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Adhesion of Microbial Cells to Porous Hydrophilic and Hydrophobic Solid Substrata
159
Adhesion of Microbial Cells to Porous Hydrophilic and Hydrophobic Solid Substrata Takayoshi Ban and Shinjiro Yamamoto Chemical Engineering Department, Shizuoka University, 5-1, Johoku 3-chome, Hamamatsu, Shizuoka, Japan 432
Abstract Adhesion of microbial cells to solid surfaces is thought to significantly influence the growth and transport of microbes in porous geological materials. The feasibility of cell adhesion to a porous solid substratum can be predicted as a function of the hydrophobicity of the substratum according to the following equation derived from the balance of surface and interfacial free energy in relation to adhesion under conditions where the electrical charge interaction is negligible:
This thermodynamic approach provides us with the following predictions: (1) the more hydrophilic microbes may be expected to adhere more favorably to the hydrophilic surfaces of a solid substratum, and 2 ) the more hydrophobic microbes will adhere more favorably to the hydrophobic surfaces of a solid substratum. To experimentally verify the theoretical predictions, a series of experimcnts were carried out to investigate the influence of hydrophobicity of solid substrata upon the adhesion of growing cells of Penicillium spiculisporum ATCC 16071 using either a typically hydrophobic or hydrophilic solid substratum. The results revealed that nearly 100% of the growing cells of P. spiculisporum adhered to the hydrophilic substrata; by contrast, most of the cells were suspended freely in the culture broth when hydrophobic polyurethane foamwas used as an adhesive substrate. These results are in reasonable agreement with the results of the theoretical prediction, and we conclude that the thermodynamic approach offers a powerful tool to predict microbial adhesion onto solid substrates.
1. INTRODUCTION Adhesion or adsorption phenomenon of microbial cells to solid surfaces is thought to significantly influence the growth and transport of microbes in or through porous geologic materials. Such a phenomenon may be regarded as a prerequisite for microbial plugging, particularly in the initial stage, associated with conventional waterflooding operations for oil recovery from reservoirs. Adhesion of bacteria or other microorganisms to surfaces of geological materials is thought to involve the physiocochemical surface characteristics of the solid surface, such as charge and hydrophobicity. A thermodynamic approach offers a powerful tool to predict microbial adhesion to a solid substratum under conditions where electrical charge interaction can be neglected.
160
In this study, a theoretical prediction was made on the basis of thermodynamics about the possibility of particular microbial cells adhering onto both hydrophilic and hydrophobic solid substrata. To verify the theoretical predictions, a series of adhesion experiments were made using either typically hydrophilic or hydrophobic solid substrata to investigate the influence of hydrophobicity, and surface and interfacial free energy of the solid substrata upon the numbers of microbial cells adhering to the substrata during their growth. 2.
THEORETICAL CONSIDERATIONS
Suppose a material, M , suspended or dispersed freely in an aqueous liquid, L, adheres to the surface of a solid substratum, S . A change in the interfacial free energy during the process of adhesion of material M to the surface of solid substratum S , AG,,,, can then be written as equation (1) based on the interfacial free energy balance before and after adhesion under conditions where the electrical charge interaction is negligible.
, and ysL are interfacial free energies between solid S and material S and liquid L, respectively. Equation (1) predicts that adhesion may be expected if AGadh is less than zero (AGadh < o ) , whereas adhesion is energetically unfavorable if AG,,, is greater than zero (AGacih > 0). To estimate AGadh, the change in free energy in the adhesion process, it is necessary to determine the interfacial free energies, three terms found on the right-hand side of equation (1) according to the surface chemical approaches. One useful approach is based on separation of the surface free energy into two components--a dispersion component y d , and a polar component yp. Surface free energy is essentially caused by attractive force acting between molecules of the material. This intermolecular attractive force can be separated into the following two components: (1) a dispersion force proposed by London in 1930, y d , [ l ] and (2) the attractive forces due to the polarity of the molecules involved, yp. Thus surface free energy of, for example, material A, yAv, can be expressed as : where
ySM, ym
M , material M and liquid L, and solid
Yav = Y":
+
YZV
Similarly, the surface free energy of material B , ysv, can be written as: YEW =
Yt"
+
Y$V
According to Fowkes' approach [2] the dispersion and polar components of interfacial free energy between any two surfaces A and B can be expressed as follows on the basis of the geometric mean equation:
161
Consequently, AGadh in equation (1) can be written as follows by using equation:
or rearranged:
The influence of the surface characteristics of the solid substratum upon the adhesion of material M is subsequently described by the following equation obtained by differentiating equation ( 4 ) with respect to y& and y z v :
