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Improvement of Escherichia coli growth by kaolinite
Applied Clay Science 44 (2009) 67–70
Contents lists available at ScienceDirect
Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l a y
Improvement of Escherichia coli growth by kaolinite Elise Courvoisier, Sam Dukan ⁎ Laboratoire de Chimie Bactérienne, UPR 9043, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France
a r t i c l e
i n f o
Article history: Received 16 July 2008 Received in revised form 15 January 2009 Accepted 22 January 2009 Available online 29 January 2009 Keywords: Kaolinite Escherichia coli Growth parameters Catabolic activity Acetate assimilation
a b s t r a c t Knowledge of the impacts of clay minerals on microorganisms is essential to a complete understanding of microbially-mediated processes. Information available in this regard remains scarce. Using Escherichia coli (E. coli) as a model bacterium, we investigated the effect of kaolinite on various growth parameters. This clay mineral significantly affected maximal growth rate and yield of the E. coli-strain (MG 1655) in minimum growth medium with 0.2% glucose, at an optimal concentration of 0.2 to 0.5 g/l. These physiological modifications were related to a decrease in catabolic activity and increased acetate assimilation via an energy transfer from acetate degradation to cell division rather than maintenance. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Clay minerals are abundant and ubiquitous in the natural environment. They have been reported to enhance the biodegradation by microorganisms of a variety of substances. Montmorillonite alone or with kaolinite improved the mineralization of organic pollutants, phenanthrene or heavy-oil, in a pure culture of Pseudomonas bacterial strains (Ortega-Calvo and Saiz-Jimenez, 1998; Chaerun et al., 2005). Clay minerals have also been claimed to have a positive impact on the growth and the metabolic activity of a variety of microorganisms ranging from bacteria to fungi. Stotzky and Rem (1966) reported that the growth of Achromobacter sp., Agrobacterium radiobacter, Bacillus subtilis, Bacillus megaterium, Escherichia coli (E. coli), Escherichia intermedia, Proteus vulgaris, Pseudomonas striata and Pseudomonas aeruginosa was stimulated by both montmorillonite and kaolinite. Filip (1967) reported beneficial effects of bentonite on the growth of soil microflora in liquid minimum medium and Novakova (1968) observed that bentonite shortened the lag phase of E. coli. The latest study reported the stimulatory effect of kaolinite and montmorillonite on the exponential growth of Bacillus thuringiensis in rich medium (Rong et al., 2007). Hypotheses have been formulated to explain these observations. One of these is that clay minerals trap metabolic inhibitors by adsorption (Martin et al., 1976). Also, clay minerals may have an effect on the growth rate and respiration by buffering the pH of the suspension at levels adequate to sustain growth (Stotzky, 1966; Stotzky and Rem, 1966), by being an important electron acceptor thus
⁎ Corresponding author. Tel.: +33 491 164 601; fax: +33 491 718 914. E-mail address: [email protected] (S. Dukan). 0169-1317/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2009.01.010
supporting bacterial growth (Kostka et al., 2002), or by acting as a microbial growth-support material (Mark van Loosdrecht et al., 1990; Chaerun et al., 2005; Rong et al., 2007). On the other hand, the presence of clay minerals has been shown to inhibit growth, microbial activity and sporulation (Novakova, 1968; Rong et al., 2007). A reduction of the transmembrane movement of nutrients, waste products and gas caused by the adhesion of clay minerals (Lavie and Stotzky, 1986) as well as an inhibitory effect of compounds such as Al3+ found within clay minerals on the bacterium (Wong et al., 2004) has been proposed. Common in all hypotheses is that clay minerals are surface-active particles, but no real mechanism of action has been described until now. In the present work we investigated the influence of kaolinite on the growth parameters and metabolic activity of a model bacterium, E. coli. Results of this study enable us to propose possible mechanisms by which clay minerals act on microorganisms.
2. Materials and methods 2.1. Clay mineral and suspension 2.1.1. Kaolinite Kaolinite was provided by EPARCO Assainissement (Paris, France) from bassin de Provins (France) and is composed of (%) SiO2 49.8, Al2O3 29.2, MgO 1.7, CaO 1.5, TiO2 1.0, Fe 0.9, K2O 0.8, Na2O 0.3 and (mg/kg) Zn 1204.7, Cu 537.5, P 230.6, Se 56.1, Mn 28.5, Ni 20.0, Co 3.3, Mo 2.1; cation exchange capacity 14 meq/100 g (analysis performed by CIRAD – Montpellier, France). Kaolinite was dry-sterilized for one hour in microtubes at 160 °C.
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E. Courvoisier, S. Dukan / Applied Clay Science 44 (2009) 67–70
2.1.2. Clay mineral suspension Clay mineral (0.025 g of the sterile powder, final concentration 0.5 g/l) was added to 50 ml of liquid minimum medium M9 with 0.2% glucose at pH 7 (Davis, 1980) and incubated for 2 h at 37 °C on a rotary shaker at 160 rpm. The larger particles of the kaolinite were harvested by centrifugation at 5500 ×g for 15 min at 4 °C. The supernatant, composed of the colloidal fraction, and the pellet were stored at 4 °C or frozen before used as culture medium or added to a fresh medium.
minimum medium M9. After an incubation of 2 h at 37 °C on a rotary shaker at 160 rpm, the larger particles were harvested by centrifugation at 3000 ×g for 2 min. The free glucose concentration was measured in the supernatant. These twelve values enabled us to determine a mean quantity of glucose adsorbed per gram of kaolinite (g/g). The same study was performed with 0.5, 1 and 2 g/l of acetate.
