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The role of electrokinetic properties on adhesion of nitrifying bacteria to solid surfaces
Studies in Surface Science and Catalysis 132 Y. Iwasawa, N. Oyama and H. Kunieda (Editors) c 2001 Elsevier Science B.V. All rights reserved.
293
The role of electrokinetic properties on adhesion of nitrifying bacteria to solid surfaces Hiroshi Hayashi", Satoshi Tsuneda'*, Akira Hirata' and Hiroshi Sasaki'' "•Department of Chemical Engineering, Waseda University ^Department of Environment and Resources Engineering, Waseda University Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan. Electrokinetic properties of nitrifying bacteria of Nitrosomonas europaea and Nitrobacter winogradskyi were investigated by electrophoretic mobility measurement and analyzed by soft particle electrophoresis theory. Also bacterial adhesion onto glass beads surface was examined by packed bed method. A^. europaea had more negative and rigid surface character compared with A^. winogradskyi. Cell adhesion properties were significantly dependent on pH and critical increase of cell attachment was observed below pH 4.3 for A^. europaea, and below pH 5.0 for N. winogradskyi, respectively.
1. INTRODUCTION Much attention has been paid to control of attachment/detachment of specific bacterial cell due to increasing demand for utilizing specific microbial cell to mediate biological reactions for technological applications. A typical example in wastewater treatment technology is immobilizing nitrifying bacteria, which is difficult to retain in bioreactor because of its low growth rate and lack of production of exopolymeric saccharides to facilitate biofilm formation. In order to develop immobilizing technique of specific bacteria onto support material, it is essential to elucidate interfacial characteristics of the strain and their influence on adhesion to solid substratum. Although many works has been made to investigate bacterial adhesion in terms of DLVO theory [1], knowledge of surface properties of nitrifying bacteria has been still limited due to complexity of environments where they usually exist. The purpose of this study is to investigate electrokinetic properties of typical nitrifying bacteria of Nitrosomonas europaea and Nitrobacter winogradskyi by electrophoretic mobility measurement and reveal its relevance to adhesion onto glass beads by packed column bed method. For the interpretation of cell electrokinetics, soft particle electrophoresis theory [2] was applied to analyze surface structure peculiar to biological cell. DLVO-type interaction energy between cell - glass was calculated and compared with collision efficiency parameter OQ.
2. MATERIALS AND METHODS 2.1 Bacterial strains and electrophoretic mobility measurement Ammonia-oxidizing bacterium of Nitrosomonas europaea IFO-14298 (A^. europaea). * Corresponding author. Tel:+81-3-5286-3210; Fax:+81-3-3209-3680, E-mail:[email protected]
294 nitrite-oxidizing bacterium of Nitrobacter winogradskyi IFO-14297 {N. winogradskyi) were used in this study. A^. europaea and A^. winogradskyi were aerobically cultured at 30 ^Q in an inorganic medium containing 1.0 gdm'^of (NH4)2S04 and NaNOj. Cells at their exponential growth phase were harvested by centrifligation (10000 g, 10 min) and washed five times by 10 mM KNO3 solution (pH 6.0) and then given to the experiments. Measurement of electrophoretic mobility was carried out by electrophoretic light scattering spectrophotometer (ELS-800, Otsuka Electronics, Japan). All of the measurement was carried out in 12 hours after harvesting from culture medium for to avoid changing cell surface properties. Mechanically ground glass particulates were used for measurement of glass beads. Supporting electrolyte used in all experiments was KNO3 and droplets of HNO3 or KOH that had the same ionic strength as the prepared suspensions were added to adjust pH. Cell suspension was dispersed in ultrasonic bath for 3 min and quickly supplied to the apparatus. For soft particle analysis, plots of mobility data were fitted with the electrophoresis formula given by Ohshima and Kondo [2]: £oS, T Q / K , + y p o ^ / A ^ ez^ 11
1/K,+1/A
(1)
TiA^
where \i is electrophoretic mobility, CQ and e^ are the permittivitiy of vacuum and relative permittivity of medium, respectively, r\ is the viscosity, K^, is Debye-Huckel parameter of surface region, e is elementary charge, ZN is special charge density in the surface region, k is a parameter characterizing the resistance to liquid flow in the surface region and T^ON is Donnan potential. ^ 0 is surface potential and written as follows. -\V2^
Izn)
Izn)
(2)
