores produced by both P. aeruginosa and P. fluorescens had affinities for all the metals, not only for Fe. This result was not surprising since these microorganisms are well known producers of several siderophore species (Cornelis and Matthijs, 2002), in particular pyoverdine and pyochelin, which show a high affinity for trivalent cations such as Fe3þ and divalent ones such as Zn2þ (Cuppels et al., 1987), respectively. Consequently, to take into account the bias in siderophore determination in case of metal-containing medium, calibration curves were established (Fig. 2) by measuring OD at 630 nm of samples with TM- and/or Fe-containing medium and by relating to OD of the metal-free samples at the same wavelength. Metal Sorption Onto Ca-Alginate Beads The concentration in metals used in our experiments represents the soluble fraction of an agricultural soil polycontaminated by Cr, Hg, and Pb (Braud et al., 2005). Resistance to TM in soil of P. Aeruginosa and P. fluorescens

Biotechnology and Bioengineering, Vol. 94, No. 6, August 20, 2006 DOI 10.1002/bit

Figure 2. Siderophore determination by measuring OD at 630 nm of samples with medium containing various concentrations of either Fe (^), a mixture of Cr, Hg, and Pb (&) at concentration used in experiments or metals all together (~) and by relating to OD of metal-free samples at the same wavelength. a: P. aeruginosa, (b) P. fluorescens. Results show mean standard deviation.

used in this study has not already been studied. Thus semiquantitative assays (data not shown), which aimed to estimate this tolerance towards cell growth, consisted in cultivating these strains on agar plates-containing TM gradients (mixture of Cr, Hg, and Pb at 1, 5, and 10 times the TM concentration in soil). The two strains were tolerant to TM at the first two concentrations (slight inhibition of cell growth at the intermediate concentration). At the highest concentration, cell growth was partly inhibited.

Table I shows the sorption properties of non-inoculated Ca-alginate beads for Cr, Fe, Hg, and Pb. Equilibrium times were reached after 30–60 min for Pb and 120–360 min for Cr which was close to those reported by Arica et al. (2003a) for Pb and Ibań˜ez and Umetsu (2004) for Cr(III). Fe (97.3%) was adsorbed after 90 min. Several authors showed that alginate beads were likely to adsorb other metals such as Cd, Co, Cu, Hg, Mn, Zn (Arica et al., 2004; Gotoh et al., 2004; Lebeau et al., 2002; Ozdemir et al., 2005). Conversely, only a maximum of 57.39% of Hg was sorbed at the end of our experiments (500 min) although Arica et al. (2003b) and Kaçar et al. (2002) showed that sorption equilibrium was established in about 60 min. However, the concentration used by these authors (200 mg Hg/L) was 1,000 times higher than in our experiments which most probably explains that result. Indeed Lin and Lin (2005) showed that the higher the initial concentration in aqueous solution, the shorter the time to reach equilibrium adsorption. Interaction between metals in the solution could also partly explain the difference in time to reach equilibrium. In our experiments time equilibrium was modified as Fe was added or not in the solution. It increased in a factor 3 for Cr and decreased in a factor 2 for Pb in the Fefree solution by comparison with the Fe-containing one. In case of Hg equilibrium was not reached after 500 min but it could be expected to reach it more quickly with the Fe-free solution than with the Fe-containing one since 57.39% were already adsorbed against only 17.29%. Immobilization of either P. aeruginosa or P. fluorescens did not modify significantly adsorption properties (data not shown) as already observed by Aksu et al. (1998) for Cu with Chlorella vulgaris. Metal adsorption on Ca-alginate beads and diffusion through the pores of immobilization matrices are two simultaneous phenomena. Because the diffusion of solutes can be restricted (Aksu et al., 1998; Xiangliang et al., 2005) and that low metal concentrations were used in our experimental conditions free-metal areas can be expected to appear in the core of beads. For example, Pandley et al. (2002) observed that only 8% of a medium containing 2.8 mg Cr/L entered the alginate beads and Sreeram et al. (2004) showed that Fe was adsorbed onto the surface of alginate beads. Thus the lower concentrations in metals into alginate beads may prevent both immobilized cells from Cr, Hg, and Pb toxicity and create favorable conditions, that is, Fe deficiency in the microorganisms environment, for siderophore synthesis.

