- Email: [email protected]
High-throughput method for a kinetics analysis of the high-pressure inactivation of microorganisms using microplates
Journal of Bioscience and Bioengineering VOL. 113 No. 6, 788 – 791, 2012 www.elsevier.com/locate/jbiosc
TECHNICAL NOTE
High-throughput method for a kinetics analysis of the high-pressure inactivation of microorganisms using microplates Toshimi Hasegawa, 1 Manabu Hayashi, 1 Kazuki Nomura, 1 Mayumi Hayashi, 1 Miyuki Kido, 2 Tsuneo Ohmori, 2 Masao Fukuda, 3 Akinori Iguchi, 1 Shigeaki Ueno, 4 Toru Shigematsu, 1, ⁎ Masao Hirayama, 1, 5 and Tomoyuki Fujii 1, 4 Department of Food Science, Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences (NUPALS), 265-1 Higashijima, Akiha-ku, Niigata, Niigata 956-8603, Japan, 1 Niigata Industrial Creation Organization (NICO), 5-1 Bandaijima, Chuou-ku, Niigata, Niigata 950-0078, Japan, 2 Department of Bioengineering, Nagaoka University of Technology, Kamitomioka 1603-1, Nagaoka, Niigata 940-2188, Japan, 3 Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan, 4 and Bourbon Corporation, 4-2-14 Matsunami, Kashiwazaki, Niigata 945-8611, Japan 5 Received 4 October 2011; accepted 1 February 2012 Available online 29 February 2012
Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the highpressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis. © 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Microplate; High-throughput; High-pressure; Microbial inactivation; Escherichia coli; Saccharomyces cerevisiae]
High-pressure (HP) treatment is a nonthermal sterilization technique that can inactivate microorganisms while preventing alterations in the flavor and nutrient contents of foods. The HP inactivation behavior of microorganisms has been shown to vary according to microbial species and strains, as well as by type and concentration of coexisting materials (1–4). We kinetically analyzed the HP inactivation of Escherichia coli in the presence of salt solutions (KCl, NaCl, and LiCl) around isotonic concentrations. As a result, the HP inactivation behavior of E. coli varied not only by the different ion species but also by their concentration (5). The cellular response against salt stress has been suggested to play an important role in microbial inactivation behavior. To understand the mechanism of the HP inactivation of microorganisms, it is necessary to carry out more comprehensive analyses on the effect of inactivation for combinations of different microorganisms and coexisting materials, such as salts. Two potential applications of the HP inactivation of microorganisms are the prevention of foodborne pathogens and the control of overfermentation (6). For the application of HP treatment to fermentation control, we obtained a barosensitive mutant strain of Saccharomyces cerevisiae (7,8). To analyze the pressure sensitivity mechanism of this mutant, as well as to obtain more barosensitive mutants, further comprehensive analyses on the HP inactivation of strains under a number of pressure conditions are still needed. ⁎ Corresponding author. Tel./fax: +81 250 25 5145. E-mail address: [email protected] (T. Shigematsu).
For further application of HP technology to sterilization and fermentation control, it is important to obtain more knowledge about the HP inactivation of microorganisms under a number of pressure conditions with coexisting materials. Therefore, we thought that a highthroughput experimental system to analyze HP microbial inactivation would be helpful. Recently, a number of methods using microplates for microbial cultivation have been developed. Based on liquid cultivation using microplates, a method that can provide accurate enumeration and simultaneous separation of microorganisms was developed (9). The growth of Listeria monocytogenes in various media could be analyzed using microplates in a short amount of time (10). Moreover, Abe and Minegishi reported a high-throughput analysis on the growth of a number of mutants of S. cerevisiae under HP conditions using microplates (11). This knowledge could allow us to develop a high-throughput experimental system analyzing the HP inactivation of microorganism. However, no reports concerning high-throughput methods have yet provided kinetics data for microbial inactivation by HP treatment. In this study, a new method for analyzing the HP inactivation kinetics of microorganisms using microplates as cultivation and pressure vessels is described. Enumeration of the viable cells was performed by liquid cultivation in microplates. In a batch-wise cultivation of a microorganism, the growth delays with decreases in the initial cell concentration (12). To analyze the relationship between the initial cell concentration and growth delay, the culture broth of E. coli strain K12 or S. cerevisiae strain KA31a, diluted with 0.145 M NaCl solution, was applied for cultivation using 96-well microplates. The viable cell concentration of the culture broth was determined by colony counting after cultivation
1389-1723/$ - see front matter © 2012, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2012.02.001
VOL. 113, 2012
TECHNICAL NOTE
A
B
12
45
y =-0.548 x + 13.080 R² = 0.999
10
789
y =-2.331 x + 46.659 R² = 0.999
40 35
8
30 25
6
20 4
15 10
2
5 0
0
5
10
15
20
0
25
Viable cell concentration [ln CFU ml-1]
0
4
8
12
16
20
Viable cell concentration [ln CFU ml-1]
FIG. 1. Relationship between the logarithmic viable cell concentrations and values of tΔ0.5 for E. coli strain K12 (A) and S. cerevisiae strain KA31a (B). Means and standard deviations were obtained for 9 experiments and 7 experiments for E. coli and for S. cerevisiae, respectively. The viable cell concentration shows the concentration of the cell suspension used as the inoculum for the microplate cultivation.