Our experimental determination showed the value of y& to be approximate1 40 mN/m for almost all solid substrata we used. Therefore, we can assumedy,,J to be zero, and equation ( 5 ) can be simplified to equation ( 6 ) :
Equation ( 6 ) is a useful theoretical expression for predictingmicrobial adhesion onto the solid substrata when material M is regarded as microbial cell. From equation ( 6 ) , it appears ( 3 1 : 1) If rPw > ~ P L v , (namely, if the microbial cell is more hydrophilic): The left-hand side of the equation ( 6 ) becomes less than zero as T~~ increases because ( y f ~ ) ~ ’ ’ is less than (yh)’” . Therefore, AGadh becomes less than zero. This finding means that the adhesion of microbial cells onto surfaces of the solid substrata may become energetically more favorable as TSV increases. In other words, adhesion may be expected as the surface of the solid substratum becomes more hydrophilic. Therefore, more hydrophilic microbes may be expected to adhere more favorably to the surface of the solid substratum if the surface
162
Table 1 Variations of parameters influencing AGadh . ySV was variable throughout Y LV
YMV
Simulation I
44 mN/m
(1) 1 0 5 mN/m (2) 8 6 mN/m (3) 6 9 mN/m ( 4 ) 5 5 mN/m
Simulation I1
5 6 mN/m
(1) 1 0 5 (2) 86 ( 3 ) 69 ( 4 ) 55
Simulation I11
6 8 mN/m
(1) 1 0 5 mN/m (2) 8 6 mN/m (3) 6 9 mN/m ( 4 ) 5 5 mN/m
mN/m mN/m mN/m mN/m
becomes more hydrophilic and vice versa. Therefore, we conclude that more hydrophilic microbes may be expected to adhere more favorably to hydrophilic surfaces of solid substrata. 2) By contrast, if y h < y b , namely, if the microbial cell is more hydrophobic, then d (AGO,,,,) / dy,, becomes more than zero as ysv increases. Therefore, more hydrophobic microbes can be expected to adhere more favorably to hydrophobic surfaces of the solid substrata. To clarify the results of the prediction from equation ( 6 ) , a simulation was made to investigate the factors influencing adhesion of microbial cells suspended freely in an aqueous liquid to surfaces of solid substrata having different surface properties. The three major parameters in equation ( 6 ) which may affect the adhesion of microbial cells are the surface free energies of solid, 7sV, the microbial cell, yMV,and the liquid, yLv. Simulations were made under conditions where these three parameters were independently varied to demonstrate the influence of each parameter upon microbial adhesion. Table 1 shows range of variation of each parameter for the simulation. The results of the simulation are illustrated schematically in Figure 1 through Figure 3 as plots of AGadh against ysv with the parameter yMV. Simulation I was made under the conditions where 1) the surface free energy of the liquid, y ~ was ~ ,44 mN/m, and 2 ) the surface free energy of the microbial cells, yMV,ranged between 1 0 5 mN/m and 5 5 mN/m, which covers microbial cells having surface properties from extremely hydrophilic to hydrophobic. The results are illustrated schematically in Figure 1, where AGadh is plotted against 7sv. This figure shows that the area where ysv is lower than 50 mN/m, or the surface of the solid substrata is relatively hydrophobic, AG,& becomes greater than zero for cells (l), ( 2 ) , and ( 3 ) , which covers cells having hydrophilic surfaces. Thus, adhesion does not occur spontaneously in this area. A s ysv increases, adhesion may become energetically more favorable Since AGadh becomes less than zero. Simulation I1 was made at ytv = 5 2 mN/m; in other words, yLv is greater than that of Simulation I, but other conditions are the same as before. S O far as
163
Figure 1. Simulation I.
20
n
.
10
E
Z
€
0
condi lions : 5 2 mNlm
yLv=
U
c
;-lo
y M v = (1) 105 rnNlm
0
a
(2) 8 6 m N l m
- 20
(3) 69rnNlrn ( 4 ) 55mN/rn
-30
20
0
8
8
n
Figure 3. Simulation I11
condi Iions :
YLV=6 0 mNlm
U
0
-a
-20 -
Figure 2. Simulation I1
yMv= (1)
1 0 5 mNlm
(2)
86 m N l m
( 3 ) 6 9 rnNlrn ( 4 ) 5 5 mNlm
164
Table 2 The size and porosity of the carriers Size
Porosity
hydrophilic carrier - 1
2.5 x 2 . 5 x 2.5 mm cubic
97%
hydrophilic carrier
5 mm diameter, spherical
97%
hydrophilic microbial cells, or cells (l), (2), and ( 3 ) are concerned, similar trends are observed as those in Simulation I (Figure 2). Figure 3 indicates the result of Simulation I11 made at -yLv = 60 mN/m, which means that the surface tension of the liquid medium is greater than those of Simulations I and 11, but other conditions are the same as before. Almost completely similar curves of AGadh as function of -ysv were obtained as those of Simulation 11. In this case, it is also apparent that for hydrophilic cells with a -yMv to be greater than 70 mN/m, adhesion may become energetically more favorable than an increase in the surface free energy of a solid surface. 3. EXPERIMENTS To verify the prediction made from the thermodynamic considerations in equation ( 6 ) , an experimental study was made to investigate the influence of hydrophobicity, or surface and interfacial free energy of solid substrata upon the number of microbial cells adhered to the substrata during microbial growth. 3.1. Microorganism Penicillium spiculisporum ATCC 16071, a fungus, was used in this experimental study. This microbe produces 4-hydroxy-4,5-dicarboxypentadecanoic acid, one of the microbial biosurfactants, during aerobic growth on glucose [ 4 ] . While developing a bioreactor system for producing the biosurfactant using immobilized P . spiculisporum and selecting porous carriers onto which the microbial cells adhered and became immobilized, a focus of attention was given to the theoretical analysis of adhesion phenomenon of microbial cells onto solid substrata.