Table I. Adsorption of Cr, Fe, Hg, and Pb on Ca-alginate beads. Cr Equilibrium time (min) Maximal adsorption (%) qe (mg/g)

120 (360) 99.13 1.7 107 (98.39 2.3 104) 0.075 2.9 106 (0.065 9.3 104)

Fe

Hga

90 97.34 1.4 103

>500 (>500) 17.28 1.8 102 (57.32 2.29 104) 3.9 104 3.2 105 (1.2 103 1.1 104)

0.047 1.4 103

Pb 60 (30) 99.90 2.0 104 (99.61 7.3 105) 0.165 1.5 103 (0.153 2.8 103)

Experiments were performed with the mixture of metals (in brackets, without Fe). Results show mean standard deviation. a Equilibrium was not reached at the end of the experiment, that is, 500 min.


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Siderophore Production by Free and Immobilized Cells of P. Aeruginosa and P. Fluorescens Cell Growth by Free and Immobilized Cells In the main, cell growth rates along with maximum biomass were low because of the poorness in nutrients of the minimal medium. Indeed the purpose of this study was to be close to the soil conditions as much as possible. Tables II and III show the growth of P. aeruginosa and P. fluorescens, respectively, cultivated as free and immobilized cells. Free cell growth started immediately whatever the bacterium, the medium, and the inoculum size might have been, except for TM-containing medium cultivated with the low inoculum size of P. aeruginosa which showed a 3-day lag phase. P. aeruginosa showed systematically the highest maximum biomass over P. fluorescens which ranged from a factor of 1.4 to 16.5. The cell concentration of P. aeruginosa reached at the maximum 5.3 108 CFU/mL and only 3.0 108 CFU/mL with P. fluorescens while maximum growth rate (mmax) of P. aeruginosa cultivated with TMcontaining media was in general higher than with P. fluorescens. mmax was quite similarly cultivated with TMfree media. In this latter case, however, we concluded that one or several nutrients in the minimal medium became limiting for P. fluorescens in the course of incubation since maximum biomass was 1.4–2.1 lower than that of P. aeruginosa. The cell growth was slightly stimulated by Fe, mainly with the low inoculum size irrespective of TM in the medium. The growth experiments showed that free cells of P. aeruginosa were not sensitive to TM toxicity except for the low inoculum size (maximal decrease of mmax and maximum biomass in a factor of 1.5 and 2.7, respectively). Conversely P. fluorescens was sensitive to TM whatever the conditions might have been. Maximum biomass decreased 2.0–0.7 times. We can notice that the sensitivity of the two bacteria to TM was lower with the high inoculum size. It especially concerned P. fluorescens with a decrease of the maximum biomass in a factor 4.4–10.7 with the low inoculum size which was only 2.0–2.6 with the high inoculum one. Toxicity depends on their amount of TM in contact with each microorganism. The difference in cell concentration between the low and the high inoculum size—in a factor 1,000 at the beginning of the incubation and 1–4.5 when maximum biomass was reached—most probably explains the results mentioned above. As for free cells, immobilized P. aeruginosa (Table II) showed a higher mmax and maximum biomass than P. fluorescens and a lag phase was observed with same modalities. Thus alginate beads did not protect the cells from TM toxicity although all Cr and Pb were adsorbed on alginate beads and consequently that they should be less bioavailable. However, at the maximum 57.4% of Hg only was adsorbed onto beads which may explain that immobilized cells also suffered from TM toxicity. Moreover, immobilized microorganisms most often multiply in the