2.2. Bacterial strain and culture conditions
Aliquots of 1 ml were fixed with 1 ml formaldehyde 4%. The tubes were mixed for 30 s and left to stand for one hour at 4 °C. Bacteria were then collected by centrifugation at 5500 ×g for 15 min at 20 °C, washed twice in the same volume of PBS and resuspended in PBS at 109 cells per ml. Fixed sample volumes of 2 µl were examined using phase contrast microscopy (Axio Imager – ZEISS) with an oil immersion objective (100× oil universal objective). Mean cell size was measured by Image J, examining on average 1000 cells per sample. Statistical analyses were performed at P = 1‰ with a student's t test.
E. coli MG1655 (K-12 wild type strain) was cultured at 37 °C on a rotary shaker at 160 rpm in M9 with 0.2% glucose. E. coli overnight culture was diluted 100-fold in 50 ml of the same medium (initial cell concentration 5–107 cfu/ml) equilibrated at 37 °C for 30 min. Growth was monitored by measuring absorbance at 600 nm with a spectrophotometer (Biochrom LibraS22) or by plating cells on Luria-BertaniAgar followed by serial dilutions in phosphate buffer (PBS: 0.05 M, pH 7, 4 °C). Colonies were counted after incubating for 24 h at 37 °C and expressed as colony forming units (CFU) per milliliter representing the mean of triplicate measurements.
2.6. Bacterial size
3. Results 3.1. Kaolinite influence on growth parameters
2.3. Growth parameters 2.3.1. Biomass (X, g/l) After an incubation of 24 h, the culture was harvested by centrifugation at 5500 ×g for 15 min at 4 °C. The cell pellet was washed twice with cold PBS and then transferred into a preweighed aluminium cupel. Cells were dried at 120 °C for 4 h before being cooled and weighed. Centrifugation supernatant was collected to quantify glucose and acetate content as described below. 2.3.2. Maximal growth rate Maximal growth rate (µmax, h− 1) was determined graphically as the growth curve slope, plotted by monitoring cell growth either in CFU or in absorbance using a logarithmic scale. These two methods led to similar results. Nevertheless, µmax determination was only realizable up to 1 g/l of kaolinite at 600 nm. 2.3.3. Molecular growth yield Molecular growth yield (YGlu, g/mol) was defined as the mass of cells produced with one mole of glucose: YGlu =X/glucose consumed (mol/l).
With dispersions containing 0–4 g kaolinite per liter, the pH of the medium remained initially constant at 7.00±0.05. No significant adsorption of glucose was observed, with 96 to 98% of glucose remaining in solution after centrifugation. About 0.014 g of glucose per gram of kaolinite were adsorbed at 37 °C. Using phase contrast microscopy, we observed that the bacteria were mainly localized around clay particles suggesting interaction between clay particles and bacteria (data not shown). The responses were dose-dependent with optimal effects between 0.2 and 0.5 g kaolinite/liter followed by a decline suggesting a toxic effect of kaolinite at high concentrations (Table 1). Growth rate, absorbance and cell concentration reached maxima respectively 1.6 fold, 3 fold and 3.5 fold higher than control. The lag time suggest an initial physiological adaptation of bacteria in these conditions. Nevertheless, an enhanced bacterial growth overcame this negative impact on the cells. Consequently, kaolinite provokes an increase in maximal growth rate, maximal absorbance and final E. coli cell concentration in minimum growth medium. 3.2. Influence of kaolinite nutritional contribution and cell size variation
2.3.4. Catabolic activity Catabolic activity (qGlu, mmol g− 1 h− 1) was defined as the degradation rate of one mole of glucose per gram of cells in one hour: qGlu = µmax/YGlu.
An apparent rise in absorbance and growth rate that could easily be explained by a kaolinite nutritional contribution, led to the hypothesis that kaolinite addition leads to medium enrichment through a readily assimilated carbon–nitrogen and/or phosphate
2.4. Glucose and acetate quantification 2.4.1. Glucose quantification (g/liter) According to the method of Trinder (1969), glucose concentration is proportional to the formation of a red quinoneimine, detected at 500 nm within the range 0.05–2 g/l (±0.01) (Glucose GOD-PAP, Biolabo, France). 2.4.2. Acetate quantification (g/l) According to Bergmeyer and Mollering (1974), acetate concentration is proportional to the formation of NADH and detected at 340 nm within the range 0.025–1.2 g/l (±0.005) (Acide acétique enzymatique/ UV KHPE036058, Seppal, France). 2.5. Glucose and acetate sorption to kaolinite Increasing kaolinite concentrations (0.5, 1, 2 and 4 g/l) were contacted with increasing glucose concentrations (1, 2 and 4 g/l) in
Table 1 E. coli growth parameters in the presence of increasing kaolinite concentrations. Kaolinite concn Maximal growth rate Maximal (g/l) (h− 1) A600 nm
Final CFU × 109 per ml Cell size (µm)b
0 0.05 0.1 0.2 0.3 0.5 1 2 4
1.0 ± 0.1 1.7 ± 0.1 3.0 ± 0.1 3.3 ± 0.2 3.6 ± 0.4 3.5 ± 0.4 3.4 ± 0.1 2.3 ± 0.1 1.4 ± 0.2
a
0.74 ± 0.03 0.87 ± 0.01 1.21 ± 0.07 1.23 ± 0.03 1.21 ± 0.02 1.16 ± 0.05 1.07 ± 0.01 NDa NDa
0.83 ± 0.03 1.28 ± 0.18 2.01 ± 0.06 2.24 ± 0.10 2.33 ± 0.08 2.54 ± 0.10 2.35 ± 0.10 1.51 ± 0.15 1.33 ± 0.12
2.04 2.05 2.03 2.07 2.05 1.91 1.65 NDa NDa
ND, not determined. Aliquots of culture sample were taken for each growth condition after 24 h. According to t test (P = 1‰), E. coli mean cell size in control culture was highly significantly different from that acquired in the presence of a kaolinite concentration higher to 0.5 g/l. b
E. Courvoisier, S. Dukan / Applied Clay Science 44 (2009) 67–70