Here, k is Boltzmann constant, T is absolute temperature, z and n are the valence and concentration of bulk ions, respectively. This theory assumes particles with a charged layer of finite thickness at its outer region and best fitted combination oiZN and MX is experimentally given from eq(l). 2.2 Packed column experiments and analytical procedure Ten grams of glass beads (GB) were packed at the bottom of glass-made column (inner diameter: 15 mm, height: 40mm, porosity: 0.52). Cell suspension (concentration: 5 X 10* cm'^) containing 10 mM KNO3 was introduced into the column at flow rate of 9.0 mLmin'^ (0.08 mm-sec'). Cell concentration of effluent was examined by 00^60 measurement. Cell deposition was analyzed by clean bed collision efficiency followed by Rijinaarts et al [3]: CyCo = e x p [ - 3 / 4 { ( l - e ) / a > a o ( l - B 0 > ]
(3)
where C, and CQ are cell concentrations of feed and effluent at time t, respectively, e is bed porosity, a, is collector radius, 0 is collector mass transfer efficiency, OQ is clean bed collision efficiency, B is the blocking factor influenced by attached cells and 6 is the fraction of surface covered. DLVO-type interaction energy Vj between cell and GB were calculated by
295 following equation [4]: K, =So£.a,[(%. +^o2)'log(l + e-"*)+(vFo, - 4 ^ 0 2 ) ' M l - e - * ) ] -
h
• + log
h + 2a,
(4) h-\-2a
where a^^ is cell radius, ^01 ^ ^ ^ 2 ^ ^ surface potential of cell and solid, respectively, h is separation distance, A is Hamaker constant and referred to as [5].
3. RESULTS AND DISCUSSION 3.1 Electrokinetic behavior of bacterial cell Figure 1 shows electrophoretic mobility of A^. europaea and A^. winogradskyi as a function of pH in 10 mM KNO3. Mobility of both strains as well as GB showed negative value while absolute value of A^. europaea (-2.75 ^im/sA^/cm) was larger than A^. winogradskyi (-1.48 \mdsNlQm) at neutral pH. Mobility significantly increased below pH 5. Figure 2 shows electrophoretic mobility of A^. europaea and A^. winogradskyi as a function of KNO3 concentration and the solid lines were theoretical curves fitted by eq (1). Both mobility data converged to nonzero values as the KNO3 concentration got higher, which was a typical property of soft particle and indicated that application of this model was reasonable. 0
i
-0.5
0 i-1.5 « > -2 |i-2.5 2 =i -3
1
w
10 11
Fig.l Electrophoretic mobility of bacterial cells and GB as a function of pH.
--; / ^ \j/ >
tt/ yf -3-5 I B_ .4
0
U-*-'
•
A^. w
nogri idsky\
0.05 0.1 0.15 KNO3 concentration, M
Fig.2 Electrophoretic mobility of bacterial cells as a function of KNO3 concentration.
Best fitted combinations of ZN and 1/A (pH 7.0) that represents cell surface character are listed in Table 1. It was found that A^. europaea had larger ZN and smaller MX values than A^. winogradskyi, indicating surface layer of A^. europaea had more negative charge density and rigid structure as compared with A^. winogradskyi based on soft electrophoresis analysis. 3.2 Adhesion assay and DLVO interaction energy Figure 3 shows breakthrough curves of A^. europaea (a) and A^. winogradskyi (b) and solid lines were theoretical curves fitted by equation (3). In the case of A^. europaea, little attachment was occurred in the pH range from 7.0 to 5.0, where both A^. europaea and GB had relatively high negative charge as observed in Fig.l. On the other hand, cell collection drastically increased below pH 4.3, probably due to suppression of electrostatic repulsive forces between cell and GB. Similar phenomenon was observed in attachment of A^. winogradskyi to GB, in which increment of cell collection took place below pH 5.0. Figure 4 shows relationship between cell - GB collision efficiency OQ and DLVO interaction maximum V^^ calculated by eq (4). Electric potential of glass was approximated as zeta potential by converting mobility with Smoluchowski formula and cell surface potential at pH 7.0 could be given from eq (3) using ZN in Table 1. In other pH conditions, lA. values
296 are assumed to be constant at pH ranging from 4.0 to 7.0 because pH has great influence on surface dissociation group, i.e., ZN, compared with lA., even though MX values are more or less affected by solution pH [6]. ZN values at any pH were derived from mobility data from Fig. 1 and converted to surface potentials. From Fig. 4, OQ got lower in proportion as the V ^ increased when OQ was less than 4 X 10'^ On the other hand, Oo substantially got higher with a slight decrease of V^^^ when OQ was above 4X lOl This critical change of OQ was observed when V^3^ is around 35 kT for both strains, probably indicating rapid heterocoagulation of cell - GB came to take place. (a) A^. europaea I 0.8 o
rn
53
5^0.6 ^"0.4 0.2 0
pH4.0
^
pH4.2
•
pH4.3
•
pH5.0
•
pH7.0
-•— -d •
pH4.0
^
pH4.5
i 0
•
Table 1 Best fitted values of ZA^and \/X for A^. europaea and N. winogradskyi at pH 7.0 l//l(nm) ZN(M) Strain 0.90 -0.0531 N. europaea -0.0227 1.23 N. winogradskyi
200 400 600 800 1000
Time, s (b) N. winogradskyi 1 0.8
\tn t t | ft" • - • C3Z TJL.