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border of beads because of the non-limiting O2 rate at their surface (Laca et al., 2000, Monbouquette and Ollis, 1988). Thus, the close contact between TM and microorganisms may also explain toxicity. By considering the maximum biomass, immobilized cells of the two bacteria were proportionally more sensitive than free cells to TM toxicity in case of the low inoculum size. It was the contrary with the high inoculum size. In the former case the diffusion resistance of beads to TM most probably increased and hence metals diffused only in the vicinity of beads without any toxicity towards all immobilized cells. Thus, a pronounced gradient in TM concentration most probably occurred within beads and consequently TM concentration in the core of beads was lower than that in medium. It was in accordance with several authors who showed that mass transfer limitation increased with cell concentration immobilized in matrix (Barranco-Florido et al., 2001; Lebeau et al., 1998a; Vilchez and Vega, 1995). Concerning TM diffusion in non-inoculated beads, opposite conclusions stood out. Although Ibań˜ez and Umetsu (2004) showed a uniform distribution of Cr(III) throughout alginate beads. Conversely Pandley et al. (2002) who measured the percentage of metal adsorbed on beads and retained in beads showed that metals were mainly adsorbed onto beads. Indeed the calculated ratios ranged from 3.6 with Fe to 7.8 with Mn (5.9 for Cr). The diffusion of Cu in Ca-alginate beads also showed that a gradient in Cu concentration appeared since the diffusion coefficient of Cu in beads was 25% of that in the solution (Potter and McFarland, 1996). Immobilization did not prevent any release of cells in the medium, as already shown by several authors for various applications (Cachon et al., 1995; Lebeau et al., 1998b; Leung et al., 2000). In the main, cell release occurred immediately after the beads were incubated in the minimal medium or after a delay of 2 days for experiments which were performed with the low inoculum size of P. aeruginosa. mmax, which included both cells released from Ca-alginate beads and their multiplication in the liquid minimal medium, was independent of the presence of Fe and/or TM. Thus, the adsorption of TM onto beads restricted their toxicity towards released ones. As for free cells, highest biomass was reached with Fe-containing medium. TM had no adverse effect on the maximum biomass and had even a positive effect which could be the result of a modification in the mineral equilibrium of the medium as a consequence of the adsorption phenomenon mentioned above. Siderophore Production by Free and Immobilized Cells Tables II and III show siderophore production with free and immobilized cells of the two bacteria. For all the modalities, that is, the culture medium without any Fe nor TM or with Fe and/or TM, free cells of P. aeruginosa (Table II) exhibited a higher siderophore production than P. fluorescens, which reached a factor 10 for the siderophore concentration, 15.8 for the volumetric productivity, and 7 for the specific


Braud et al.: Siderophore Production by Using Free and Immobilized Cells of Pseudomonads

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2.4 e 0.12 15.3 d 1.25 (1.2 e 0.11) 5.2 e 0.07 17.7 d 2.26 (4.7 b 0.25) 1.4 e 0.05 0.6 e 0.28 (2.8 d 0.41) 3.8 e 0.16 1.9 e 0.54 (3.8 c 0.25) 4.0 e 0.15 22.0 c 2.54 (1.6 e 0.25) 5.2 e 0.06 28.8 a 5.16 (5.6 a 0.63) 3.2 e 0.03 23.7 bc 0.8 (3.1 d 0.52) 5.3 e 0.25 26.7 ab 1.62 (4.6 b 0.62)

0.46 c 0.005 0.49 bc 0.005 (1.08 b 0.015) 0.54 a 0.001 0.50 b 0.008 (1.05 c 0.008) 0.38 d 0.012 0.14 f 0.054 (1.08 b 0.006) 0.36 de 0.032 0.34 e 0.017 (1.06 bc 0.01)

0.12 f 0.002 0.14 f 0.007 (1.04 c 0.02) 0.14 f 0.001 0.15 f 0.011 (1.13 a 0.02) 0.11 f 0.001 0.14 f 0.015 (1.14 a 0.001) 0.14 f 0.003 0.15 f 0.004 (1.14 a 0.023)