source. Table 2 presents the final cell concentration of E. coli following 24 h cultivation M9 medium lacking a carbon, nitrogen or phosphate source. The addition of the kaolinite showed no evidence of enriching the medium, and we even observed a cell growth inversely proportional to kaolinite concentration with phosphate and nitrogen starvation. In these conditions, the kaolinite may have adsorbed all phosphate and ammonia thus rendering the medium even more starved of these two compounds, thus explaining these phenomena. Phase contrast images of 24-hour culture samples and measured bacterial size at each kaolinite concentration tested (0–1 g/l) revealed a statistically significant decrease in bacterial size at kaolinite contents upper than 0.6 g/l. However, a maximal decrease of 20% in the size of bacteria could not explain the 3.5 fold rise in cell number, indicating that variations in cell size did not account for the observed increase. Consequently, these modifications were neither caused by a kaolinite nutritional contribution nor by a decrease in cell size. 3.3. Kaolinite suspension To investigate whether the kaolinite effect is due to colloidal particles and/or larger setting particles we separated the two phases by centrifugation at an optimal kaolinite concentration (0.5 g/l). As demonstrated in Fig. 1, the colloidal particles did not show any effect whereas the larger particles revealed the same trend as the unfractionated kaolinite. Chemical analysis of the stable colloidal fraction revealed that the mineral elements found in kaolinite predominated in the larger particles (data not shown). Thus, the kaolinite effect arises from the larger particles. 3.4. Assimilation and degradation of glucose and acetate
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Fig. 1. E. coli MG1655 growth curve (i) with 0.5 g/l of kaolinite rinsed once in M9 medium with 0.2% glucose (Δ) or (ii) not rinsed (●), (iii) in the rinse medium containing the stable colloidal particles (▲) and (iv) in M9 medium with 0.2% glucose (○). Growth was followed using A600 nm measurements. Experiments were repeated at least three times and the standard deviation was always below 10%.
However, part of the energy outcome from acetate degradation due to anabolism may explain the observed increase in growth yield. 3.5. Kaolinite influence on catabolic activity We tested the hypothesis that kaolinite influences glucose assimilation and determined final E. coli biomass at several kaolinite concentrations and calculated the growth yield. Biomass and molecular growth yield increased to a maximum with at least 0.3 g/l of kaolinite (Table 3) and also revealed a close relationship between
We have demonstrated that the larger particles of kaolinite were responsible for increased bacterial growth altough the dispersion did not contain assimilated nutrients. One way of understanding this phenomenon is to consider a differing assimilation and/or degradation of glucose and/or acetate in the presence or absence of kaolinite. We thus measured the concentration of glucose and its degradation by-product (i.e. acetate) during E. coli growth in the presence or absence (control) of an optimal kaolinite concentration (0.5 g/l). Fig. 2A clearly shows a difference in the evolution of glucose and acetate concentrations between the two conditions. Without kaolinite, total glucose consumption and maximum observed acetate concentration occurred three hours later than with kaolinite. Interestingly, acetate was half-consumed after 24 h (data not shown). To compare the substrate degradation rate for the same bacterial concentration, we plotted the concentration of cells (A600) versus that of glucose or acetate (mg/liter). Fig. 2B reveals a slower glucose consumption rate per unit of A600 with 0.5 g/l of kaolinite. Unlike the control culture, the acetate consumption was linked to absorbance increase while the glucose had already been degraded. With only 0.063 g of acetate per gram of kaolinite (at 37 °C), the adsorption of acetate on kaolinite was not significant and therefore could not explain the increased acetate consumption rate per unit of A600.
Table 2 E. coli cultures limited by a nutrient in the presence of increasing kaolinite additions. Kaolinite concn CFU × 106 per mla (g/l) M9 without glucose M9 without phosphateb M9 without nitrogen 0 0.2 0.5 1 a
17.8 ± 0.8 17.0 ± 0.6 16.9 ± 0.4 18.1 ± 0.8
55.8 ± 2.5 38.8 ± 3.2 13.5 ± 1.4 7.5 ± 0.7
49.7 ± 1.4 32.8 ± 1.9 32.8 ± 2.5 24.3 ± 1.8
CFU in 24 hour culture samples. b The buffer power is maintained by the addition of NaCl and KCl. The cellular density of the overnight culture was 1.5 ± 0.1 × 109 per ml.
Fig. 2. Glucose (○) and acetate (▲) concentrations during E. coli growth in a medium containing 0.5 g/l of kaolinite (– –) or without kaolinite (—). (A) Profile against time, (B) profile against absorbance. The standard deviation, indicated by the error bars, was always below 10%.
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Table 3 E. coli growth parameters in the presence of increasing kaolinite concentrations. Kaolinite Maximal In 24 hour concn Growth Biomass (g/l) rate (g/l) (h− 1) 0 0.05 0.1 0.2 0.3 0.5 1 a b
0.74 ± 0.030.50 ± 0.05 0.87 ± 0.010.61 ± 0.04 1.21 ± 0.070.85 ± 0.02 1.23 ± 0.030.88 ± 0.04 1.21 ± 0.020.93 ± 0.07 1.16 ± 0.050.98 ± 0.09 1.07 ± 0.010.98 ± 0.08
culture samples
Molecular
Catabolic
Avg Avg glucose acetate (g/l) (g/l)
Growth yield (g/mol)
Activityb (mmol g−1 h−1)
b 0.05a b 0.05a b 0.05a b 0.05a b 0.05a b 0.05a b 0.05a
51 ± 5 62 ± 4 86 ± 2 89 ± 4 95 ± 7 99 ± 9 99 ± 8
14.4 ± 0.5 14.0 ± 0.2 14.0 ± 0.8 13.8 ± 0.4 12.9 ± 0.2 11.6 ± 0.5 10.8 ± 0.1
0.24±0.02 0.21±0.02 b 0.025a b 0.025a b 0.025a b 0.025a b 0.025a
Below the detection limit. qGlu (catabolic activity) = μmax (maximal growth rate)/YGlu (molecular growth yield).
