Biotechnol. Prog. 1992, 8, 165-166
185
An Improved Method for Disruption of Microbial Cells with Pressurized Carbon Dioxide Ho-Mu Lin,*Zhiying Yang, and Li-Fu Chen Department of Food Science, Smith Hall,Purdue University, West Lafayette, Indiana 47907 Disruption of microbial cells by pressurized carbon dioxide at both subcritical and supercritical temperatures has been previously investigated. This method differs in principle from other disruption techniques and was found to have potential applications for rupture of a variety of microorganisms. However, it is not as effective for some of the microbial cells, including yeast, of which the cell walls are extremely robust and rigid. This work suggests an alternative operation to improve the disruption rates of cells by repeatedly releasing the applied fluid pressure within the cells in the midst of a disruption process. The improvement is substantial at aU the experimental conditions studied.
Introduction Microbial cells have long been recognized as an important source of commercially useful biochemicals, antibiotics, and enzymes (1). With increasing demand for microbial products in industry and medicine, considerable effort has recently been committed to develop technologies for production of intracellular microbial materials from microorganisms and various genetically altered cultures. Release of cell contents is vital also to many investigations of bacterial metabolism. In any case, the cell disruption is a necessary operation for isolation or recovery of intracellular enzymes and organelles. A variety of disruption techniques have been developed (1-3) and some are available commercially. Mechanical methods appear to be favored at the present time for their economic advantages, although several nonmechanical methods, particularly enzymatic lysis, have attracted great attention. Nevertheless, many of these methods often ensue with degradation or denaturation of proteins in the process. A technique to exploit supercritical fluid (SCF) for disruption of microbial cells has recently been developed in this laboratory (4). The process involves a sudden release of the applied fluid pressure that follows penetration of SCF into cells. The expansion of SCF within the cells, when it is released, forces the breakage of microorganisms. It is simple, inexpensive, and, more importantly, noninjurious to enzyme activities. The functional properties of proteins are all preserved (4).This technique has also been applied to carbon dioxide at subcritical temperatures. Liquid COz expands to a gas state within the cells, when the process pressure is reduced to below its vapor pressure at the temperature of interest, to prompt the action of cell rupture. Disruption rates are sensitive to such process variables as temperature, pressure, and addition of entrainers to the fluid. Under optimum conditions, over 80% of cells can easily be ruptured within an hour for a variety of microbial cells. The disruption is, however, not as effective for some other cells. A typical example is yeast cells, which have been described as having one of the most robust and rigid of all microbial cell walls (5,6). The present work suggestsa simple operation to improve the disruption rates by repeated release of the applied fluid pressure within the cells in the midst of a disruption process. The effectiveness of this operation is illustrated by the experimental results of rupturing yeast cells with 8756-7938/92/3008-0165$03.00/0
3
*O[
4
2
0
6
8
10
12
14
Time (hour)
Figure 1. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 35 O C and lo00 psi. 3
0
2
4
6
6
10
12
14
16
Time (hour)
Figure 2. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 25 OC and lo00 psi.
COZas a primary fluid. Carbon dioxide was chosen in this work because it has advantages over many other fluids in food and pharmaceutical applications (4). The same technique can be applied to other microbial cells (or other fluids).
Experimental Procedures Saccharomyces cerevisiae cells used in this work were bakers’ yeast obtained from Red Star (Milwaukee, WI). The preparation of yeast samples was detailed by Lin and co-workers (4). The yeast pellets of this work contain 10% less moisture than those used in our previous study. Carbon dioxide was purchased from Matheson Gas Products with a minimum purity of 99.99 %.
0 1992 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. prog., 1992, Vol. 8, No. 2
166 80 r
Y : 5
20
[
0
,
,
,
,
2
0
,
,
6
4
1
,
8
1
,
,
10
,
1
12
,
,
14
,
16
,
1
18
Time (hour)
Figure 3. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 35 "C and 3000 psi.
Y
0
2
4
6
8
10
12
14
Time (hour)
Figure 4. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 25 "C and 3000 psi.