Maximum biomass (108 CFU/mL)a

0.93 a 0.01 0.12 f 0.06 0.41 cde 0.005 0.37 de 0.14 0.95 a 0.003 0.25 e 0.08 0.53 bcd 0.06 0.57 bc 0.02

0.97 a 0.01 0.10 f 0.05 0.39 de 0.01 0.40 de 0.03 0.90 a 0.01 0.60 b 0.02 0.44 cd 0.2 0.45 cd 0.06

Maximum concentration (OD630 nm)

0.93 a 0.01 (0.92 a 0.01) 0.03 g 0.01 (0.07 g 0.02) 0.21 c 0.003 (0.19 f 0.06) 0.04 g 0.01 (0.20 f 0.04) 0.95 a 0.003 (0.95 a 0.003) 0.25 c 0.08 (0.25 ef 0.08) 0.52 b 0.06 (0.53 c 0.06) 0.57 b 0.02 (0.57 c 0.02)

0.14 de 0.001 (0.66 b 0.13) 0.02 g 0.01 (0.06 g 0.05) 0.19 cd 0.007 (0.26 def 0.04) 0.13 de 0.009 (0.17 f 0.05) 0.15 de 0.001 (0.38 d 0.02) 0.10 ef 0.004 (0.30 def 0.01) 0.07 fg 0.03 (0.33 de 0.04) 0.23 c 0.03 (0.33 de 0.009)

Volumetric productivity (OD630 nm/day)b

0.88 c 0.15 0.40 c 0.10 0.13 c 0.005 0.19 c 0.07 0.46 c 0.07 0.50 c 0.28 0.20 c 0.06 0.43 c 0.19

3.91 a 1.14 0.17 c 0.09 0.21 c 0.04 0.23 c 0.06 1.79 b 0.13 0.37 c 0.19 1.88 b 0.75 1.57 b 0.17

Maximum specific production [OD630 nm/(108 CFU/mL)]

Cultures were batch incubated in minimal medium with or without toxic metals (TM) and/or iron (Fe). Results show mean standard deviation and homogenous groups are designated by letters (a, b, c, etc.). FC, free cells; IC, immobilized cells; TM, toxic metals (mixture of Cr, Hg, and Pb). a For IC, the values corresponded to cell growth in beads. In brackets, to cell leakage which included both cells released from the Ca-alginate beads and their multiplication in the liquid minimal medium. b Average and maximun (in brackets) values are given.

Low inoculum size FC IC Fe-FC Fe-IC TM-FC TM-IC Fe-TM-FC Fe-TM-IC High inoculum size FC IC Fe-FC Fe-IC TM-FC TM-IC Fe-TM-FC Fe-TM-IC

mmax (per h)a

Table II. Growth and siderophore production (concentration, volumetric productivity, and specific production) by free and immobilized cells of P. aeruginosa.

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DOI 10.1002/bit

1.39 e 0.29 4.38 bc 0.31 (0.46 c 0.02) 2.46 d 0.06 6.75 a 1.03 (1.56 b 0.65) 0.31 f 0.04 0.03 f 0.01 (0.50 c 0.21) 0.23 f 0.006 0.03 f 0.05 (0.34 c 0.01) 2.93 d 0.03 6.73 a 0.56 (0.44 c 0.06) 2.99 d 0.04 6.39 a 0.40 (2.10 ab 0.69) 1.11 e 0.20 4.62 b 0.32 (1.43 b 0.08) 1.51 e 0.26 3.85 c 0.32 (2.36 a 0.14)

0.46 ab 0.013 0.39 cd 0.004 (1.02 a 0.008) 0.49 a 0.002 0.41 bc 0.009 (1.06 a 0.017) 0.34 d 0.011 0.09 ef 0.003 (1.06 a 0.001) 0.35 cd 0.002 0.06 ef 0.01 (1.04 a 0.003)