this increase in growth yield and a significant decrease in catabolic activity. In light of these results, we propose that kaolinite reduces the glucose degradation rate, associated with increasing maximal growth rate and growth yield. 4. Discussion Under the experimental conditions outlined in this study, we have shown that kaolinite has a significant effect on E. coli maximal growth rate and growth yield. These physiological effects are related to a decrease in catabolic activity and improved acetate assimilation. Previous studies have reported the effects of several clay minerals (kaolinite and other minerals) on biomass production and biodegradation of various organic matters (Ortega-Calvo and Saiz-Jimenez, 1998; Chaerun et al., 2005; Chaerun and Tazaki, 2005). Mechanisms by which several clay minerals act are notably explained either (i) by interaction phenomena with organic molecules (e.g. humic acid (Ortega-Calvo and Saiz-Jimenez, 1998)), inorganic ions or microorganisms, or (ii) through their silicate content leading to C–O–Na–Si complex formation on the surface of bacterial cell walls (e.g. on hydrocarbon-degrading bacteria) (Inagaki et al., 2003; Chaerun et al., 2005). Kaolinites have previously been shown to act as a microbial growth-support material (Chaerun et al., 2005). Adsorption phenomena may result in higher substrate concentrations in the vicinity of the bacterial cells, thereby increasing their bioavailability (Mark van Loosdrecht et al., 1990; Ortega-Calvo and Saiz-Jimenez, 1998; Chaerun and Tazaki, 2005). One recent report suggests that by adsorption, clay minerals could mitigate the toxic effects of compounds highly concentrated in the medium (Chaerun et al., 2005). Even though experiments were performed over a period of 24 h and not several days as in other studies, the present study clearly demonstrates that the adsorption of glucose and acetate on the kaolinite particles (0.014 g and 0.063 g per gram kaolinite) was insignificant and that kaolinite promoted interactions with bacteria. We postulate that kaolinite-dependant effects on E. coli are not directly due to its surface-active properties. It seems more likely that
kaolinite leads to a decrease in catabolic activity and an increase in acetate assimilation by transferring a part of the energy outcome from acetate degradation to cell division rather than cell maintenance. So we can explain the increase in the final cell concentration observed in the presence of kaolinite but not the rise in maximal growth rate. Further experiments are necessary to elucidate the mechanisms involved in this process and the reasons why maximal growth rate is affected by kaolinite concentration. Acknowledgements We thank J. P. Belaïch, Bioénergétique et Ingénierie des Protéines, Marseille, France for helpful comments on the manuscript. We thank EPARCO Assainissement that kindly provided the kaolinite. References Bergmeyer, H.U., Mollering, H., 1974. In: Bergmeyer, H.U. (Ed.), second edition. Methods of Enzymatic Analysis, vol. 3. Academic Press, New York, pp. 1196–1201. Chaerun, S.K., Tazaki, K., 2005. How kaolinite plays an essential role in remediating oilpolluted seawater. Clay Minerals 40, 481–491. Chaerun, S.K., Tazaki, K., Asada, R., Kogure, K., 2005. Interaction between clay minerals and hydrocarbon-utilizing indigenous microorganisms in high concentrations of heavy oil: implications for bioremediation. Clay Minerals 40, 105–114. Davis, R.W., 1980. A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor, NY. 204 pp. Filip, Z., 1967. Effect of small additions of bentonite on the development of some groups of soil microorganisms in liquid culture. Folia Microbiologica 12 (4), 396. Inagaki, F., Motomura, Y., Ogata, S., 2003. Microbial silica deposition in geothermal hot waters. Applied Microbiology and Biotechnology 60, 605–611. Kostka, J.E., Dalton, D.D., Skelton, H., Dollhopf, S., Stucki, J.W., 2002. Growth of iron(III)reducing bacteria on clay minerals as the sole electron acceptor and comparison of growth yields on a variety of oxidized iron forms. Applied and Environmental Microbiology 68 (12), 6256–6262. Lavie, S., Stotzky, G., 1986. Adhesion of the clay minerals montmorillonite, kaolinite, and attapulgite reduces respiration of histoplasma capsulatum. Applied and Environmental Microbiology 51 (1), 61–73. Mark van Loosdrecht, C.M., Lyklema, J., Norde, W., Zehnder, A.J.B., 1990. Influence of interfaces on microbial activity. Microbiological Reviews 54 (1), 75–87. Martin, J.P., Filip, Z., Haider, K., 1976. Effect of montmorillonite and humate on growth and metabolic activity of some actinomycetes. Soil Biology and Biochemistry 8, 409–413. Novakova, J., 1968. Effect of bentonite and kaolinite on the growth curve of E. coli. Folia Microbiologica 13 (6), 543. Ortega-Calvo, J.J., Saiz-Jimenez, C., 1998. Effect of humic fractions and clay on biodegradation of phenanthrene by a Pseudomonas fluorescens strain isolated from soil. Applied Environmental Microbiology 64 (8), 3123–3126. Rong, X., Huang, Q., Chen, W., 2007. Microcalorimetric investigation on the metabolic activity of Bacillus thuringiensis as influenced by kaolinite, montmorillonite and goethite. Applied Clay Science 38, 97–103. Stotzky, G., 1966. Influence of clay minerals on microorganisms. II. Effect of various clay species, homoionic clays, and other particles on bacteria. Canadian Journal of Microbiology 12, 831–848. Stotzky, G., Rem, L.T., 1966. Influence of clay minerals on microorganisms. I. Montmorillonite and kaolinite on bacteria. Canadian Journal of Microbiology 12, 547–563. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6, 24–28. Wong, D., Suflita, J.M., McKinley, J.P., Krumholz, L.R., 2004. Impact of clay minerals on sulfate-reducinf activity in aquifers. Microbial Ecology 47, 80–86.