The apparatus is a static type that consists of a piston injector to feed C02 at the experimental pressure (1000 or 3000 psi) into a pressure vessel containing 1g of wet yeast cells. The vessel was immersed in a thermostated water bath to maintain a constant temperature (25 or 35 "C). After yeast cells in the vessel were exposed to C02 for a designated length of time, COZwas released and the cells were collected for subsequent assay of total protein and enzyme activities, as described by Lin et al. (4). In the experiments of repeated release of applied COZpressure, COZwas recharged into the vessel at the experimental pressure immediately after it was released. The operation was repeated more than once in some experiments with a time interval that was divided evenly over the duration of a complete run.
Results and Discussion Experimental results were obtained at regions of both supercritical and subcritical temperatures of COZ for comparison. Figure 1shows the release of soluble proteins as a function of exposure time to C02 at 35 "C, while Figure 2 presents similar results a t 25 "C. Both are under pressure of lo00 psi. R(2) and R(1) in the figures denote that carbon dioxide in the pressure vessel was released/repressurized twice and once, respectively, during a complete disruption process. R(0) serves as a control operation (i.e., without any pressure release in the midst of an experimental run). The improvement in disruption rates with repeated release of applied COZ pressure is evident. Cell disruption
approaches its maximum level a t a significantly faster rate with the repetition of pressure release under any of the experimental conditions studied. At 35 "C, the length of exposure time for rupture of 80% of cells was reduced from 7 to 3.5 h for R(1) and 2 h for R(2), while the time reduction at 25 "C was from 7.5 to 4 and 2.5 h for R(1) and R(2), respectively. Similar results were observed at 3000 psi, as shown in Figures 3 and 4. The disruption rates are sensitive to process temperature and pressure. An increase in temperature and/or pressure will facilitate penetration of COz into cells. Higher temperatures appear to enhance the transfer rate of COz and also relax the cell walls to ease the penetration. Cell breakage comes as a result of gas expansion within the microbial cells when the vessel pressure is suddenly released. The action is strengthened under higher pressures. Another effective means to reduce the resistance of microorganisms to disruption is by addition of cell lytic enzymes as entrainers to COZfluid (4). The operation of repetitious pressure release can be used in combination with lytic enzymes to further improve the efficiency of cell disruption without denaturation of proteins. Activities of various enzymes (alcohol dehydrogenase, invertase, glucose-6-phosphate dehydrogenase, and fumarase) in the ruptured cell suspension were assayed to ensure that the process preserved the functional properties of proteins in the presence of COz. The results were similar to those presented previously by Lin et al. ( 4 ) and need not be elaborated.
Acknowledgment Financial support from Biomass Energy Producer of Savannah, GA, is gratefully acknowledged. This work was also partially supported (to H.-M.L.) by BIOS International, Mississauga, Ontario, Canada.
Literature Cited (1) Chisti, Y.; Moo-Young, M. Disruption of Microbial Cells for Intracellular Products. Enzyme Microb. Technol. 1986,8,194204. (2) Dunnill, P.; Lilly, M. D. Protein Extraction and Recovery
from Microbial Cells. InSingle-CellProteinII; Tannenbaum, S. R., Wang, D. I. C., Eds.; MIT Press: Cambridge, MA, 1975; pp 179-207. (3) Cota-Robles, E. H.;Stein, S. M. Bacterial Cell Breakage of Lysis. In CRC Handbook ofMicrobiology;2nd ed.; Laskin, A. I., Lechevalier, H. A,, Eds.; CRC Press, Inc.: Boca Raton, FL, 1982;Vol. IV, pp 601-611. (4) Lin, H.M.; Chan, E. C.; Chen, C.; Chen, L. F. Disintegration of Yeast Cells by Pressurized Carbon Dioxide. Biotechnol. Prog. 1991,7,201-204. (5) Phaff, H.J. Enzymatic Yeast Cell Wall Degradation. In Food Proteins; Feeney, R. E., Whitaker, J. R., Eds.; Advances in Chemistry Series 160; American Chemical Society: Washington, DC, 1977;pp 244-282. (6)Hunter, J. B.; Asenjo, J. A. A Structured Mechanistic Model of the Kinetics of Enzymatic Lysis and Disruption of Yeast Cells. Biotechnol. Bioeng. 1988,31, 929-943. Accepted August 8,1991. Registry No. COz, 124-38-9.
