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Application of cryo-cut method for measurements of biofilm thickness
Water Res. Vol. 16, pp. 1619 to 1621, 1982 Printed in Great Britain. All rights reserved
0043-1354/82/121619-03503.00/0 Copyright © 1982 Pergamon Press Ltd
TECHNICAL NOTE A P P L I C A T I O N O F C R Y O - C U T M E T H O D FOR MEASUREMENTS OF BIOFILM THICKNESS GERT HOLM KRISTENSEN1. and FREDE R. CHRISTENSEN2 t Department of Sanitary Engineering and 2Department of Biochemistry and Nutrition, The Technical University of Denmark, 2800 Lyngby, Copenhagen, Denmark (Received May 1981)
Abstraet--A general histological embedding and cutting technique has proved successful for determination of biofilm thickness. Microscope pictures of cross-sections of the biofilms are taken and they yield information about the micro-structure of the films. Examples of this are given.
INTRODUCTION
crometer screw. Recognition of the surface and basal part of the biofilm is, in some investigations, made by microscopic focusing (Sanders, 1966; Kornegay &
For several years investigations on biofilm kinetics have taken place at the Department of Sanitary Engineering, The Technical University of Denmark. The first experiments made for verification of the halforder model (Harremo~s, 1976) were performed with denitrification in a down-flow pilot plant filter (Riemer & Harremo~s, 1978). However such filters are inadequate for fundamental investigations on biofilm kinetics, mainly because measurements of significant parameters, e.g. biofilm thickness, were impossible. In order to establish well defined circumstances for investigation of the different processes in bacterial films, a laboratory scale reactor was constructed. The reactor is based on the same principles as those described by Kornegay & Andrews (1968) and La Motta (1976). Details of the reactor construction and performance are given in Kristensen & Jansen (1980). The reactor volume ( - 1 l.) is situated between two cylinders of which the inner cylinder is rotating to ensure total mixing in the reactor. The biofilm is grown on the cylinder walls. As integrated parts of the outer, stationary cylinder wall, four dovetailed pieces are constructed. They can be taken out and used for measurements of the biofilm thickness, The biofilm thickness gives the maximum distance for diffusional transportation of substrates in the biofilm. With this parameter measured an explicit determination can be made of biofilm parameters such as an intrinsic reaction rate and a diffusion coefficient in the biofilm (see Harremo~s, 1976). In the literature different methods for measurements of the biofilm thickness are given, C o m m o n to the above methods is the measuring of the biofilm thickness by means of a calibrated mi*Present address: Akvadan Inc., Kroegshoegvej 29, 2880 Bagsvaerd, Denmark. tLab.-Tek. Products, Division Miles Laboratories Inc., Naperville IL 60540, U.S.A.
Andrews, 1968; La Motta, 1976). Another way is an electrical current initiated by contact with the biofilm surface and increased by contact with the support media (Hoehn & Ray, 1973). When applying these methods, difficulties in exact focusing or the presence of a liquid layer on the biofilm surface, may lead to significant uncertainties in the determinations. These problems are overcome with the cryo-cut method.
DESCRIPTION OF THE CRYO-CUT METHOD The fixed film reactor used for laboratory experiments is provided with four pieces integrated in the outer tube wall. The pieces can be taken out of the reactor through holes in the top cover. The four tube pieces are covered with a waterproof transparent tape before innoculation of the reactor walls. The thickness of the tape is very small ( < 100 #m) which means that the hydraulic conditions around the pieces are unaffected by the tape. This is in agreement with observations from growth experiments. When the biofilm thickness is to be measured a bit of tape is cut off one of the tube pieces. This biofilm sample is placed in a 49/o formaldehyde solution for preservation. The determination of the biofilm thickness is made as shown in Fig. 1. From the biofilm sample a piece of 2 x 10 mm is taken and is embedded in Tissue-Tek II* in a cryo-cut microtome at - 2 5 ° C . Thin slices (approx. 50-150 p m ) a r e cut and photographed in an interference contrast microscope, where the biofilm can be seen very distinctly without staining. The biofilm thickness is measured at the photos. Examples of cross-sections of biofilms are shown in Figs 2 and 3. The bubbles shown in the photographs appear in the embedding media as a result of the procedure.