3.2. Composition of growth medium The growth medium used for P. spiculisporum consisted of 10% glucose, 0.1% N H 4 C 1 , 0.1% KHzP04, 0.02% Mg S 0 4 7 H z 0 , 0.1% peptone, and 0.1%yeast extract. 3.3. Porous carriers Two types of porous polymers were used as solid substrata or microbe adhesion carriers for P. spiculisporum. One was a commercial polyurethane foam, which is a typical hydrophobic porous carrier, hereafter called the hydrophobic carrier. The other was a typical hydrophilic porous carrier prepared from natural cellulose, and referred to as the hydrophilic carrier - 1. The size and porosity of both the above porous carriers are shown in Table 2. Gelatin-treated polyurethane foam also was used as a hydrophilic carrier, which was prepared by coating the surface of the commercial polyurethane foam with gelatin film, and thus giving a relatively hydrophilic surface: We called this hydrophilic carrier - 2.
165
3.4. Determination of surface free energy 3.4.1. Surface free energy of liquid The surface free energy, or surface tension of liquid, rLv,can be easily and directly measured by using a proper tensiometer. In this study we used Wilhelmy’s vertical plate method. 3.4.2. Surface free energy of hydrophobic solid substratum The surface free energy, or surface tension of the hydrophobic solid substratum (the hydrophobic carrier), rSv, was determined as critical surface tension, -yc, estimated from a Zisman plot [ 5 ] by measuring the contact angle. 3.4.3. Surface free energy of hydrophilic solid substrata and microbial cells Determination of the surface free energy of both the hydrophilic solid substrata, rSv, and the microbial cells, y M v , was made using the method developed by Busscher and Arends [ 6 ] on the basis of the following equation: cos
e
= -16.74 (yps,jl/’
02
+
2(y&)11’
+
3.996
( ~ p s ~ ) ~ / D~ ’ +
0.002 (ypgV)l’’
-
1
(7)
where,
3.5. Growth and adhesion experiments Growth experiments with P. spiculisporum were made using a 5 0 0 m l flask containing 100 m l of growth medium and a specified amount of pieces of either hydrophobic or hydrophilic carrier. The flask was autoclaved, inoculated, and then placed on a reciprocal shaker operated at 120 strokes per minute at 3OoC to allow the microbe to grow. After reaching almost stationary phase of growth, after 12 days cultivation, the amount of cells adhering to the carriers, and those suspended freely in the culture broth were weighed. 4. EXPERIMENTAL RESULTS
4.1. Experimental determination of surface free energies 4.1.1. Zisman plot Figure 4 shows Zisman plots of the hydrophobic carrier used in this experiment and of several commercial synthetic polymers including polyethylene, which is a typical non-polar compound. The value of the surface free energy, rSv, of the hydrophobic carrier obtained from the Zisman plot, in other words, the value of its critical surface free energy, rC, was estimated to be 44 mN/m. 4.1.2. Results of experimentally determined surface free energies of liquid and solid materials Table 3 shows the experimentally determined surface free energies of the culture broths, of the solid substrata includinghydrophilic carriers, and of the microbial cells. Surface free energies of culture broth at the initial and final stages of cultivation measured directly by using Wilhelmy’s vertical plate method were 60 mN/m and 44 mN/m, respectively.
166
Table 3 Experimentally determined surface free energies
(1) initial stage (2) final stage
Culture Broth
7Lv = 60 mN/m YLV
- 44
Hydrophobic Carrier
Ysv
- 4 4 mN/m
Hydrophilic Carrier 1
ySv
Hydrophilic Carrier 2
ySv
Microbial Cells
yMY = 101 mN/m
- 123 mN/m - 110 mN/m
The ySv values of the hydrophilic carriers 1 and 2 were determined from equation (7) by measuring the contact angle, and were 123 mN/m, and 110 mN/m, respectively. The ySv value of the hydrophobic carrier was much less than those of hydrophilic carriers, and was estimated to be 44 mN/m from the Zisman plot (Figure 4 ) .