Contents lists available at ScienceDirect
Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l a y
Improvement of Escherichia coli growth by kaolinite Elise Courvoisier, Sam Dukan ⁎ Laboratoire de Chimie Bactérienne, UPR 9043, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France
a r t i c l e
i n f o
Article history: Received 16 July 2008 Received in revised form 15 January 2009 Accepted 22 January 2009 Available online 29 January 2009 Keywords: Kaolinite Escherichia coli Growth parameters Catabolic activity Acetate assimilation
a b s t r a c t Knowledge of the impacts of clay minerals on microorganisms is essential to a complete understanding of microbially-mediated processes. Information available in this regard remains scarce. Using Escherichia coli (E. coli) as a model bacterium, we investigated the effect of kaolinite on various growth parameters. This clay mineral significantly affected maximal growth rate and yield of the E. coli-strain (MG 1655) in minimum growth medium with 0.2% glucose, at an optimal concentration of 0.2 to 0.5 g/l. These physiological modifications were related to a decrease in catabolic activity and increased acetate assimilation via an energy transfer from acetate degradation to cell division rather than maintenance. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Clay minerals are abundant and ubiquitous in the natural environment. They have been reported to enhance the biodegradation by microorganisms of a variety of substances. Montmorillonite alone or with kaolinite improved the mineralization of organic pollutants, phenanthrene or heavy-oil, in a pure culture of Pseudomonas bacterial strains (Ortega-Calvo and Saiz-Jimenez, 1998; Chaerun et al., 2005). Clay minerals have also been claimed to have a positive impact on the growth and the metabolic activity of a variety of microorganisms ranging from bacteria to fungi. Stotzky and Rem (1966) reported that the growth of Achromobacter sp., Agrobacterium radiobacter, Bacillus subtilis, Bacillus megaterium, Escherichia coli (E. coli), Escherichia intermedia, Proteus vulgaris, Pseudomonas striata and Pseudomonas aeruginosa was stimulated by both montmorillonite and kaolinite. Filip (1967) reported beneficial effects of bentonite on the growth of soil microflora in liquid minimum medium and Novakova (1968) observed that bentonite shortened the lag phase of E. coli. The latest study reported the stimulatory effect of kaolinite and montmorillonite on the exponential growth of Bacillus thuringiensis in rich medium (Rong et al., 2007). Hypotheses have been formulated to explain these observations. One of these is that clay minerals trap metabolic inhibitors by adsorption (Martin et al., 1976). Also, clay minerals may have an effect on the growth rate and respiration by buffering the pH of the suspension at levels adequate to sustain growth (Stotzky, 1966; Stotzky and Rem, 1966), by being an important electron acceptor thus
⁎ Corresponding author. Tel.: +33 491 164 601; fax: +33 491 718 914. E-mail address: [email protected] (S. Dukan). 0169-1317/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2009.01.010
supporting bacterial growth (Kostka et al., 2002), or by acting as a microbial growth-support material (Mark van Loosdrecht et al., 1990; Chaerun et al., 2005; Rong et al., 2007). On the other hand, the presence of clay minerals has been shown to inhibit growth, microbial activity and sporulation (Novakova, 1968; Rong et al., 2007). A reduction of the transmembrane movement of nutrients, waste products and gas caused by the adhesion of clay minerals (Lavie and Stotzky, 1986) as well as an inhibitory effect of compounds such as Al3+ found within clay minerals on the bacterium (Wong et al., 2004) has been proposed. Common in all hypotheses is that clay minerals are surface-active particles, but no real mechanism of action has been described until now. In the present work we investigated the influence of kaolinite on the growth parameters and metabolic activity of a model bacterium, E. coli. Results of this study enable us to propose possible mechanisms by which clay minerals act on microorganisms.
2. Materials and methods 2.1. Clay mineral and suspension 2.1.1. Kaolinite Kaolinite was provided by EPARCO Assainissement (Paris, France) from bassin de Provins (France) and is composed of (%) SiO2 49.8, Al2O3 29.2, MgO 1.7, CaO 1.5, TiO2 1.0, Fe 0.9, K2O 0.8, Na2O 0.3 and (mg/kg) Zn 1204.7, Cu 537.5, P 230.6, Se 56.1, Mn 28.5, Ni 20.0, Co 3.3, Mo 2.1; cation exchange capacity 14 meq/100 g (analysis performed by CIRAD – Montpellier, France). Kaolinite was dry-sterilized for one hour in microtubes at 160 °C.
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E. Courvoisier, S. Dukan / Applied Clay Science 44 (2009) 67–70
2.1.2. Clay mineral suspension Clay mineral (0.025 g of the sterile powder, final concentration 0.5 g/l) was added to 50 ml of liquid minimum medium M9 with 0.2% glucose at pH 7 (Davis, 1980) and incubated for 2 h at 37 °C on a rotary shaker at 160 rpm. The larger particles of the kaolinite were harvested by centrifugation at 5500 ×g for 15 min at 4 °C. The supernatant, composed of the colloidal fraction, and the pellet were stored at 4 °C or frozen before used as culture medium or added to a fresh medium.
minimum medium M9. After an incubation of 2 h at 37 °C on a rotary shaker at 160 rpm, the larger particles were harvested by centrifugation at 3000 ×g for 2 min. The free glucose concentration was measured in the supernatant. These twelve values enabled us to determine a mean quantity of glucose adsorbed per gram of kaolinite (g/g). The same study was performed with 0.5, 1 and 2 g/l of acetate.