1619
1620
Technical Note
biofilrn
embedding
Q~ Biofilm specimen
f~J
giofilrn specimen placed ~n a drop of embedding media (at the freezing tabte)
-
Biofilrn specimen embedded at the tapestde
f--'J
Biof~m specimen totally embedded
embedded biofilm
~ - ~ " ~
pecimen
freezing table
/
~
,~,~7
embedding 1 media
Knife and freezing table
Schematical magnification of a thin slice of the biotilm
seen from top
specimen
Fig. 1. Embedding and cutting of the biofilm specimen. The optimum thickness of the slices is not the same for all biofilms. As a main principle there has to be a "reasonable" ratio between the biofilm thickness and the thickness of the slice. If thick slices are cut of a
thin biofilm there is a risk that the biofilm slice will capsize on the object glass. For biofilm thicknesses greater than around 2001tm slice thicknesses of 100-150/~m have given good results. Biofilms with
Fig. 2. Cross-section of a biofilm, biofilm thickness approx. 4501~m. Calcium phosphate precipitation in the rear, dark zone.
Fig. 3. The biotilm section from Fig. 2 after treatment wit,, 10",, acetic acid. Dissolution of the calcium phosphate is observed.
Technical Note
Fig. 4. Illustration of a hollow floc from fluidized bed illter. "Shell thickness" approx. 400/~m. smaller thicknesses will have to be cut with a smaller slice thickness (smaller than the biofilm thickness). EXAMPLES OF MICRO-STRUCTURE INFORMATION One method currently in progress at the Department of Sanitary Engineering, deals with precipitation of calcium phosphate in biofilms. As a consequence of biological denitrification, alkalinity is produced in the biofilm. Caused by diffusional resistance the alkalinity is built up in the biofilm, thereby creating a pH-rise with increasing distance from the biofilm surface (Riemer & Harremo~s, 1978). Experimental results have shown that the pH-rise might induce precipitation of calcium phosphate in the biofilm (Arvin & Kristensen, 1982). Cross-sections from these biofilms showed a dense layer in the rear part of the biofilm (Fig. 2). After adding a few drops of 10% acetic acid to the slices, the dark (dense) zone disappeared after some minutes corresponding to dissolution of the calcium phosphate. Figure 3 shows the slice from Fig. 2 after treatment with acetic acid. This is interpreted as a demonstration of a precipitation zone in the rear part of the biofilm in agreement with what would be expected from theoretical considerations. Another method deals with performance of biological denitrification in a fluidized bed reactor. The flui-
1621
dized bed reactor is run without any support media, thus fluidizing biological flocs only. Experiments have shown that the flocs grow to several mm in diameter (Hansen & Kirkegaard, 1980). When investigating the micro-structure of these ftocs by embedding and cryocutting the flocs, it was revealed that the flocs were hollow. Figure 4 shows a sector of one of these flocs illustrating the shell structure. The cavity is believed to be a result of endogeneous decomposition of the innermost organisms, because all nutrients are consumed by the outer organisms. Evidence of the microstructure of the flocs is considered a piece of valuable information when modelling kinetics of the reactor performance.
CONCLUSION The cryo-cut method has proved successfull in determination of the biofilm thickness. As illustrated there is potential of applying the method for investigation of various fixed film micro-structured phenomena.
REFERENCES Arvin E. & Kristensen G. H. (1982) Phosphate precipitation in biofilms and flocs. Water Sci. Technol. In press. Hansen J. & Kirkegaard C. (1980) Denitrification in a fluidized bed filter without support media. M.S. rhesis, Department of Sanitary Engineering, Technical University of Denmark (in Danish). Harremo~s P. (1976) The significance of pore diffusion to filter denitrification. J. War. Pollut. Control Fed. 48, 2. Hoehn R. & Ray A. D. (1973) Effects of thickness on bacterial film, J. Wat. Pollut. Control Fed. 45, 11. Kornegay B. H. & Andrews J. F. (1968) Kinetics of fixed film biological reactors. J. Wat. Pollut. Control Fed. 40, 11. Kristensen G. H. & la Cour Jansen J. (1980) Fixed film kinetics-description of laboratory equipment. Department of Sanitary Engineering, Technical University of Denmark. La Motta E. J. (1976) Internal diffusional and reaction in biological films. Envir. Sci. Technol. 10, 8. Riemer M. & Harremo6s P. (1978) Multi-component diffusion in denitrifying biofilms. Prog. Wat. Technol. 10. Sanders W. M. (1966) Oxygen utilization by slime organisms in continuous culture. Air War. Pollut. 10.