1
30 40 50 60 70 80 Y L V CmN/m>
ysv=
pol yet hy Iene
(
polyvinyl chloride
(y,,
polyacryl resin
(
= 40 mNlm
pol ycarbona te
ysv= ( ysv=
hydrophobic carrier
(
y,,
3 9 mNlm
41 mNlm 4 3 mNlm
= 4 4 mNlm
Figure 4 . Zisman plots of various hydrophobic polymers including the one used in this study.
167
The surface free energy of the microbial cells used in this study was 101 mN/m, as determined by measurement of the contact angle. Thus, the cells of P. spiculisporum are extremely hydrophilic.
4.2. Simulation By using the experimentally determined values of surface free energies, a theoretical prediction of the feasibility of adhesion of the microbial cells to both hydrophobic andhydrophilic carriers was made. The result ofthe simulation is illustrated in Figure 5 . The shaded curve indicates the resultant prediction under our experimental conditions. Thus, AG,& for the hydrophobic carrier (ysv 44 mN/m) is estimated to be greater than zero. Therefore, the microbial cells may be expected to adhere less successfully to the hydrophobic carrier. However, in the case of both hydrophilic carriers, the surface free energies were 123 mN/m and 110 mN/m, respectively, and thus AGadh becomes much lower than zero for both carriers, and the adhesion of the microbe becomes energetically more favorable. A series of growth and adhesion experiments were carried out to verify the above theoretical prediction experimentally.
-
4.3.
Results of the growth and adhesion experiment The growth of P. spiculisporum and adhesion of the microbial cells to hydrophobic or hydrophilic carriers are illustrated in Figure 6 , which gives the % adhesion of cells on either carrier. The % adhesion is plotted as a function of the amount of carrier added in the culture broth before starting cultivation. The % adhesion in defined in the following equation:
where, Madh is the amount of microbial cells adhered to the carrier, Mfree is the amount of microbial cells suspended freely in the broth, and Mtotal is the total Madh + Mfree. amount of cells grown, expressed as: Mtotal A s the amount of carrier added to the culture broth increased, the adhesion increased steadily, finally approaching 10%. Under these conditions, almost all the microbial cells adhere to the hydrophilic carriers, and very few cells remain suspended in the culture broth when there is more than log of carrier per liter of the medium. For the hydrophobic carrier, the % adhesion similarly increased as the amount of carrier increased. However, it was interesting to note that the amount of microbial cells adhering to the carrier was restricted, and significant number of microbial cells were suspended freely in the culture broth. From these adhesion experiments we conclude that the experimental results are in good agreement with the theoretical predictions.
-
5.
CONCLUSIONS
While developing a bioreactor system to produce a microbial biosurfactant using immobilized P. spiculisporum, particularly selecting porous carriers to
168
n
20
E
E
V
c
0
-0
2%
0
-20
-30
0
100
50
Figure 5. Theoretical prediction on the feasibility of adhesion of the cells of Penicillum spiculisporum to the hydrophobic and hydrophilic carriers.
n
100
-
A
u
Pn
A -
-
./”’
A
0
-A-
hydrophilic carrier
-1
11- hydrophilic carrier-0-hydrophobic carrier
\ 0
2
-
0 0
5
10
15
amount o f carriers added C g / I > Figure 6. Experimental results of cell adhesion onto both hydrophobic and hydrophilic carriers.
169 which the microbial cells can adhere and become immobilized, we gave particular attention to the theoretical analysis of the adhesion. The feasibility of cell adhesion or immobilization to porous solid carriers could be predicted as a function of the hydrophobicity of the solid surface according to the equation (6); this equation is derived from surface and interfacial free energy balance in relation to the adhesion under conditions where electrical charge interaction is negligible. We believe that the thermodynamic approach may offer a powerful tool to predict adhesion or adsorption of microbial cells onto porous geological materials encountered in common waterflooding operations. 6.
REFERENCES
1. F. London, Z. Physik. 63 (1930) 245; Z. Phys. Chem., B11 (1930) 222. 2 . F.M. Fowkes, Ind. Eng. Chem. 12 (1964) 40. 3 . H.J. Busscher, A.H. Weerkamp, H.C. van der Mei, A.W.J. van Pelt, H.P. de Jong and J. Arends, Appl. Environ. Microbiol., 48 (1984) 980. 4. J.E. Zajic and T. Ban, Microbes and Oil Recovery, J.E. Zajic and E.C.Donaldson (eds.), Bioresource Publ., El Paso, Texas, 1985. 5. H.W. Fox and W.A. Zisman, J. Colloid Sci., 5 (1950) 514. 6. H.J. Busscher and J. Arends, J. Colloid Interface Sci., 81 (1981) 75.