2.2. Bacterial strain and culture conditions
Aliquots of 1 ml were fixed with 1 ml formaldehyde 4%. The tubes were mixed for 30 s and left to stand for one hour at 4 °C. Bacteria were then collected by centrifugation at 5500 ×g for 15 min at 20 °C, washed twice in the same volume of PBS and resuspended in PBS at 109 cells per ml. Fixed sample volumes of 2 µl were examined using phase contrast microscopy (Axio Imager – ZEISS) with an oil immersion objective (100× oil universal objective). Mean cell size was measured by Image J, examining on average 1000 cells per sample. Statistical analyses were performed at P = 1‰ with a student's t test.
E. coli MG1655 (K-12 wild type strain) was cultured at 37 °C on a rotary shaker at 160 rpm in M9 with 0.2% glucose. E. coli overnight culture was diluted 100-fold in 50 ml of the same medium (initial cell concentration 5–107 cfu/ml) equilibrated at 37 °C for 30 min. Growth was monitored by measuring absorbance at 600 nm with a spectrophotometer (Biochrom LibraS22) or by plating cells on Luria-BertaniAgar followed by serial dilutions in phosphate buffer (PBS: 0.05 M, pH 7, 4 °C). Colonies were counted after incubating for 24 h at 37 °C and expressed as colony forming units (CFU) per milliliter representing the mean of triplicate measurements.
2.6. Bacterial size
3. Results 3.1. Kaolinite influence on growth parameters
2.3. Growth parameters 2.3.1. Biomass (X, g/l) After an incubation of 24 h, the culture was harvested by centrifugation at 5500 ×g for 15 min at 4 °C. The cell pellet was washed twice with cold PBS and then transferred into a preweighed aluminium cupel. Cells were dried at 120 °C for 4 h before being cooled and weighed. Centrifugation supernatant was collected to quantify glucose and acetate content as described below. 2.3.2. Maximal growth rate Maximal growth rate (µmax, h− 1) was determined graphically as the growth curve slope, plotted by monitoring cell growth either in CFU or in absorbance using a logarithmic scale. These two methods led to similar results. Nevertheless, µmax determination was only realizable up to 1 g/l of kaolinite at 600 nm. 2.3.3. Molecular growth yield Molecular growth yield (YGlu, g/mol) was defined as the mass of cells produced with one mole of glucose: YGlu =X/glucose consumed (mol/l).
With dispersions containing 0–4 g kaolinite per liter, the pH of the medium remained initially constant at 7.00±0.05. No significant adsorption of glucose was observed, with 96 to 98% of glucose remaining in solution after centrifugation. About 0.014 g of glucose per gram of kaolinite were adsorbed at 37 °C. Using phase contrast microscopy, we observed that the bacteria were mainly localized around clay particles suggesting interaction between clay particles and bacteria (data not shown). The responses were dose-dependent with optimal effects between 0.2 and 0.5 g kaolinite/liter followed by a decline suggesting a toxic effect of kaolinite at high concentrations (Table 1). Growth rate, absorbance and cell concentration reached maxima respectively 1.6 fold, 3 fold and 3.5 fold higher than control. The lag time suggest an initial physiological adaptation of bacteria in these conditions. Nevertheless, an enhanced bacterial growth overcame this negative impact on the cells. Consequently, kaolinite provokes an increase in maximal growth rate, maximal absorbance and final E. coli cell concentration in minimum growth medium. 3.2. Influence of kaolinite nutritional contribution and cell size variation
2.3.4. Catabolic activity Catabolic activity (qGlu, mmol g− 1 h− 1) was defined as the degradation rate of one mole of glucose per gram of cells in one hour: qGlu = µmax/YGlu.
An apparent rise in absorbance and growth rate that could easily be explained by a kaolinite nutritional contribution, led to the hypothesis that kaolinite addition leads to medium enrichment through a readily assimilated carbon–nitrogen and/or phosphate
2.4. Glucose and acetate quantification 2.4.1. Glucose quantification (g/liter) According to the method of Trinder (1969), glucose concentration is proportional to the formation of a red quinoneimine, detected at 500 nm within the range 0.05–2 g/l (±0.01) (Glucose GOD-PAP, Biolabo, France). 2.4.2. Acetate quantification (g/l) According to Bergmeyer and Mollering (1974), acetate concentration is proportional to the formation of NADH and detected at 340 nm within the range 0.025–1.2 g/l (±0.005) (Acide acétique enzymatique/ UV KHPE036058, Seppal, France). 2.5. Glucose and acetate sorption to kaolinite Increasing kaolinite concentrations (0.5, 1, 2 and 4 g/l) were contacted with increasing glucose concentrations (1, 2 and 4 g/l) in
Table 1 E. coli growth parameters in the presence of increasing kaolinite concentrations. Kaolinite concn Maximal growth rate Maximal (g/l) (h− 1) A600 nm
Final CFU × 109 per ml Cell size (µm)b
0 0.05 0.1 0.2 0.3 0.5 1 2 4
1.0 ± 0.1 1.7 ± 0.1 3.0 ± 0.1 3.3 ± 0.2 3.6 ± 0.4 3.5 ± 0.4 3.4 ± 0.1 2.3 ± 0.1 1.4 ± 0.2
a
0.74 ± 0.03 0.87 ± 0.01 1.21 ± 0.07 1.23 ± 0.03 1.21 ± 0.02 1.16 ± 0.05 1.07 ± 0.01 NDa NDa
0.83 ± 0.03 1.28 ± 0.18 2.01 ± 0.06 2.24 ± 0.10 2.33 ± 0.08 2.54 ± 0.10 2.35 ± 0.10 1.51 ± 0.15 1.33 ± 0.12
2.04 2.05 2.03 2.07 2.05 1.91 1.65 NDa NDa
ND, not determined. Aliquots of culture sample were taken for each growth condition after 24 h. According to t test (P = 1‰), E. coli mean cell size in control culture was highly significantly different from that acquired in the presence of a kaolinite concentration higher to 0.5 g/l. b