y*o.6 iisM ^^0.4
• • • WTT r •
-T—
:M
•
•
T
0
•
pH5.0
•
pH6.0
EI50 ^ 100
•
pH7.0
50 0
"A
0.2 0
350 300 ^250 , ;200 I-
200 400 600 800 1000
Time, s Fig. 3 Breakthrough curves of A^. europaea (a) and N. winogradskyi (b) at various pH in 10 mM KNO3 as a function of time.
101-2
N. europaea N. winogradsky.
Collision efficiency tto
Fig.4 Relationship between collision efficiency Oo and potential energy maximum V„^.
4. CONCLUSION Both A^. europaea and A^. winogradskyi revealed soft particle behavior by electrophoretic mobility measurement. A^. europaea showed more negative and rigid surface layer compared with A^. winogradskyi based on soft particle analysis. Electrokinetics and adhesion of cells were influenced by pH of the suspension and significant increase of cell - GB attachment was observed around V ^ , o f 35 kT for both strains.
Reference 1. M Hemiansson, Collokis Surf. B: Biointei&es, 14 (1999) 105. 2. R OishimaandT. Kondo, J. CoUokilnterfeceSci., 130(1989)281. 3. R R Nl Rijinarrts, W. Noide, E. J. Bouvv«; J. Lyklema, A. J. ZdindCT, Enviioa Sc^ 4. R. J. Hunter, Foundatiais of Colloid Science, Vol.1 (1987) Oxford, New York 5. R R NL Rijinants, W. Node, E. J. L)4dema, A. J. B. Zdmdei; Colbkls Surf B: Biointe^^ 6. R ScMxtora, N. Muramatsu, R CAishimaandT. Kondo, Bioph^^
293
The role of electrokinetic properties on adhesion of nitrifying bacteria to solid surfaces Hiroshi Hayashi", Satoshi Tsuneda'*, Akira Hirata' and Hiroshi Sasaki'' "•Department of Chemical Engineering, Waseda University ^Department of Environment and Resources Engineering, Waseda University Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan. Electrokinetic properties of nitrifying bacteria of Nitrosomonas europaea and Nitrobacter winogradskyi were investigated by electrophoretic mobility measurement and analyzed by soft particle electrophoresis theory. Also bacterial adhesion onto glass beads surface was examined by packed bed method. A^. europaea had more negative and rigid surface character compared with A^. winogradskyi. Cell adhesion properties were significantly dependent on pH and critical increase of cell attachment was observed below pH 4.3 for A^. europaea, and below pH 5.0 for N. winogradskyi, respectively.
1. INTRODUCTION Much attention has been paid to control of attachment/detachment of specific bacterial cell due to increasing demand for utilizing specific microbial cell to mediate biological reactions for technological applications. A typical example in wastewater treatment technology is immobilizing nitrifying bacteria, which is difficult to retain in bioreactor because of its low growth rate and lack of production of exopolymeric saccharides to facilitate biofilm formation. In order to develop immobilizing technique of specific bacteria onto support material, it is essential to elucidate interfacial characteristics of the strain and their influence on adhesion to solid substratum. Although many works has been made to investigate bacterial adhesion in terms of DLVO theory [1], knowledge of surface properties of nitrifying bacteria has been still limited due to complexity of environments where they usually exist. The purpose of this study is to investigate electrokinetic properties of typical nitrifying bacteria of Nitrosomonas europaea and Nitrobacter winogradskyi by electrophoretic mobility measurement and reveal its relevance to adhesion onto glass beads by packed column bed method. For the interpretation of cell electrokinetics, soft particle electrophoresis theory [2] was applied to analyze surface structure peculiar to biological cell. DLVO-type interaction energy between cell - glass was calculated and compared with collision efficiency parameter OQ.