using the dry sheet media Compact Dry EC and YM (Nissui Pharmaceutical, Tokyo, Japan) for E. coli and S. cerevisiae, respectively. A serial dilution of culture broth of 102 to 108 CFU ml− 1 was prepared. A 15-μl sample of each diluted cell suspension was inoculated into 135 μl of cultivation media to give the initial cell concentration of 101 to 107 CFU ml− 1 in the wells of a 96-well microplate (Asahi Glass, Tokyo, Japan). The media for E. coli and S. cerevisiae were LB medium (Bacto tryptone, 10 g L− 1; Bacto yeast extract, 5 g L− 1; NaCl, 10 g L− 1; pH 7.2) and 2% YPD medium (Bacto yeast extract, 10 g L− 1; Bacto peptone, 20 g L− 1; glucose, 20 g L− 1), respectively. The microplate was set in the Bio Microplate Reader “HiTS” (High Throughput Screening) (Scinics, Tokyo, Japan), and cultivation was carried out. Before cultivation, the absorbance values at 660 nm of the cell suspensions, measured by HiTS, were 0.01 and 0.02 for 107 CFU ml− 1 of E. coli and 106 CFU ml− 1 of S. cerevisiae. Microplate cultivation was carried out with shaking at a speed of 10 (approximately 220 rpm) at 37°C for E. coli or at 30°C for S. cerevisiae. The
absorbance value at 660 nm in each well was monitored automatically at 30 min intervals. The absorbance values in each well during cultivation were used to generate the growth curve. The tΔ0.5 value, which is the cultivation time when the absorbance increased by 0.5 from the initial absorbance, was calculated using software (HiTS reader for Windows version 1.71-RK, Scinics). Using the growth curve, the viable cell concentration X [CFU ml− 1] can be described as a function of the cultivation time t as follows: X ¼ X 0 e½μ ðtl Þ
ð1Þ
where X0 [CFU ml− 1], μ [h− 1] and l [h] are the initial cell concentration, specific growth rate and lag time, respectively. A culture of a homogeneous population can show a uniform lag time. If the cell concentration at tΔ0.5 was defined as XΔ0.5, Eq. 1 can be written as follows: X Δ0:5 ¼ X 0 e½ μðt Δ0:5 lÞ
ð2Þ
Cell suspension Cap mat (silicone rubber)
Vacuum-packed polyethylene bag 8-well microplate strips
Colony counting High pressure treatment
Standard curve
Up to 80 samples Standards
(standards)
Blank
Viable cell concentration
Microplate cultivation
9
-1
(samples)
OD 660
10 CFU ml
Bio Microplate Reader
107CFU ml-1
Incubation time [h] Cultivation time
Viable cell concentration FIG. 2. Schematic diagram of the microplate method.
790
HASEGAWA ET AL.