Adhesion of Microbial Cells to Porous Hydrophilic and Hydrophobic Solid Substrata Takayoshi Ban and Shinjiro Yamamoto Chemical Engineering Department, Shizuoka University, 5-1, Johoku 3-chome, Hamamatsu, Shizuoka, Japan 432
Abstract Adhesion of microbial cells to solid surfaces is thought to significantly influence the growth and transport of microbes in porous geological materials. The feasibility of cell adhesion to a porous solid substratum can be predicted as a function of the hydrophobicity of the substratum according to the following equation derived from the balance of surface and interfacial free energy in relation to adhesion under conditions where the electrical charge interaction is negligible:
This thermodynamic approach provides us with the following predictions: (1) the more hydrophilic microbes may be expected to adhere more favorably to the hydrophilic surfaces of a solid substratum, and 2 ) the more hydrophobic microbes will adhere more favorably to the hydrophobic surfaces of a solid substratum. To experimentally verify the theoretical predictions, a series of experimcnts were carried out to investigate the influence of hydrophobicity of solid substrata upon the adhesion of growing cells of Penicillium spiculisporum ATCC 16071 using either a typically hydrophobic or hydrophilic solid substratum. The results revealed that nearly 100% of the growing cells of P. spiculisporum adhered to the hydrophilic substrata; by contrast, most of the cells were suspended freely in the culture broth when hydrophobic polyurethane foamwas used as an adhesive substrate. These results are in reasonable agreement with the results of the theoretical prediction, and we conclude that the thermodynamic approach offers a powerful tool to predict microbial adhesion onto solid substrates.
1. INTRODUCTION Adhesion or adsorption phenomenon of microbial cells to solid surfaces is thought to significantly influence the growth and transport of microbes in or through porous geologic materials. Such a phenomenon may be regarded as a prerequisite for microbial plugging, particularly in the initial stage, associated with conventional waterflooding operations for oil recovery from reservoirs. Adhesion of bacteria or other microorganisms to surfaces of geological materials is thought to involve the physiocochemical surface characteristics of the solid surface, such as charge and hydrophobicity. A thermodynamic approach offers a powerful tool to predict microbial adhesion to a solid substratum under conditions where electrical charge interaction can be neglected.
160
In this study, a theoretical prediction was made on the basis of thermodynamics about the possibility of particular microbial cells adhering onto both hydrophilic and hydrophobic solid substrata. To verify the theoretical predictions, a series of adhesion experiments were made using either typically hydrophilic or hydrophobic solid substrata to investigate the influence of hydrophobicity, and surface and interfacial free energy of the solid substrata upon the numbers of microbial cells adhering to the substrata during their growth. 2.
THEORETICAL CONSIDERATIONS
Suppose a material, M , suspended or dispersed freely in an aqueous liquid, L, adheres to the surface of a solid substratum, S . A change in the interfacial free energy during the process of adhesion of material M to the surface of solid substratum S , AG,,,, can then be written as equation (1) based on the interfacial free energy balance before and after adhesion under conditions where the electrical charge interaction is negligible.
, and ysL are interfacial free energies between solid S and material S and liquid L, respectively. Equation (1) predicts that adhesion may be expected if AGadh is less than zero (AGadh < o ) , whereas adhesion is energetically unfavorable if AG,,, is greater than zero (AGacih > 0). To estimate AGadh, the change in free energy in the adhesion process, it is necessary to determine the interfacial free energies, three terms found on the right-hand side of equation (1) according to the surface chemical approaches. One useful approach is based on separation of the surface free energy into two components--a dispersion component y d , and a polar component yp. Surface free energy is essentially caused by attractive force acting between molecules of the material. This intermolecular attractive force can be separated into the following two components: (1) a dispersion force proposed by London in 1930, y d , [ l ] and (2) the attractive forces due to the polarity of the molecules involved, yp. Thus surface free energy of, for example, material A, yAv, can be expressed as : where
ySM, ym
M , material M and liquid L, and solid
Yav = Y":
+
YZV
Similarly, the surface free energy of material B , ysv, can be written as: YEW =
Yt"
+
Y$V
According to Fowkes' approach [2] the dispersion and polar components of interfacial free energy between any two surfaces A and B can be expressed as follows on the basis of the geometric mean equation:
161
Consequently, AGadh in equation (1) can be written as follows by using equation:
or rearranged:
The influence of the surface characteristics of the solid substratum upon the adhesion of material M is subsequently described by the following equation obtained by differentiating equation ( 4 ) with respect to y& and y z v :
Our experimental determination showed the value of y& to be approximate1 40 mN/m for almost all solid substrata we used. Therefore, we can assumedy,,J to be zero, and equation ( 5 ) can be simplified to equation ( 6 ) :