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source. Table 2 presents the final cell concentration of E. coli following 24 h cultivation M9 medium lacking a carbon, nitrogen or phosphate source. The addition of the kaolinite showed no evidence of enriching the medium, and we even observed a cell growth inversely proportional to kaolinite concentration with phosphate and nitrogen starvation. In these conditions, the kaolinite may have adsorbed all phosphate and ammonia thus rendering the medium even more starved of these two compounds, thus explaining these phenomena. Phase contrast images of 24-hour culture samples and measured bacterial size at each kaolinite concentration tested (0–1 g/l) revealed a statistically significant decrease in bacterial size at kaolinite contents upper than 0.6 g/l. However, a maximal decrease of 20% in the size of bacteria could not explain the 3.5 fold rise in cell number, indicating that variations in cell size did not account for the observed increase. Consequently, these modifications were neither caused by a kaolinite nutritional contribution nor by a decrease in cell size. 3.3. Kaolinite suspension To investigate whether the kaolinite effect is due to colloidal particles and/or larger setting particles we separated the two phases by centrifugation at an optimal kaolinite concentration (0.5 g/l). As demonstrated in Fig. 1, the colloidal particles did not show any effect whereas the larger particles revealed the same trend as the unfractionated kaolinite. Chemical analysis of the stable colloidal fraction revealed that the mineral elements found in kaolinite predominated in the larger particles (data not shown). Thus, the kaolinite effect arises from the larger particles. 3.4. Assimilation and degradation of glucose and acetate
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Fig. 1. E. coli MG1655 growth curve (i) with 0.5 g/l of kaolinite rinsed once in M9 medium with 0.2% glucose (Δ) or (ii) not rinsed (●), (iii) in the rinse medium containing the stable colloidal particles (▲) and (iv) in M9 medium with 0.2% glucose (○). Growth was followed using A600 nm measurements. Experiments were repeated at least three times and the standard deviation was always below 10%.
However, part of the energy outcome from acetate degradation due to anabolism may explain the observed increase in growth yield. 3.5. Kaolinite influence on catabolic activity We tested the hypothesis that kaolinite influences glucose assimilation and determined final E. coli biomass at several kaolinite concentrations and calculated the growth yield. Biomass and molecular growth yield increased to a maximum with at least 0.3 g/l of kaolinite (Table 3) and also revealed a close relationship between
We have demonstrated that the larger particles of kaolinite were responsible for increased bacterial growth altough the dispersion did not contain assimilated nutrients. One way of understanding this phenomenon is to consider a differing assimilation and/or degradation of glucose and/or acetate in the presence or absence of kaolinite. We thus measured the concentration of glucose and its degradation by-product (i.e. acetate) during E. coli growth in the presence or absence (control) of an optimal kaolinite concentration (0.5 g/l). Fig. 2A clearly shows a difference in the evolution of glucose and acetate concentrations between the two conditions. Without kaolinite, total glucose consumption and maximum observed acetate concentration occurred three hours later than with kaolinite. Interestingly, acetate was half-consumed after 24 h (data not shown). To compare the substrate degradation rate for the same bacterial concentration, we plotted the concentration of cells (A600) versus that of glucose or acetate (mg/liter). Fig. 2B reveals a slower glucose consumption rate per unit of A600 with 0.5 g/l of kaolinite. Unlike the control culture, the acetate consumption was linked to absorbance increase while the glucose had already been degraded. With only 0.063 g of acetate per gram of kaolinite (at 37 °C), the adsorption of acetate on kaolinite was not significant and therefore could not explain the increased acetate consumption rate per unit of A600.
Table 2 E. coli cultures limited by a nutrient in the presence of increasing kaolinite additions. Kaolinite concn CFU × 106 per mla (g/l) M9 without glucose M9 without phosphateb M9 without nitrogen 0 0.2 0.5 1 a
17.8 ± 0.8 17.0 ± 0.6 16.9 ± 0.4 18.1 ± 0.8
55.8 ± 2.5 38.8 ± 3.2 13.5 ± 1.4 7.5 ± 0.7
49.7 ± 1.4 32.8 ± 1.9 32.8 ± 2.5 24.3 ± 1.8
CFU in 24 hour culture samples. b The buffer power is maintained by the addition of NaCl and KCl. The cellular density of the overnight culture was 1.5 ± 0.1 × 109 per ml.
Fig. 2. Glucose (○) and acetate (▲) concentrations during E. coli growth in a medium containing 0.5 g/l of kaolinite (– –) or without kaolinite (—). (A) Profile against time, (B) profile against absorbance. The standard deviation, indicated by the error bars, was always below 10%.
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Table 3 E. coli growth parameters in the presence of increasing kaolinite concentrations. Kaolinite Maximal In 24 hour concn Growth Biomass (g/l) rate (g/l) (h− 1) 0 0.05 0.1 0.2 0.3 0.5 1 a b
0.74 ± 0.030.50 ± 0.05 0.87 ± 0.010.61 ± 0.04 1.21 ± 0.070.85 ± 0.02 1.23 ± 0.030.88 ± 0.04 1.21 ± 0.020.93 ± 0.07 1.16 ± 0.050.98 ± 0.09 1.07 ± 0.010.98 ± 0.08
culture samples
Molecular
Catabolic
Avg Avg glucose acetate (g/l) (g/l)
Growth yield (g/mol)
Activityb (mmol g−1 h−1)
b 0.05a b 0.05a b 0.05a b 0.05a b 0.05a b 0.05a b 0.05a
51 ± 5 62 ± 4 86 ± 2 89 ± 4 95 ± 7 99 ± 9 99 ± 8
14.4 ± 0.5 14.0 ± 0.2 14.0 ± 0.8 13.8 ± 0.4 12.9 ± 0.2 11.6 ± 0.5 10.8 ± 0.1
0.24±0.02 0.21±0.02 b 0.025a b 0.025a b 0.025a b 0.025a b 0.025a
Below the detection limit. qGlu (catabolic activity) = μmax (maximal growth rate)/YGlu (molecular growth yield).