J. BIOSCI. BIOENG.,
This equation gives the following equation: t Δ0:5 ¼ −1=μ lnX 0 þ 1=μ lnX Δ0:5 þ l
ð3Þ
From Eq. 3, tΔ0.5 and ln X0 should be negatively linearly proportional. Actually, the tΔ0.5 was negatively linearly proportional to the logarithmic concentration of the diluted cell suspension, which was used as the inoculum for the microplate cultivation (Fig. 1). The specific growth rates μ of E. coli and S. cerevisiae were calculated from the slopes of the curves, 1.82 h− 1 and 0.43 h− 1, respectively. These values were equivalent to the μ values calculated from the growth curves based on Eq. 1, 1.38 ± 0.05 h− 1 and 0.46 ± 0.04 h− 1 for E. coli and S. cerevisiae, respectively. Thus, the curve shown in Fig. 1 can be used as a standard curve for enumeration of the cell suspension, which is applied to microplate cultivation. The new experimental method for analyzing HP microbial inactivation kinetics (the microplate method) constructed in this study is summarized in Fig. 2. The HP treatment of the cells was carried out using 8-well microplate strips (8-well stripwell, Corning, NY, USA). The culture broths of E. coli and S. cerevisiae were prepared by aerobic cultivation in LB medium for 24 h at 37°C and in 2% YPD medium for 48 h at 30°C, respectively. A 20-ml sample of each culture broth was mixed with 180 ml of 0.145 M NaCl solution. A 360-μl sample of the cell suspension was introduced into each well of the microplate strip. A silicon capmat (Capmats 96-well round silicone rubber, Whatman, NJ, USA) was cut into strips and was carefully put on the microplate strip to exclude air from the wells. The microplate strip with the capmat was soaked with distilled water, vacuum-packed in a polyethylene bag and subjected to the HP treatment using an HP apparatus (WIP, Kobe Steel, Kobe, Japan) at ambient temperature. For E. coli, HP treatments at 200, 250, 300 and 350 MPa for 0 to 360 s were carried out. For S. cerevisiae, HP treatments at 200, 225 and 250 MPa for 0 to 360 s were carried out. After the HP treatment, 15 μl of pressurized cell suspension was mixed with 135 μl of medium (LB medium for E. coli or 2% YPD medium for S. cerevisiae) in a new
0 -1 -2 -3 -4 -5 -6 -7 -8 -9 -10
N ¼ N 0 ⋅e–kt
B
0
100
200
300
400
0 -1 -2 -3 -4 -5 -6 -7 -8 -9 -10
0
Time [s]
C
0
y = 0.009 x - 5.866 R² = 0.981
400
0
microplate method conventional method
-3
y = 0.033 x - 11.643 R² = 0.979
-4 -5 y = 0.041 x - 13.776 R² = 0.995
-6
-6 250
300
-2
-4 -5
200
-1
y = 0.008x - 5.396 R² = 0.936
-3
-7 150
D
ln k
ln k
-2
100
Time [s]
microplate method conventional method
-1
ð4Þ
where N is the viable cell concentration [CFU ml− 1] at time t [s], N0 is the viable cell concentration [CFU ml− 1] at the time 0 [s] and k is the inactivation rate constant [s− 1]. The HP inactivation curves were compared with those obtained by the conventional method (dotted lines in Figs. 3A and 3B) (5,7,8), which was based on colony counting after HP treatment of the cell suspension in a polyethylene bag. For both strains, N/N0 values obtained via the microplate method at all pressure levels showed no significant difference from those obtained via the conventional method. Takano and Tsuchido demonstrated that the cell damage by heat treatment on E. coli showed larger values of the survival ratio determined by colony counting, compared with those determined by the growth delay method (11). This discrepancy was explained by the increase of the lag time in the heated cells, depending on the length of treatment. In contrast, they also reported that the heat treatment on Bacillus subtilis strain 168 showed no significant differences in the survival ratios, which were calculated by the colony-counting method and the growth delay method. Strain 168
ln (N/N0)
ln (N/N0)
A
96-well microplate. The microplate cultivation was carried out to obtain the tΔ0.5 values. At the same time, the cell suspensions without HP treatment, serial diluted with 0.145 M NaCl solution, were applied for microplate cultivation. The tΔ0.5 values obtained and the viable cell concentration of the cell suspensions by colony counting using the dry sheet media were used for generating the standard curve. Using the standard curve, the viable cell concentration of each pressurized cell suspension was calculated from the tΔ0.5 value. For both strains, the logarithmic values of the survival ratio N/N0, which is the viable cell concentration after HP treatment (N) divided by the viable cell concentration after HP treatment for 0 s (N0), were negatively proportional with the pressuring time (solid lines in Figs. 3A and 3B), indicating that the inactivation of both strains followed first-order kinetics, as expressed in the following equation:
350
Pressure [MPa]
450
-7 150
200
250
300
Pressure [MPa]
FIG. 3. Survival curves (A, B) and pressure dependency of the pressure inactivation rate constants (C, D) of E. coli strain K12 (A, C) and S. cerevisiae strain KA31a (B, D) for highpressure treatment assayed using the microplate method (closed symbols with solid lines) or the conventional method (open symbols with dotted lines). The pressure levels in panels A and B were 200 (diamonds), 225 (inverted triangles), 250 (squares), 300 (triangles) and 350 MPa (circles). Means and standard deviations were obtained for at least 3 experiments. Linear curves are shown based on Eq. 4 (A, B) and Eq. 5 (C, D).
