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Improvement of Escherichia coli growth by kaolinite

Elise Courvoisier, Sam Dukan

Laboratoire de Chimie Bactrienne, UPR 9043, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France

a b s t r a c ta 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

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 minimumgrowth 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 aloneor with kaolinite improved the mineralization of organic pollutants,

phenanthrene or heavy-oil, in a pure culture ofPseudomonas 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 montmorilloniteon 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

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 metabolicactivityof a model bacterium, E.

coli. Results of this study enableus 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 (%) SiO 2 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.

Applied Clay Science 44 (2009) 6770

Corresponding author. Tel.: +33 491 164 601; fax: +33 491 718 914.

E-mail address: sdukan@ibsm.cnrs-mrs.fr (S. Dukan).

0169-1317/$ see front matter 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.clay.2009.01.010

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 e v i e r. c o m / l o c a t e / c l a y

mailto:sdukan@ibsm.cnrs-mrs.frhttp://dx.doi.org/10.1016/j.clay.2009.01.010http://www.sciencedirect.com/science/journal/01691317http://www.sciencedirect.com/science/journal/01691317http://dx.doi.org/10.1016/j.clay.2009.01.010mailto:sdukan@ibsm.cnrs-mrs.fr

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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 atpH 7 (Davis,1980) and incubated for 2 h at37 Con 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.

2.2. Bacterial strain and culture conditions

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 5107 cfu/ml) equilibrated at 37 C for 30 min. Growth

was monitored by measuring absorbance at 600 nm with a spectro-

photometer (Biochrom LibraS22) or by plating cells on Luria-Bertani-

Agar 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 represent-

ing the mean of triplicate measurements.

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

aluminiumcupel. Cells were dried at 120 C for4 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 (max, h1) was determined graphically as

the growth curve slope, plotted by monitoring cell growth either in

CFUor in absorbance using a logarithmic scale. These twomethods led

to similar results. Nevertheless, max determination was only realiz-

able 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).

2.3.4. Catabolic activity

Catabolic activity (qGlu, mmol g1 h1) was defined as the deg-

radation rate of one mole of glucose per gram of cells in one hour:

qGlu = max/YGlu.

2.4. Glucose and acetate quantification

2.4.1. Glucose quantification (g/liter)

According to the method ofTrinder (1969), glucose concentrationis proportional to the formation of a red quinoneimine, detected at

500 nm within the range 0.052 g/l (0.01) (Glucose GOD-PAP,

Biolabo, France).

2.4.2. Acetate quantification (g/l)

According to Bergmeyer and Mollering (1974), acetate concentra-

tion is proportional to the formation of NADH and detected at 340 nm

within the range 0.0251.2 g/l (0.005) (Acide actique 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

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 centrifuga-

tion 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.

2.6. Bacterial size

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 gfor 15 min at 20 C, washed

twice in the same volume of PBS and resuspended in PBS at 10 9 cells

per ml. Fixed sample volumes of 2 l 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 ttest.

3. Results

3.1. Kaolinite influence on growth parameters

With dispersions containing 04 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. Never-

theless, an enhanced bacterial growth overcame this negative impacton 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

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 carbonnitrogen and/or phosphate

Table 1

E. coli growth parameters in the presence of increasing kaolinite concentrations.

Kaolinite concn

(g/l)

Maximal growth rate

(h1)

Maximal

A600 nm

Final CFU109 per ml Cell size

(m)b

0 0.74 0.03 0.83 0.03 1.0 0.1 2.04

0.05 0.87 0.01 1.28 0.18 1.7 0.1 2.05

0.1 1.21 0.07 2.01 0.06 3.0 0.1 2.03

0.2 1.23 0.03 2.24 0.10 3.3 0.2 2.07

0.3 1.21 0.02 2.33 0.08 3.6 0.4 2.05

0.5 1.16 0.05 2.54 0.10 3.5 0.4 1.91

1 1.07 0.01 2.35 0.10 3.4 0.1 1.65

2 NDa 1.51 0.15 2.3 0.1 NDa

4 NDa 1.33 0.12 1.4 0.2 NDa

a ND, not determined.b 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.