0.13 e 0.001 0.06 ef 0.005 (1.04 a 0.013) 0.13 e 0.001 0.06 ef 0.004 (1.11 a 0.008) 0.06 ef 0.004 0.03 f 0.011 (1.09 a 0.007) 0.06 ef 0.003 0.02 f 0.003 (1.07 a 0.008)

Maximum biomass (108 CFU/mL)a

0.10 e 0.05 0.20 de 0.03 0.06 e 0.03 0.12 e 0.04 0.11 e 0.12 0.29 bcd 0.05 0.51 a 0.02 0.38 abc 0.06

0.40 ab 0.06 0.19 de 0.03 0.06 e 0.07 0.10 e 0.01 0.09 e 0.03 0.13 e 0.11 0.24 cde 0.07 0.35 bcd 0.10

Maximum concentration (OD630 nm)

0.02 e 0.01 (0.06 de 0.04) 0.10 c 0.01 (0.13 bcd 0.02) 0.02 e 0.01 (0.04 de 0.03) 0.04 e 0.01 (0.06 de 0.02) 0.02 e 0.02 (0.06 de 0.04) 0.15 b 0.03 (0.16 abc 0.03) 0.08 cd 0.004 (0.13 bcd 0.03) 0.06 de 0.01 (0.23 a 0.05)

0.20 a 0.03 (0.20 ab 0.02) 0.10 c 0.01 (0.15 abc 0.006) 0.02 e 0.01 (0.03 e 0.03) 0.03 e 0.005 (0.07 cde 0.01) 0.03 e 0.01 (0.06 de 0.04) 0.02 e 0.02 (0.06 de 0.05) 0.04 e 0.01 (0.13 bcd 0.05) 0.06 de 0.02 (0.17 ab 0.06)

Volumetric productivity (OD630 nm/day)b

0.08 b 0.04 0.58 b 0.18 0.03 b 0.01 0.08 b 0.04 0.13 b 0.05 0.27 b 0.04 0.49 b 0.24 0.21 b 0.04

0.42 b 0.07 0.56 b 0.09 0.03 b 0.01 0.09 b 0.01 0.41 b 0.15 0.32 b 0.25 1.97 a 0.88 1.59 a 0.55

Maximum specific production [OD630 nm/(108 CFU/mL)]

Cultures were batch incubated in minimal medium with or without toxic metals (TM) and/or iron (Fe). Results show mean standard deviation and homogenous groups are designated by letters (a, b, c, etc.). FC, free cells; IC, immobilized cells; TM, toxic metals (mixture of Cr, Hg and Pb). a For IC, the values corresponded to cell growth in beads. In brackets, to cell leakage which included both cells released from the Ca-alginate beads and their multiplication in the liquid minimal medium. b Average and maximun (in brackets) values are given.

Low inoculum size FC IC Fe-FC Fe-IC TM-FC TM-IC Fe-TM-FC Fe-TM-IC High inoculum size FC IC Fe-FC Fe-IC TM-FC TM-IC Fe-TM-FC Fe-TM-IC

mmax (per h)a

Table III. Growth and siderophore production (concentration, volumetric productivity, and specific production) by free and immobilized cells of P. fluorescens.

Table IV. Comparison of the siderophore production by free and immobilized cells of P. aeruginosa and P. fluorescens in the minimal medium by measuring the specific production of immobilized cells to their free counterpart ratio. Low inoculum size Fe P. aeruginosa 0.05 f 0.03 1.11 def 0.12 P. fluorescens 1.39 def 0.49 1.72 de 0.09

TM

High inoculum size Fe-TM

0.21 f 0.10 0.94 def 0.41 0.70 def 0.42 0.87 def 0.44

0.47 ef 0.18 4.34 a 0.68

Fe

TM

Fe-TM

1.49 def 0.60 2.87 bc 0.18

1.01 def 0.01 3.69 ab 0.90

2.11 cd 0.43 0.46 ef 0.12

Cultures were batch incubated. Results show mean standard deviation and homogenous groups are designated by letters (a, b, c, etc.). TM, toxic metals (mixture of Cr, Hg and Pb).