Equation ( 6 ) is a useful theoretical expression for predictingmicrobial adhesion onto the solid substrata when material M is regarded as microbial cell. From equation ( 6 ) , it appears ( 3 1 : 1) If rPw > ~ P L v , (namely, if the microbial cell is more hydrophilic): The left-hand side of the equation ( 6 ) becomes less than zero as T~~ increases because ( y f ~ ) ~ ’ ’ is less than (yh)’” . Therefore, AGadh becomes less than zero. This finding means that the adhesion of microbial cells onto surfaces of the solid substrata may become energetically more favorable as TSV increases. In other words, adhesion may be expected as the surface of the solid substratum becomes more hydrophilic. Therefore, more hydrophilic microbes may be expected to adhere more favorably to the surface of the solid substratum if the surface
162
Table 1 Variations of parameters influencing AGadh . ySV was variable throughout Y LV
YMV
Simulation I
44 mN/m
(1) 1 0 5 mN/m (2) 8 6 mN/m (3) 6 9 mN/m ( 4 ) 5 5 mN/m
Simulation I1
5 6 mN/m
(1) 1 0 5 (2) 86 ( 3 ) 69 ( 4 ) 55
Simulation I11
6 8 mN/m
(1) 1 0 5 mN/m (2) 8 6 mN/m (3) 6 9 mN/m ( 4 ) 5 5 mN/m
mN/m mN/m mN/m mN/m
becomes more hydrophilic and vice versa. Therefore, we conclude that more hydrophilic microbes may be expected to adhere more favorably to hydrophilic surfaces of solid substrata. 2) By contrast, if y h < y b , namely, if the microbial cell is more hydrophobic, then d (AGO,,,,) / dy,, becomes more than zero as ysv increases. Therefore, more hydrophobic microbes can be expected to adhere more favorably to hydrophobic surfaces of the solid substrata. To clarify the results of the prediction from equation ( 6 ) , a simulation was made to investigate the factors influencing adhesion of microbial cells suspended freely in an aqueous liquid to surfaces of solid substrata having different surface properties. The three major parameters in equation ( 6 ) which may affect the adhesion of microbial cells are the surface free energies of solid, 7sV, the microbial cell, yMV,and the liquid, yLv. Simulations were made under conditions where these three parameters were independently varied to demonstrate the influence of each parameter upon microbial adhesion. Table 1 shows range of variation of each parameter for the simulation. The results of the simulation are illustrated schematically in Figure 1 through Figure 3 as plots of AGadh against ysv with the parameter yMV. Simulation I was made under the conditions where 1) the surface free energy of the liquid, y ~ was ~ ,44 mN/m, and 2 ) the surface free energy of the microbial cells, yMV,ranged between 1 0 5 mN/m and 5 5 mN/m, which covers microbial cells having surface properties from extremely hydrophilic to hydrophobic. The results are illustrated schematically in Figure 1, where AGadh is plotted against 7sv. This figure shows that the area where ysv is lower than 50 mN/m, or the surface of the solid substrata is relatively hydrophobic, AG,& becomes greater than zero for cells (l), ( 2 ) , and ( 3 ) , which covers cells having hydrophilic surfaces. Thus, adhesion does not occur spontaneously in this area. A s ysv increases, adhesion may become energetically more favorable Since AGadh becomes less than zero. Simulation I1 was made at ytv = 5 2 mN/m; in other words, yLv is greater than that of Simulation I, but other conditions are the same as before. S O far as
163
Figure 1. Simulation I.
20
n
.
10
E
Z
€
0
condi lions : 5 2 mNlm
yLv=
U
c
;-lo
y M v = (1) 105 rnNlm
0
a
(2) 8 6 m N l m
- 20
(3) 69rnNlrn ( 4 ) 55mN/rn
-30
20
0
8
8
n
Figure 3. Simulation I11
condi Iions :
YLV=6 0 mNlm
U
0
-a
-20 -
Figure 2. Simulation I1
yMv= (1)
1 0 5 mNlm
(2)
86 m N l m
( 3 ) 6 9 rnNlrn ( 4 ) 5 5 mNlm
164
Table 2 The size and porosity of the carriers Size
Porosity
hydrophilic carrier - 1
2.5 x 2 . 5 x 2.5 mm cubic
97%
hydrophilic carrier
5 mm diameter, spherical
97%
hydrophilic microbial cells, or cells (l), (2), and ( 3 ) are concerned, similar trends are observed as those in Simulation I (Figure 2). Figure 3 indicates the result of Simulation I11 made at -yLv = 60 mN/m, which means that the surface tension of the liquid medium is greater than those of Simulations I and 11, but other conditions are the same as before. Almost completely similar curves of AGadh as function of -ysv were obtained as those of Simulation 11. In this case, it is also apparent that for hydrophilic cells with a -yMv to be greater than 70 mN/m, adhesion may become energetically more favorable than an increase in the surface free energy of a solid surface. 3. EXPERIMENTS To verify the prediction made from the thermodynamic considerations in equation ( 6 ) , an experimental study was made to investigate the influence of hydrophobicity, or surface and interfacial free energy of solid substrata upon the number of microbial cells adhered to the substrata during microbial growth. 3.1. Microorganism Penicillium spiculisporum ATCC 16071, a fungus, was used in this experimental study. This microbe produces 4-hydroxy-4,5-dicarboxypentadecanoic acid, one of the microbial biosurfactants, during aerobic growth on glucose [ 4 ] . While developing a bioreactor system for producing the biosurfactant using immobilized P . spiculisporum and selecting porous carriers onto which the microbial cells adhered and became immobilized, a focus of attention was given to the theoretical analysis of adhesion phenomenon of microbial cells onto solid substrata.