0043-1354/82/121619-03503.00/0 Copyright © 1982 Pergamon Press Ltd
TECHNICAL NOTE A P P L I C A T I O N O F C R Y O - C U T M E T H O D FOR MEASUREMENTS OF BIOFILM THICKNESS GERT HOLM KRISTENSEN1. and FREDE R. CHRISTENSEN2 t Department of Sanitary Engineering and 2Department of Biochemistry and Nutrition, The Technical University of Denmark, 2800 Lyngby, Copenhagen, Denmark (Received May 1981)
Abstraet--A general histological embedding and cutting technique has proved successful for determination of biofilm thickness. Microscope pictures of cross-sections of the biofilms are taken and they yield information about the micro-structure of the films. Examples of this are given.
INTRODUCTION
crometer screw. Recognition of the surface and basal part of the biofilm is, in some investigations, made by microscopic focusing (Sanders, 1966; Kornegay &
For several years investigations on biofilm kinetics have taken place at the Department of Sanitary Engineering, The Technical University of Denmark. The first experiments made for verification of the halforder model (Harremo~s, 1976) were performed with denitrification in a down-flow pilot plant filter (Riemer & Harremo~s, 1978). However such filters are inadequate for fundamental investigations on biofilm kinetics, mainly because measurements of significant parameters, e.g. biofilm thickness, were impossible. In order to establish well defined circumstances for investigation of the different processes in bacterial films, a laboratory scale reactor was constructed. The reactor is based on the same principles as those described by Kornegay & Andrews (1968) and La Motta (1976). Details of the reactor construction and performance are given in Kristensen & Jansen (1980). The reactor volume ( - 1 l.) is situated between two cylinders of which the inner cylinder is rotating to ensure total mixing in the reactor. The biofilm is grown on the cylinder walls. As integrated parts of the outer, stationary cylinder wall, four dovetailed pieces are constructed. They can be taken out and used for measurements of the biofilm thickness, The biofilm thickness gives the maximum distance for diffusional transportation of substrates in the biofilm. With this parameter measured an explicit determination can be made of biofilm parameters such as an intrinsic reaction rate and a diffusion coefficient in the biofilm (see Harremo~s, 1976). In the literature different methods for measurements of the biofilm thickness are given, C o m m o n to the above methods is the measuring of the biofilm thickness by means of a calibrated mi*Present address: Akvadan Inc., Kroegshoegvej 29, 2880 Bagsvaerd, Denmark. tLab.-Tek. Products, Division Miles Laboratories Inc., Naperville IL 60540, U.S.A.
Andrews, 1968; La Motta, 1976). Another way is an electrical current initiated by contact with the biofilm surface and increased by contact with the support media (Hoehn & Ray, 1973). When applying these methods, difficulties in exact focusing or the presence of a liquid layer on the biofilm surface, may lead to significant uncertainties in the determinations. These problems are overcome with the cryo-cut method.
DESCRIPTION OF THE CRYO-CUT METHOD The fixed film reactor used for laboratory experiments is provided with four pieces integrated in the outer tube wall. The pieces can be taken out of the reactor through holes in the top cover. The four tube pieces are covered with a waterproof transparent tape before innoculation of the reactor walls. The thickness of the tape is very small ( < 100 #m) which means that the hydraulic conditions around the pieces are unaffected by the tape. This is in agreement with observations from growth experiments. When the biofilm thickness is to be measured a bit of tape is cut off one of the tube pieces. This biofilm sample is placed in a 49/o formaldehyde solution for preservation. The determination of the biofilm thickness is made as shown in Fig. 1. From the biofilm sample a piece of 2 x 10 mm is taken and is embedded in Tissue-Tek II* in a cryo-cut microtome at - 2 5 ° C . Thin slices (approx. 50-150 p m ) a r e cut and photographed in an interference contrast microscope, where the biofilm can be seen very distinctly without staining. The biofilm thickness is measured at the photos. Examples of cross-sections of biofilms are shown in Figs 2 and 3. The bubbles shown in the photographs appear in the embedding media as a result of the procedure.