185
An Improved Method for Disruption of Microbial Cells with Pressurized Carbon Dioxide Ho-Mu Lin,*Zhiying Yang, and Li-Fu Chen Department of Food Science, Smith Hall,Purdue University, West Lafayette, Indiana 47907 Disruption of microbial cells by pressurized carbon dioxide at both subcritical and supercritical temperatures has been previously investigated. This method differs in principle from other disruption techniques and was found to have potential applications for rupture of a variety of microorganisms. However, it is not as effective for some of the microbial cells, including yeast, of which the cell walls are extremely robust and rigid. This work suggests an alternative operation to improve the disruption rates of cells by repeatedly releasing the applied fluid pressure within the cells in the midst of a disruption process. The improvement is substantial at aU the experimental conditions studied.
Introduction Microbial cells have long been recognized as an important source of commercially useful biochemicals, antibiotics, and enzymes (1). With increasing demand for microbial products in industry and medicine, considerable effort has recently been committed to develop technologies for production of intracellular microbial materials from microorganisms and various genetically altered cultures. Release of cell contents is vital also to many investigations of bacterial metabolism. In any case, the cell disruption is a necessary operation for isolation or recovery of intracellular enzymes and organelles. A variety of disruption techniques have been developed (1-3) and some are available commercially. Mechanical methods appear to be favored at the present time for their economic advantages, although several nonmechanical methods, particularly enzymatic lysis, have attracted great attention. Nevertheless, many of these methods often ensue with degradation or denaturation of proteins in the process. A technique to exploit supercritical fluid (SCF) for disruption of microbial cells has recently been developed in this laboratory (4). The process involves a sudden release of the applied fluid pressure that follows penetration of SCF into cells. The expansion of SCF within the cells, when it is released, forces the breakage of microorganisms. It is simple, inexpensive, and, more importantly, noninjurious to enzyme activities. The functional properties of proteins are all preserved (4).This technique has also been applied to carbon dioxide at subcritical temperatures. Liquid COz expands to a gas state within the cells, when the process pressure is reduced to below its vapor pressure at the temperature of interest, to prompt the action of cell rupture. Disruption rates are sensitive to such process variables as temperature, pressure, and addition of entrainers to the fluid. Under optimum conditions, over 80% of cells can easily be ruptured within an hour for a variety of microbial cells. The disruption is, however, not as effective for some other cells. A typical example is yeast cells, which have been described as having one of the most robust and rigid of all microbial cell walls (5,6). The present work suggestsa simple operation to improve the disruption rates by repeated release of the applied fluid pressure within the cells in the midst of a disruption process. The effectiveness of this operation is illustrated by the experimental results of rupturing yeast cells with 8756-7938/92/3008-0165$03.00/0
3
*O[
4
2
0
6
8
10
12
14
Time (hour)
Figure 1. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 35 O C and lo00 psi. 3
0
2
4
6
6
10
12
14
16
Time (hour)
Figure 2. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 25 OC and lo00 psi.
COZas a primary fluid. Carbon dioxide was chosen in this work because it has advantages over many other fluids in food and pharmaceutical applications (4). The same technique can be applied to other microbial cells (or other fluids).
Experimental Procedures Saccharomyces cerevisiae cells used in this work were bakers’ yeast obtained from Red Star (Milwaukee, WI). The preparation of yeast samples was detailed by Lin and co-workers (4). The yeast pellets of this work contain 10% less moisture than those used in our previous study. Carbon dioxide was purchased from Matheson Gas Products with a minimum purity of 99.99 %.
0 1992 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. prog., 1992, Vol. 8, No. 2
166 80 r
Y : 5
20
[
0
,
,
,
,
2
0
,
,
6
4
1
,
8
1
,
,
10
,
1
12
,
,
14
,
16
,
1
18
Time (hour)
Figure 3. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 35 "C and 3000 psi.
Y
0
2
4
6
8
10
12
14
Time (hour)
Figure 4. Release of total proteins from yeast cells as a function of repeated release of fluid pressure at 25 "C and 3000 psi.