this increase in growth yield and a significant decrease in catabolic activity. In light of these results, we propose that kaolinite reduces the glucose degradation rate, associated with increasing maximal growth rate and growth yield. 4. Discussion Under the experimental conditions outlined in this study, we have shown that kaolinite has a significant effect on E. coli maximal growth rate and growth yield. These physiological effects are related to a decrease in catabolic activity and improved acetate assimilation. Previous studies have reported the effects of several clay minerals (kaolinite and other minerals) on biomass production and biodegradation of various organic matters (Ortega-Calvo and Saiz-Jimenez, 1998; Chaerun et al., 2005; Chaerun and Tazaki, 2005). Mechanisms by which several clay minerals act are notably explained either (i) by interaction phenomena with organic molecules (e.g. humic acid (Ortega-Calvo and Saiz-Jimenez, 1998)), inorganic ions or microorganisms, or (ii) through their silicate content leading to C–O–Na–Si complex formation on the surface of bacterial cell walls (e.g. on hydrocarbon-degrading bacteria) (Inagaki et al., 2003; Chaerun et al., 2005). Kaolinites have previously been shown to act as a microbial growth-support material (Chaerun et al., 2005). Adsorption phenomena may result in higher substrate concentrations in the vicinity of the bacterial cells, thereby increasing their bioavailability (Mark van Loosdrecht et al., 1990; Ortega-Calvo and Saiz-Jimenez, 1998; Chaerun and Tazaki, 2005). One recent report suggests that by adsorption, clay minerals could mitigate the toxic effects of compounds highly concentrated in the medium (Chaerun et al., 2005). Even though experiments were performed over a period of 24 h and not several days as in other studies, the present study clearly demonstrates that the adsorption of glucose and acetate on the kaolinite particles (0.014 g and 0.063 g per gram kaolinite) was insignificant and that kaolinite promoted interactions with bacteria. We postulate that kaolinite-dependant effects on E. coli are not directly due to its surface-active properties. It seems more likely that
kaolinite leads to a decrease in catabolic activity and an increase in acetate assimilation by transferring a part of the energy outcome from acetate degradation to cell division rather than cell maintenance. So we can explain the increase in the final cell concentration observed in the presence of kaolinite but not the rise in maximal growth rate. Further experiments are necessary to elucidate the mechanisms involved in this process and the reasons why maximal growth rate is affected by kaolinite concentration. Acknowledgements We thank J. P. Belaïch, Bioénergétique et Ingénierie des Protéines, Marseille, France for helpful comments on the manuscript. We thank EPARCO Assainissement that kindly provided the kaolinite. References Bergmeyer, H.U., Mollering, H., 1974. In: Bergmeyer, H.U. (Ed.), second edition. Methods of Enzymatic Analysis, vol. 3. Academic Press, New York, pp. 1196–1201. Chaerun, S.K., Tazaki, K., 2005. How kaolinite plays an essential role in remediating oilpolluted seawater. Clay Minerals 40, 481–491. Chaerun, S.K., Tazaki, K., Asada, R., Kogure, K., 2005. Interaction between clay minerals and hydrocarbon-utilizing indigenous microorganisms in high concentrations of heavy oil: implications for bioremediation. Clay Minerals 40, 105–114. Davis, R.W., 1980. A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor, NY. 204 pp. Filip, Z., 1967. Effect of small additions of bentonite on the development of some groups of soil microorganisms in liquid culture. Folia Microbiologica 12 (4), 396. Inagaki, F., Motomura, Y., Ogata, S., 2003. Microbial silica deposition in geothermal hot waters. Applied Microbiology and Biotechnology 60, 605–611. Kostka, J.E., Dalton, D.D., Skelton, H., Dollhopf, S., Stucki, J.W., 2002. Growth of iron(III)reducing bacteria on clay minerals as the sole electron acceptor and comparison of growth yields on a variety of oxidized iron forms. Applied and Environmental Microbiology 68 (12), 6256–6262. Lavie, S., Stotzky, G., 1986. Adhesion of the clay minerals montmorillonite, kaolinite, and attapulgite reduces respiration of histoplasma capsulatum. Applied and Environmental Microbiology 51 (1), 61–73. Mark van Loosdrecht, C.M., Lyklema, J., Norde, W., Zehnder, A.J.B., 1990. Influence of interfaces on microbial activity. Microbiological Reviews 54 (1), 75–87. Martin, J.P., Filip, Z., Haider, K., 1976. Effect of montmorillonite and humate on growth and metabolic activity of some actinomycetes. Soil Biology and Biochemistry 8, 409–413. Novakova, J., 1968. Effect of bentonite and kaolinite on the growth curve of E. coli. Folia Microbiologica 13 (6), 543. Ortega-Calvo, J.J., Saiz-Jimenez, C., 1998. Effect of humic fractions and clay on biodegradation of phenanthrene by a Pseudomonas fluorescens strain isolated from soil. Applied Environmental Microbiology 64 (8), 3123–3126. Rong, X., Huang, Q., Chen, W., 2007. Microcalorimetric investigation on the metabolic activity of Bacillus thuringiensis as influenced by kaolinite, montmorillonite and goethite. Applied Clay Science 38, 97–103. Stotzky, G., 1966. Influence of clay minerals on microorganisms. II. Effect of various clay species, homoionic clays, and other particles on bacteria. Canadian Journal of Microbiology 12, 831–848. Stotzky, G., Rem, L.T., 1966. Influence of clay minerals on microorganisms. I. Montmorillonite and kaolinite on bacteria. Canadian Journal of Microbiology 12, 547–563. Trinder, P., 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6, 24–28. Wong, D., Suflita, J.M., McKinley, J.P., Krumholz, L.R., 2004. Impact of clay minerals on sulfate-reducinf activity in aquifers. Microbial Ecology 47, 80–86.