2. MATERIALS AND METHODS 2.1 Bacterial strains and electrophoretic mobility measurement Ammonia-oxidizing bacterium of Nitrosomonas europaea IFO-14298 (A^. europaea). * Corresponding author. Tel:+81-3-5286-3210; Fax:+81-3-3209-3680, E-mail:[email protected]
294 nitrite-oxidizing bacterium of Nitrobacter winogradskyi IFO-14297 {N. winogradskyi) were used in this study. A^. europaea and A^. winogradskyi were aerobically cultured at 30 ^Q in an inorganic medium containing 1.0 gdm'^of (NH4)2S04 and NaNOj. Cells at their exponential growth phase were harvested by centrifligation (10000 g, 10 min) and washed five times by 10 mM KNO3 solution (pH 6.0) and then given to the experiments. Measurement of electrophoretic mobility was carried out by electrophoretic light scattering spectrophotometer (ELS-800, Otsuka Electronics, Japan). All of the measurement was carried out in 12 hours after harvesting from culture medium for to avoid changing cell surface properties. Mechanically ground glass particulates were used for measurement of glass beads. Supporting electrolyte used in all experiments was KNO3 and droplets of HNO3 or KOH that had the same ionic strength as the prepared suspensions were added to adjust pH. Cell suspension was dispersed in ultrasonic bath for 3 min and quickly supplied to the apparatus. For soft particle analysis, plots of mobility data were fitted with the electrophoresis formula given by Ohshima and Kondo [2]: £oS, T Q / K , + y p o ^ / A ^ ez^ 11
1/K,+1/A
(1)
TiA^
where \i is electrophoretic mobility, CQ and e^ are the permittivitiy of vacuum and relative permittivity of medium, respectively, r\ is the viscosity, K^, is Debye-Huckel parameter of surface region, e is elementary charge, ZN is special charge density in the surface region, k is a parameter characterizing the resistance to liquid flow in the surface region and T^ON is Donnan potential. ^ 0 is surface potential and written as follows. -\V2^
Izn)
Izn)
(2)
Here, k is Boltzmann constant, T is absolute temperature, z and n are the valence and concentration of bulk ions, respectively. This theory assumes particles with a charged layer of finite thickness at its outer region and best fitted combination oiZN and MX is experimentally given from eq(l). 2.2 Packed column experiments and analytical procedure Ten grams of glass beads (GB) were packed at the bottom of glass-made column (inner diameter: 15 mm, height: 40mm, porosity: 0.52). Cell suspension (concentration: 5 X 10* cm'^) containing 10 mM KNO3 was introduced into the column at flow rate of 9.0 mLmin'^ (0.08 mm-sec'). Cell concentration of effluent was examined by 00^60 measurement. Cell deposition was analyzed by clean bed collision efficiency followed by Rijinaarts et al [3]: CyCo = e x p [ - 3 / 4 { ( l - e ) / a > a o ( l - B 0 > ]
(3)
where C, and CQ are cell concentrations of feed and effluent at time t, respectively, e is bed porosity, a, is collector radius, 0 is collector mass transfer efficiency, OQ is clean bed collision efficiency, B is the blocking factor influenced by attached cells and 6 is the fraction of surface covered. DLVO-type interaction energy Vj between cell and GB were calculated by
295 following equation [4]: K, =So£.a,[(%. +^o2)'log(l + e-"*)+(vFo, - 4 ^ 0 2 ) ' M l - e - * ) ] -
h
• + log
h + 2a,
(4) h-\-2a
where a^^ is cell radius, ^01 ^ ^ ^ 2 ^ ^ surface potential of cell and solid, respectively, h is separation distance, A is Hamaker constant and referred to as [5].
3. RESULTS AND DISCUSSION 3.1 Electrokinetic behavior of bacterial cell Figure 1 shows electrophoretic mobility of A^. europaea and A^. winogradskyi as a function of pH in 10 mM KNO3. Mobility of both strains as well as GB showed negative value while absolute value of A^. europaea (-2.75 ^im/sA^/cm) was larger than A^. winogradskyi (-1.48 \mdsNlQm) at neutral pH. Mobility significantly increased below pH 5. Figure 2 shows electrophoretic mobility of A^. europaea and A^. winogradskyi as a function of KNO3 concentration and the solid lines were theoretical curves fitted by eq (1). Both mobility data converged to nonzero values as the KNO3 concentration got higher, which was a typical property of soft particle and indicated that application of this model was reasonable. 0
i
-0.5
0 i-1.5 « > -2 |i-2.5 2 =i -3
1
w
10 11
Fig.l Electrophoretic mobility of bacterial cells and GB as a function of pH.