3.2. Composition of growth medium The growth medium used for P. spiculisporum consisted of 10% glucose, 0.1% N H 4 C 1 , 0.1% KHzP04, 0.02% Mg S 0 4 7 H z 0 , 0.1% peptone, and 0.1%yeast extract. 3.3. Porous carriers Two types of porous polymers were used as solid substrata or microbe adhesion carriers for P. spiculisporum. One was a commercial polyurethane foam, which is a typical hydrophobic porous carrier, hereafter called the hydrophobic carrier. The other was a typical hydrophilic porous carrier prepared from natural cellulose, and referred to as the hydrophilic carrier - 1. The size and porosity of both the above porous carriers are shown in Table 2. Gelatin-treated polyurethane foam also was used as a hydrophilic carrier, which was prepared by coating the surface of the commercial polyurethane foam with gelatin film, and thus giving a relatively hydrophilic surface: We called this hydrophilic carrier - 2.
165
3.4. Determination of surface free energy 3.4.1. Surface free energy of liquid The surface free energy, or surface tension of liquid, rLv,can be easily and directly measured by using a proper tensiometer. In this study we used Wilhelmy’s vertical plate method. 3.4.2. Surface free energy of hydrophobic solid substratum The surface free energy, or surface tension of the hydrophobic solid substratum (the hydrophobic carrier), rSv, was determined as critical surface tension, -yc, estimated from a Zisman plot [ 5 ] by measuring the contact angle. 3.4.3. Surface free energy of hydrophilic solid substrata and microbial cells Determination of the surface free energy of both the hydrophilic solid substrata, rSv, and the microbial cells, y M v , was made using the method developed by Busscher and Arends [ 6 ] on the basis of the following equation: cos
e
= -16.74 (yps,jl/’
02
+
2(y&)11’
+
3.996
( ~ p s ~ ) ~ / D~ ’ +
0.002 (ypgV)l’’
-
1
(7)
where,
3.5. Growth and adhesion experiments Growth experiments with P. spiculisporum were made using a 5 0 0 m l flask containing 100 m l of growth medium and a specified amount of pieces of either hydrophobic or hydrophilic carrier. The flask was autoclaved, inoculated, and then placed on a reciprocal shaker operated at 120 strokes per minute at 3OoC to allow the microbe to grow. After reaching almost stationary phase of growth, after 12 days cultivation, the amount of cells adhering to the carriers, and those suspended freely in the culture broth were weighed. 4. EXPERIMENTAL RESULTS
4.1. Experimental determination of surface free energies 4.1.1. Zisman plot Figure 4 shows Zisman plots of the hydrophobic carrier used in this experiment and of several commercial synthetic polymers including polyethylene, which is a typical non-polar compound. The value of the surface free energy, rSv, of the hydrophobic carrier obtained from the Zisman plot, in other words, the value of its critical surface free energy, rC, was estimated to be 44 mN/m. 4.1.2. Results of experimentally determined surface free energies of liquid and solid materials Table 3 shows the experimentally determined surface free energies of the culture broths, of the solid substrata includinghydrophilic carriers, and of the microbial cells. Surface free energies of culture broth at the initial and final stages of cultivation measured directly by using Wilhelmy’s vertical plate method were 60 mN/m and 44 mN/m, respectively.
166
Table 3 Experimentally determined surface free energies
(1) initial stage (2) final stage
Culture Broth
7Lv = 60 mN/m YLV
- 44
Hydrophobic Carrier
Ysv
- 4 4 mN/m
Hydrophilic Carrier 1
ySv
Hydrophilic Carrier 2
ySv
Microbial Cells
yMY = 101 mN/m
- 123 mN/m - 110 mN/m
The ySv values of the hydrophilic carriers 1 and 2 were determined from equation (7) by measuring the contact angle, and were 123 mN/m, and 110 mN/m, respectively. The ySv value of the hydrophobic carrier was much less than those of hydrophilic carriers, and was estimated to be 44 mN/m from the Zisman plot (Figure 4 ) .