VOL. 113, 2012
TECHNICAL NOTE
was suggested not to increase in the lag time by the heat treatment. Our results suggested that no apparent damage causing an increase in the lag time for E. coli and S. cerevisiae occurred as the result of the HP treatment tested. Thus, we did not consider the effect of the HP treatment on the lag time in this study. Koseki and Yamamoto reported recovery of the injured cells by preservation of the cells under low temperature after HP treatment (13). Because the pressurized cell suspensions were immediately applied for microplate cultivation in this study, we did not consider the alteration of the viable cell concentration caused by the cell recovery. For both strains at all pressure levels tested, the k values calculated from the slope of the survival curves obtained by the microplate method were equivalent with those obtained by the conventional method. For E. coli, the k values for the microplate and conventional methods were 0.017 ± 0.003 and 0.021 ± 0.005 s− 1 (200 MPa), 0.031 ± 0.001 and 0.040 ± 0.012 s− 1 (250 MPa), 0.042 ± 0.003 and 0.046 ± 0.006 s− 1 (300 MPa), and 0.068 ± 0.011 and 0.075 ± 0.014 s− 1 (350 MPa), respectively. For S. cerevisiae, the k values for the microplate and conventional methods were 0.006 ± 0.002 and 0.004 ± 0.001 s− 1 (200 MPa), 0.016 ± 0.003 and 0.010 ± 0.001 s− 1 (225 MPa), and 0.030 ± 0.004 and 0.033 ± 0.008 s− 1 (250 MPa), respectively. The pressure dependencies of the k values of E. coli and S. cerevisiae were then analyzed. For both strains, the ln k values obtained for the microplate and conventional methods were positively proportional to the pressure levels (Figs. 3C and 3D). These results indicated that the HP inactivation reaction follows the basic equation for pressure dependency of the reaction velocity (14): k ¼ k 0 eðP ΔV
=RTÞ
ð5Þ
where k0 is a pre-exponential factor [s− 1], P is pressure [MPa], ΔV* is the activation volume [cm 3 mol − 1 ], R is the gas constant [J K− 1 mol− 1], and T is the absolute temperature [K]. The ΔV* values for E. coli, as determined by the microplate and conventional methods, were −25.0 ±2.1 and −20.1 ±4.4 cm3 mol− 1, respectively. The ΔV* values for S. cerevisiae, as determined by the microplate and conventional methods, were − 88.3 ± 1.3 and − 96.5 ± 17.5 cm3 mol− 1, respectively. The kinetics parameters obtained by the microplate method were equivalent with those values obtained by the microplate method. These results indicated apparent reproducibility of the HP inactivation behaviors analyzed by the microplate method and the conventional method. The microplate method was proven to be an alternative for the conventional method based on colony counting for a kinetics analysis of the pressure inactivation of microorganisms. In this study, we demonstrated how the HP treatment depends on the inactivation kinetics of E. coli and S. cerevisiae using the newly developed microplate method and compared the microplate method with the conventional colony-counting method. One of the critical characteristics of the new method is the high-throughput property. If one determines the viable cell concentration of a cell suspension using the conventional colony-counting method, serial dilutions of 101 to 108 usually need to be prepared. At least four dilutions from the serial dilution are used for agar plate cultivation to obtain the appropriate number of colonies formed on the medium plates. These steps are quite time consuming in the conventional method. In contrast, the microplate method does not require this serial dilution. Each cell suspension after the HP treatment was inoculated into a well of a 96-well microplate without dilution. Of the 96 wells, 16 wells are usually occupied by dilutions of cells without HP treatment for the standard curve, and the remaining 80 wells can be used for the treated cells. The viable cell concentration of 80 cell suspensions can thus be determined by only one cultivation. This method proved a remarkable high-throughput property, compared with the conventional method of colony counting.