1619
1620
Technical Note
biofilrn
embedding
Q~ Biofilm specimen
f~J
giofilrn specimen placed ~n a drop of embedding media (at the freezing tabte)
-
Biofilrn specimen embedded at the tapestde
f--'J
Biof~m specimen totally embedded
embedded biofilm
~ - ~ " ~
pecimen
freezing table
/
~
,~,~7
embedding 1 media
Knife and freezing table
Schematical magnification of a thin slice of the biotilm
seen from top
specimen
Fig. 1. Embedding and cutting of the biofilm specimen. The optimum thickness of the slices is not the same for all biofilms. As a main principle there has to be a "reasonable" ratio between the biofilm thickness and the thickness of the slice. If thick slices are cut of a
thin biofilm there is a risk that the biofilm slice will capsize on the object glass. For biofilm thicknesses greater than around 2001tm slice thicknesses of 100-150/~m have given good results. Biofilms with
Fig. 2. Cross-section of a biofilm, biofilm thickness approx. 4501~m. Calcium phosphate precipitation in the rear, dark zone.
Fig. 3. The biotilm section from Fig. 2 after treatment wit,, 10",, acetic acid. Dissolution of the calcium phosphate is observed.
Technical Note
Fig. 4. Illustration of a hollow floc from fluidized bed illter. "Shell thickness" approx. 400/~m. smaller thicknesses will have to be cut with a smaller slice thickness (smaller than the biofilm thickness). EXAMPLES OF MICRO-STRUCTURE INFORMATION One method currently in progress at the Department of Sanitary Engineering, deals with precipitation of calcium phosphate in biofilms. As a consequence of biological denitrification, alkalinity is produced in the biofilm. Caused by diffusional resistance the alkalinity is built up in the biofilm, thereby creating a pH-rise with increasing distance from the biofilm surface (Riemer & Harremo~s, 1978). Experimental results have shown that the pH-rise might induce precipitation of calcium phosphate in the biofilm (Arvin & Kristensen, 1982). Cross-sections from these biofilms showed a dense layer in the rear part of the biofilm (Fig. 2). After adding a few drops of 10% acetic acid to the slices, the dark (dense) zone disappeared after some minutes corresponding to dissolution of the calcium phosphate. Figure 3 shows the slice from Fig. 2 after treatment with acetic acid. This is interpreted as a demonstration of a precipitation zone in the rear part of the biofilm in agreement with what would be expected from theoretical considerations. Another method deals with performance of biological denitrification in a fluidized bed reactor. The flui-
1621
dized bed reactor is run without any support media, thus fluidizing biological flocs only. Experiments have shown that the flocs grow to several mm in diameter (Hansen & Kirkegaard, 1980). When investigating the micro-structure of these ftocs by embedding and cryocutting the flocs, it was revealed that the flocs were hollow. Figure 4 shows a sector of one of these flocs illustrating the shell structure. The cavity is believed to be a result of endogeneous decomposition of the innermost organisms, because all nutrients are consumed by the outer organisms. Evidence of the microstructure of the flocs is considered a piece of valuable information when modelling kinetics of the reactor performance.
CONCLUSION The cryo-cut method has proved successfull in determination of the biofilm thickness. As illustrated there is potential of applying the method for investigation of various fixed film micro-structured phenomena.
REFERENCES Arvin E. & Kristensen G. H. (1982) Phosphate precipitation in biofilms and flocs. Water Sci. Technol. In press. Hansen J. & Kirkegaard C. (1980) Denitrification in a fluidized bed filter without support media. M.S. rhesis, Department of Sanitary Engineering, Technical University of Denmark (in Danish). Harremo~s P. (1976) The significance of pore diffusion to filter denitrification. J. War. Pollut. Control Fed. 48, 2. Hoehn R. & Ray A. D. (1973) Effects of thickness on bacterial film, J. Wat. Pollut. Control Fed. 45, 11. Kornegay B. H. & Andrews J. F. (1968) Kinetics of fixed film biological reactors. J. Wat. Pollut. Control Fed. 40, 11. Kristensen G. H. & la Cour Jansen J. (1980) Fixed film kinetics-description of laboratory equipment. Department of Sanitary Engineering, Technical University of Denmark. La Motta E. J. (1976) Internal diffusional and reaction in biological films. Envir. Sci. Technol. 10, 8. Riemer M. & Harremo6s P. (1978) Multi-component diffusion in denitrifying biofilms. Prog. Wat. Technol. 10. Sanders W. M. (1966) Oxygen utilization by slime organisms in continuous culture. Air War. Pollut. 10.