--; / ^ \j/ >
tt/ yf -3-5 I B_ .4
0
U-*-'
•
A^. w
nogri idsky\
0.05 0.1 0.15 KNO3 concentration, M
Fig.2 Electrophoretic mobility of bacterial cells as a function of KNO3 concentration.
Best fitted combinations of ZN and 1/A (pH 7.0) that represents cell surface character are listed in Table 1. It was found that A^. europaea had larger ZN and smaller MX values than A^. winogradskyi, indicating surface layer of A^. europaea had more negative charge density and rigid structure as compared with A^. winogradskyi based on soft electrophoresis analysis. 3.2 Adhesion assay and DLVO interaction energy Figure 3 shows breakthrough curves of A^. europaea (a) and A^. winogradskyi (b) and solid lines were theoretical curves fitted by equation (3). In the case of A^. europaea, little attachment was occurred in the pH range from 7.0 to 5.0, where both A^. europaea and GB had relatively high negative charge as observed in Fig.l. On the other hand, cell collection drastically increased below pH 4.3, probably due to suppression of electrostatic repulsive forces between cell and GB. Similar phenomenon was observed in attachment of A^. winogradskyi to GB, in which increment of cell collection took place below pH 5.0. Figure 4 shows relationship between cell - GB collision efficiency OQ and DLVO interaction maximum V^^ calculated by eq (4). Electric potential of glass was approximated as zeta potential by converting mobility with Smoluchowski formula and cell surface potential at pH 7.0 could be given from eq (3) using ZN in Table 1. In other pH conditions, lA. values
296 are assumed to be constant at pH ranging from 4.0 to 7.0 because pH has great influence on surface dissociation group, i.e., ZN, compared with lA., even though MX values are more or less affected by solution pH [6]. ZN values at any pH were derived from mobility data from Fig. 1 and converted to surface potentials. From Fig. 4, OQ got lower in proportion as the V ^ increased when OQ was less than 4 X 10'^ On the other hand, Oo substantially got higher with a slight decrease of V^^^ when OQ was above 4X lOl This critical change of OQ was observed when V^3^ is around 35 kT for both strains, probably indicating rapid heterocoagulation of cell - GB came to take place. (a) A^. europaea I 0.8 o
rn
53
5^0.6 ^"0.4 0.2 0
pH4.0
^
pH4.2
•
pH4.3
•
pH5.0
•
pH7.0
-•— -d •
pH4.0
^
pH4.5
i 0
•
Table 1 Best fitted values of ZA^and \/X for A^. europaea and N. winogradskyi at pH 7.0 l//l(nm) ZN(M) Strain 0.90 -0.0531 N. europaea -0.0227 1.23 N. winogradskyi
200 400 600 800 1000
Time, s (b) N. winogradskyi 1 0.8
\tn t t | ft" • - • C3Z TJL.
y*o.6 iisM ^^0.4
• • • WTT r •
-T—
:M
•
•
T
0
•
pH5.0
•
pH6.0
EI50 ^ 100
•
pH7.0
50 0
"A
0.2 0
350 300 ^250 , ;200 I-
200 400 600 800 1000
Time, s Fig. 3 Breakthrough curves of A^. europaea (a) and N. winogradskyi (b) at various pH in 10 mM KNO3 as a function of time.
101-2
N. europaea N. winogradsky.
Collision efficiency tto
Fig.4 Relationship between collision efficiency Oo and potential energy maximum V„^.
4. CONCLUSION Both A^. europaea and A^. winogradskyi revealed soft particle behavior by electrophoretic mobility measurement. A^. europaea showed more negative and rigid surface layer compared with A^. winogradskyi based on soft particle analysis. Electrokinetics and adhesion of cells were influenced by pH of the suspension and significant increase of cell - GB attachment was observed around V ^ , o f 35 kT for both strains.
Reference 1. M Hemiansson, Collokis Surf. B: Biointei&es, 14 (1999) 105. 2. R OishimaandT. Kondo, J. CoUokilnterfeceSci., 130(1989)281. 3. R R Nl Rijinarrts, W. Noide, E. J. Bouvv«; J. Lyklema, A. J. ZdindCT, Enviioa Sc^ 4. R. J. Hunter, Foundatiais of Colloid Science, Vol.1 (1987) Oxford, New York 5. R R NL Rijinants, W. Node, E. J. L)4dema, A. J. B. Zdmdei; Colbkls Surf B: Biointe^^ 6. R ScMxtora, N. Muramatsu, R CAishimaandT. Kondo, Bioph^^