791
In addition, using an 8-well microplate strip as the pressure vessel, 8-cell suspensions with different strains and/or under different conditions of coexisting materials can be separately applied for HP treatment at the same time. This is also one of the high-throughput properties of the microplate method. In our previous study using the conventional method, the pressure inactivation kinetics analysis on E. coli were carried out with 6 different concentrations of NaCl solution (0.07 to 0.30 M) at 3 pressure levels (250 to 350 MPa) for 4 to 5 pressuring times (0 to 180 s) (5). Using the microplate method, duplicated data can be obtained by only 3 cultivations. In conclusion, we used microplates as the pressure vessels and cultivation vessels for the development of a new high-throughput microbial pressure inactivation kinetics analysis system (HT-PIKAS). This new system would facilitate an accelerated accumulation of useful knowledge on the HP-dependent inactivation of microorganisms. The accumulation of such knowledge could reveal the mechanism of the HP inactivation of microorganisms and could lead to better application of HP technology to sterilization and fermentation control. This work was supported by the program Niigata Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the Japan Science and Technology Agency and by the Salt Science Research Foundation. We thank Dr. Yoichi Noda (The University of Tokyo) for kindly providing the S. cerevisiae strain KA31a.
References 1. Michiels, C., Barlett, D. H., and Aertsen, A.: High-pressure microbiology. ASM Press, Washington, D.C. (2008). 2. Patterson, M. F. and Linton, M.: Factors affecting inactivation of food-borne bacteria by high pressure, p. 181–193, in: Michiels, C., Barltlett, D. H., and Aertsen, A. (Eds.), High-pressure microbiology. ASM Press, Washington, D.C. (2008). 3. Simpson, R. K. and Gilmour, A.: The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems, J. Appl. Microbiol., 83, 181–188 (1997). 4. Hauben, K. J. A., Bernaerts, K., and Michiels, C. W.: Protective effect of calcium on inactivation of Escherichia coli by high hydrostatic pressure, J. Appl. Microbiol., 85, 678–684 (1998). 5. Ueno, S., Shigematsu, T., Hasegawa, T., Higashi, J., Anzai, M., Hayashi, M., and Fujii, T.: Kinetic analysis of E. coli inactivation by high hydrostatic pressure with salts, J. Food Sci., 76, M47‐53 (2011). 6. Joo, W. L., Dong, S. C., Keum, T. H., and Hyun, J. P.: Effects of CO2 absorbent and high-pressure treatment on the shelf-life of packaged Kimchi products, Int. J. Food Sci. Technol., 38, 519–524 (2003). 7. Shigematsu, T., Nasuhara, Y., Nagai, G., Nomura, K., Ikarashi, K., Hirayama, M., Hayashi, M., Ueno, S., and Fujii, T.: Isolation and characterization of barosensitive mutants of Saccharomyces cerevisiae obtained by UV mutagenesis, J. Food Sci., 75, M509–M514 (2010). 8. Shigematsu, T., Nomura, K., Nasuhara, Y., Ikarashi, K., Nagai, G., Hirayama, M., Hayashi, M., Ueno, S., and Fujii, T.: Thermosensitivity of a barosensitive Saccharomyces cerevisiae mutant obtained by UV mutagenesis, High Pressure Res., 30, 524–529 (2010). 9. Shigematsu, T., Hayashi, M., Kikuchi, I., Ueno, S., Masaki, H., and Fujii, T.: A culture-dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment, FEMS Microbiol. Lett., 293, 240–247 (2009). 10. Horáková, K., Greifová, M., Seemannová, Z., Gondová, B., and Wyatt, G. M.: A comparison of the traditional method of counting viable cells and a quick microplate method for monitoring the growth characteristics of Listeria monocytogenes, Lett. Appl. Microbiol., 38, 181–184 (2004). 11. Abe, F. and Minegishi, H.: Global screening of genes essential for growth in highpressure and cold environments: searching for basic adaptive strategies using a yeast deletion library, Genetics, 178, 851–872 (2008). 12. Takano, M. and Tsuchido, T.: Availability of growth delay analysis for the evaluation of total injury of stressed bacterial populations, J. Ferment. Technol., 60, 189–198 (1982). 13. Koseki, S. and Yamamoto, K.: Recovery of Escherichia coli ATCC 25922 in phosphate buffered saline after treatment with high hydrostatic pressure, Int. J. Food Microbiol., 110, 108–111 (2006). 14. Laidler, K. H.: The influence of pressure on the rates of biological reactions, Arch. Biochem., 30, 226–236 (1951).