The apparatus is a static type that consists of a piston injector to feed C02 at the experimental pressure (1000 or 3000 psi) into a pressure vessel containing 1g of wet yeast cells. The vessel was immersed in a thermostated water bath to maintain a constant temperature (25 or 35 "C). After yeast cells in the vessel were exposed to C02 for a designated length of time, COZwas released and the cells were collected for subsequent assay of total protein and enzyme activities, as described by Lin et al. (4). In the experiments of repeated release of applied COZpressure, COZwas recharged into the vessel at the experimental pressure immediately after it was released. The operation was repeated more than once in some experiments with a time interval that was divided evenly over the duration of a complete run.
Results and Discussion Experimental results were obtained at regions of both supercritical and subcritical temperatures of COZ for comparison. Figure 1shows the release of soluble proteins as a function of exposure time to C02 at 35 "C, while Figure 2 presents similar results a t 25 "C. Both are under pressure of lo00 psi. R(2) and R(1) in the figures denote that carbon dioxide in the pressure vessel was released/repressurized twice and once, respectively, during a complete disruption process. R(0) serves as a control operation (i.e., without any pressure release in the midst of an experimental run). The improvement in disruption rates with repeated release of applied COZ pressure is evident. Cell disruption
approaches its maximum level a t a significantly faster rate with the repetition of pressure release under any of the experimental conditions studied. At 35 "C, the length of exposure time for rupture of 80% of cells was reduced from 7 to 3.5 h for R(1) and 2 h for R(2), while the time reduction at 25 "C was from 7.5 to 4 and 2.5 h for R(1) and R(2), respectively. Similar results were observed at 3000 psi, as shown in Figures 3 and 4. The disruption rates are sensitive to process temperature and pressure. An increase in temperature and/or pressure will facilitate penetration of COz into cells. Higher temperatures appear to enhance the transfer rate of COz and also relax the cell walls to ease the penetration. Cell breakage comes as a result of gas expansion within the microbial cells when the vessel pressure is suddenly released. The action is strengthened under higher pressures. Another effective means to reduce the resistance of microorganisms to disruption is by addition of cell lytic enzymes as entrainers to COZfluid (4). The operation of repetitious pressure release can be used in combination with lytic enzymes to further improve the efficiency of cell disruption without denaturation of proteins. Activities of various enzymes (alcohol dehydrogenase, invertase, glucose-6-phosphate dehydrogenase, and fumarase) in the ruptured cell suspension were assayed to ensure that the process preserved the functional properties of proteins in the presence of COz. The results were similar to those presented previously by Lin et al. ( 4 ) and need not be elaborated.
Acknowledgment Financial support from Biomass Energy Producer of Savannah, GA, is gratefully acknowledged. This work was also partially supported (to H.-M.L.) by BIOS International, Mississauga, Ontario, Canada.
Literature Cited (1) Chisti, Y.; Moo-Young, M. Disruption of Microbial Cells for Intracellular Products. Enzyme Microb. Technol. 1986,8,194204. (2) Dunnill, P.; Lilly, M. D. Protein Extraction and Recovery
from Microbial Cells. InSingle-CellProteinII; Tannenbaum, S. R., Wang, D. I. C., Eds.; MIT Press: Cambridge, MA, 1975; pp 179-207. (3) Cota-Robles, E. H.;Stein, S. M. Bacterial Cell Breakage of Lysis. In CRC Handbook ofMicrobiology;2nd ed.; Laskin, A. I., Lechevalier, H. A,, Eds.; CRC Press, Inc.: Boca Raton, FL, 1982;Vol. IV, pp 601-611. (4) Lin, H.M.; Chan, E. C.; Chen, C.; Chen, L. F. Disintegration of Yeast Cells by Pressurized Carbon Dioxide. Biotechnol. Prog. 1991,7,201-204. (5) Phaff, H.J. Enzymatic Yeast Cell Wall Degradation. In Food Proteins; Feeney, R. E., Whitaker, J. R., Eds.; Advances in Chemistry Series 160; American Chemical Society: Washington, DC, 1977;pp 244-282. (6)Hunter, J. B.; Asenjo, J. A. A Structured Mechanistic Model of the Kinetics of Enzymatic Lysis and Disruption of Yeast Cells. Biotechnol. Bioeng. 1988,31, 929-943. Accepted August 8,1991. Registry No. COz, 124-38-9.