production (volumetric productivity to the biomass ratio). The specific production of P. fluorescens (Table III) was higher only for the medium enriched with Fe and TM. As already shown by Page (1993), the enrichment of the culture medium with iron reduced significantly the siderophore concentration of free cells of P. aeruginosa. The maximum siderophore concentration decreased at the maximum in a factor 2.5 and 2.0 with the medium enriched or not with TM, respectively. Same conclusion was given with P. fluorescens by considering only the TM-free medium. Conversely, siderophore synthesis was stimulated in the presence of Fe combined with TM in medium. We suggested that TM toxicity was partially offset in the presence of iron in case of the low inoculum size, and that it even stimulated siderophore production (high inoculum size). By considering the specific production which gives some global information about cellular metabolism, trend was quite the same. The maximum decrease with Fe-containing medium was in a factor of 18.6 for P. aeruginosa. Immobilization of bacterial cells significantly modified siderophore production. The differences in siderophore production by the two immobilized bacteria were not as much high as for cells cultivated in a free mode. They did not exceed a factor 4.6, 9.5, and 2.4 for the maximum concentration, the volumetric and the specific production, respectively. In some cases, (Fe-free medium), P. fluorescens even exhibited higher siderophore production. By comparison with free cells, siderophore production by immobilized cells of P. aeruginosa was generally lower (up to a factor 9.7). In some cases, the contrary was observed with P. fluorescens (up to a factor 2.6). By comparing Fe-containing medium with Fe-free one, immobilized cells unlike their free counterparts led only to a low decrease and even to an increase in siderophore production. For free cells, the decrease reached at the maximum a factor of 6.7, 10.0, and 18.6 the concentration, the volumetric and the specific production, respectively, while it did not exceed a factor 1.9, 3.3, and 7.2 with immobilized cells. As above set out, the combination of metal adsorption onto Ca-alginate matrix of beads along with the limited diffusion of metals inside beads most probably modified the environment in the vicinity of immobilized cells. Concentrations gradient appeared with an impoverishment in metals when we go away from beads surface, as already discussed above. Besides the positive effect on siderophores due to the lower availability of iron for bacterial cells, microbial toxicity of

TM was then expected to be lower although some authors demonstrated a positive effect of Cd, Cu, and Zn on siderophore synthesis which act as stress factors (Dao et al., 1999; Sharma and Johri, 2003a). Siderophore-specific production of immobilized cells to their free counterparts ratio is given in Table IV. By comparison with free cells, immobilization of P. aeruginosa strongly reduced the negative effect of Fe and/or TM on siderophore synthesis. Additionally ratios were higher than one which means that specific production of immobilized cells was higher than for free cells, except for the low inoculum cultivated with the medium enriched with TM and Fe-TM. Positive effect of immobilization on the specific productivity was less evident with P. fluorescens. CONCLUSIONS Immobilization allowed both to protect cells against TM toxicity and above all to minimize all inhibition of siderophore synthesis in Fe-containing medium. Immobilization of P. fluorescens even stimulated siderophore production. This gives us hope for a steady siderophore production in soil, in spite of continuous physico-chemical changes of soil conditions. Since it was also demonstrated the positive function of alginate beads in immobilized cells survival (Cassidy et al., 1996; Leung et al., 2000; McLoughlin, 1994), this culture technique seems to be appropriate for bioremediation processes, especially bioaugmentation, to improve TM solubilization in soils thanks to microbial siderophore synthesis coupled together with phytoextraction. However, an appropriate formulation of microbial inocula requires further investigation. Indeed survival and consequently siderophore production can most likely be enhanced thanks to the supply of carbon substrates in the immobilization matrix (Duquenne et al., 1999), which could at the same time selectively promote immobilized cell growth and not indigenous microbial population in soil.

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