1
30 40 50 60 70 80 Y L V CmN/m>
ysv=
pol yet hy Iene
(
polyvinyl chloride
(y,,
polyacryl resin
(
= 40 mNlm
pol ycarbona te
ysv= ( ysv=
hydrophobic carrier
(
y,,
3 9 mNlm
41 mNlm 4 3 mNlm
= 4 4 mNlm
Figure 4 . Zisman plots of various hydrophobic polymers including the one used in this study.
167
The surface free energy of the microbial cells used in this study was 101 mN/m, as determined by measurement of the contact angle. Thus, the cells of P. spiculisporum are extremely hydrophilic.
4.2. Simulation By using the experimentally determined values of surface free energies, a theoretical prediction of the feasibility of adhesion of the microbial cells to both hydrophobic andhydrophilic carriers was made. The result ofthe simulation is illustrated in Figure 5 . The shaded curve indicates the resultant prediction under our experimental conditions. Thus, AG,& for the hydrophobic carrier (ysv 44 mN/m) is estimated to be greater than zero. Therefore, the microbial cells may be expected to adhere less successfully to the hydrophobic carrier. However, in the case of both hydrophilic carriers, the surface free energies were 123 mN/m and 110 mN/m, respectively, and thus AGadh becomes much lower than zero for both carriers, and the adhesion of the microbe becomes energetically more favorable. A series of growth and adhesion experiments were carried out to verify the above theoretical prediction experimentally.
-
4.3.
Results of the growth and adhesion experiment The growth of P. spiculisporum and adhesion of the microbial cells to hydrophobic or hydrophilic carriers are illustrated in Figure 6 , which gives the % adhesion of cells on either carrier. The % adhesion is plotted as a function of the amount of carrier added in the culture broth before starting cultivation. The % adhesion in defined in the following equation:
where, Madh is the amount of microbial cells adhered to the carrier, Mfree is the amount of microbial cells suspended freely in the broth, and Mtotal is the total Madh + Mfree. amount of cells grown, expressed as: Mtotal A s the amount of carrier added to the culture broth increased, the adhesion increased steadily, finally approaching 10%. Under these conditions, almost all the microbial cells adhere to the hydrophilic carriers, and very few cells remain suspended in the culture broth when there is more than log of carrier per liter of the medium. For the hydrophobic carrier, the % adhesion similarly increased as the amount of carrier increased. However, it was interesting to note that the amount of microbial cells adhering to the carrier was restricted, and significant number of microbial cells were suspended freely in the culture broth. From these adhesion experiments we conclude that the experimental results are in good agreement with the theoretical predictions.
-
5.
CONCLUSIONS
While developing a bioreactor system to produce a microbial biosurfactant using immobilized P. spiculisporum, particularly selecting porous carriers to
168
n
20
E
E
V
c
0
-0
2%
0
-20
-30
0
100
50
Figure 5. Theoretical prediction on the feasibility of adhesion of the cells of Penicillum spiculisporum to the hydrophobic and hydrophilic carriers.
n
100
-
A
u
Pn
A -
-
./”’
A
0
-A-
hydrophilic carrier
-1
11- hydrophilic carrier-0-hydrophobic carrier
\ 0
2
-
0 0
5
10
15
amount o f carriers added C g / I > Figure 6. Experimental results of cell adhesion onto both hydrophobic and hydrophilic carriers.
169 which the microbial cells can adhere and become immobilized, we gave particular attention to the theoretical analysis of the adhesion. The feasibility of cell adhesion or immobilization to porous solid carriers could be predicted as a function of the hydrophobicity of the solid surface according to the equation (6); this equation is derived from surface and interfacial free energy balance in relation to the adhesion under conditions where electrical charge interaction is negligible. We believe that the thermodynamic approach may offer a powerful tool to predict adhesion or adsorption of microbial cells onto porous geological materials encountered in common waterflooding operations. 6.
REFERENCES
1. F. London, Z. Physik. 63 (1930) 245; Z. Phys. Chem., B11 (1930) 222. 2 . F.M. Fowkes, Ind. Eng. Chem. 12 (1964) 40. 3 . H.J. Busscher, A.H. Weerkamp, H.C. van der Mei, A.W.J. van Pelt, H.P. de Jong and J. Arends, Appl. Environ. Microbiol., 48 (1984) 980. 4. J.E. Zajic and T. Ban, Microbes and Oil Recovery, J.E. Zajic and E.C.Donaldson (eds.), Bioresource Publ., El Paso, Texas, 1985. 5. H.W. Fox and W.A. Zisman, J. Colloid Sci., 5 (1950) 514. 6. H.J. Busscher and J. Arends, J. Colloid Interface Sci., 81 (1981) 75.