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source. Table 2 presents the final cell concentration ofE. 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 measuredbacterial size at each kaolinite concentration tested (01 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

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 degrada-

tion 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 orabsence (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 kaoli-

nite, total glucose consumption and maximum observed acetate

concentration occurred three hours later than with kaolinite. Inter-

estingly, 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 notexplain the increased acetate consumption rate per unit of A600.

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 mole-

cular growth yield increased to a maximum with at least 0.3 g/l

of kaolinite (Table 3) and also revealed a close relationship between

Table 2

E. coli cultures limited by a nutrient in the presence of increasing kaolinite additions.

Kaolinite concn

(g/l)

CFU106 per mla

M9 without glucose M9 without phosphateb M9 without nitrogen

0 17.8 0.8 55.8 2.5 49.7 1.4

0.2 17.0 0.6 38.8 3.2 32.8 1.9

0.5 16.9 0.4 13.5 1.4 32.8 2.5

1 18.1 0.8 7.5 0.7 24.3 1.8

a CFU in 24 hour culture samples.b Thebufferpoweris maintained bythe additionof NaCl andKCl. Thecellular density

of the overnight culture was 1.50.1109

per ml.

Fig. 1. E. coli MG1655 growth curve (i) with 0.5 g/l of kaolinite rinsed once in M9

medium with 0.2% glucose () 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%.

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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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 biodegra-

dation 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 micro-

organisms, or (ii) through their silicate content leading to CONaSi

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 microbialgrowth-support material (Chaerun et al., 2005). Adsorption phenom-

ena mayresult in highersubstrate 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 activityand 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. Belach, Bionergtique et Ingnierie des Protines,

Marseille, France for helpful comments on the manuscript.

We thank EPARCO Assainissement that kindly provided the

kaolinite.

References

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Chaerun, S.K., Tazaki, K., 2005. How kaolinite plays an essential role in remediating oil-polluted seawater. Clay Minerals 40, 481491.

Chaerun, S.K., Tazaki, K., Asada, R., Kogure, K., 2005. Interaction between clay mineralsand hydrocarbon-utilizing indigenous microorganisms in high concentrations ofheavy oil: implications for bioremediation. Clay Minerals 40, 105114.

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Filip, Z., 1967. Effect of small additions of bentonite on the development of some groupsof soil microorganisms in liquid culture. Folia Microbiologica 12 (4), 396.

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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 ofgrowth yields on a variety of oxidized iron forms. Applied and EnvironmentalMicrobiology 68 (12), 62566262.

Lavie, S., Stotzky,G., 1986. Adhesion of the clayminerals montmorillonite, kaolinite, andattapulgite reduces respiration of histoplasma capsulatum. Applied and Environ-mental Microbiology 51 (1), 6173.

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Table 3

E. coli growth parameters in the presence of increasing kaolinite concentrations.

Kaolinite

concn

(g/l)

Maximal In 24 hour culture samples Molecular Catabolic

Growth

rate

Biomass

(g/l)

Avg

glucose

Avg

acetate

Growth

yield

Activityb

(mmol g1 h1)

(h1) (g/l) (g/l) (g/mol)

0 0.74 0 .0 30 .5 0 0.05 b0.05a 0.24 0.02 51 5 14.4 0.5

0.05 0.870.010.610.04 b0.05a 0.21 0.02 62 4 14.0 0.2

0.1 1.210.070.850.02 b0.05a b0.025a 86 2 14.0 0.8

0.2 1.230.030.880.04 b0.05a

b0.025a

89 4 13.8 0.40.3 1.210.020.930.07 b0.05a b0.025a 95 7 12.9 0.2

0.5 1.160.050.980.09 b0.05a b0.025a 99 9 11.6 0.5

1 1.07 0 .010 .9 8 0.08 b0.05a b0.025a 99 8 10.8 0.1

a Below the detection limit.b qGlu (catabolicactivity)= max (maximalgrowth rate)/YGlu (molecular growthyield).

70 E. Courvoisier, S. Dukan / Applied Clay Science 44 (2009) 6770