TECHNICAL NOTE
High-throughput method for a kinetics analysis of the high-pressure inactivation of microorganisms using microplates Toshimi Hasegawa, 1 Manabu Hayashi, 1 Kazuki Nomura, 1 Mayumi Hayashi, 1 Miyuki Kido, 2 Tsuneo Ohmori, 2 Masao Fukuda, 3 Akinori Iguchi, 1 Shigeaki Ueno, 4 Toru Shigematsu, 1, ⁎ Masao Hirayama, 1, 5 and Tomoyuki Fujii 1, 4 Department of Food Science, Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences (NUPALS), 265-1 Higashijima, Akiha-ku, Niigata, Niigata 956-8603, Japan, 1 Niigata Industrial Creation Organization (NICO), 5-1 Bandaijima, Chuou-ku, Niigata, Niigata 950-0078, Japan, 2 Department of Bioengineering, Nagaoka University of Technology, Kamitomioka 1603-1, Nagaoka, Niigata 940-2188, Japan, 3 Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan, 4 and Bourbon Corporation, 4-2-14 Matsunami, Kashiwazaki, Niigata 945-8611, Japan 5 Received 4 October 2011; accepted 1 February 2012 Available online 29 February 2012
Using microplates as pressure and cultivation vessels, a high-throughput method was developed for analyzing the highpressure inactivation kinetics of microorganisms. The loss of viability from a high-pressure treatment, measured based on the growth delay during microplate cultivation, showed reproducibility with the conventional agar plate method and was applicable for the kinetics analysis. © 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Microplate; High-throughput; High-pressure; Microbial inactivation; Escherichia coli; Saccharomyces cerevisiae]
High-pressure (HP) treatment is a nonthermal sterilization technique that can inactivate microorganisms while preventing alterations in the flavor and nutrient contents of foods. The HP inactivation behavior of microorganisms has been shown to vary according to microbial species and strains, as well as by type and concentration of coexisting materials (1–4). We kinetically analyzed the HP inactivation of Escherichia coli in the presence of salt solutions (KCl, NaCl, and LiCl) around isotonic concentrations. As a result, the HP inactivation behavior of E. coli varied not only by the different ion species but also by their concentration (5). The cellular response against salt stress has been suggested to play an important role in microbial inactivation behavior. To understand the mechanism of the HP inactivation of microorganisms, it is necessary to carry out more comprehensive analyses on the effect of inactivation for combinations of different microorganisms and coexisting materials, such as salts. Two potential applications of the HP inactivation of microorganisms are the prevention of foodborne pathogens and the control of overfermentation (6). For the application of HP treatment to fermentation control, we obtained a barosensitive mutant strain of Saccharomyces cerevisiae (7,8). To analyze the pressure sensitivity mechanism of this mutant, as well as to obtain more barosensitive mutants, further comprehensive analyses on the HP inactivation of strains under a number of pressure conditions are still needed. ⁎ Corresponding author. Tel./fax: +81 250 25 5145. E-mail address: [email protected] (T. Shigematsu).
For further application of HP technology to sterilization and fermentation control, it is important to obtain more knowledge about the HP inactivation of microorganisms under a number of pressure conditions with coexisting materials. Therefore, we thought that a highthroughput experimental system to analyze HP microbial inactivation would be helpful. Recently, a number of methods using microplates for microbial cultivation have been developed. Based on liquid cultivation using microplates, a method that can provide accurate enumeration and simultaneous separation of microorganisms was developed (9). The growth of Listeria monocytogenes in various media could be analyzed using microplates in a short amount of time (10). Moreover, Abe and Minegishi reported a high-throughput analysis on the growth of a number of mutants of S. cerevisiae under HP conditions using microplates (11). This knowledge could allow us to develop a high-throughput experimental system analyzing the HP inactivation of microorganism. However, no reports concerning high-throughput methods have yet provided kinetics data for microbial inactivation by HP treatment. In this study, a new method for analyzing the HP inactivation kinetics of microorganisms using microplates as cultivation and pressure vessels is described. Enumeration of the viable cells was performed by liquid cultivation in microplates. In a batch-wise cultivation of a microorganism, the growth delays with decreases in the initial cell concentration (12). To analyze the relationship between the initial cell concentration and growth delay, the culture broth of E. coli strain K12 or S. cerevisiae strain KA31a, diluted with 0.145 M NaCl solution, was applied for cultivation using 96-well microplates. The viable cell concentration of the culture broth was determined by colony counting after cultivation
1389-1723/$ - see front matter © 2012, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2012.02.001
VOL. 113, 2012
TECHNICAL NOTE
A
B
12
45
y =-0.548 x + 13.080 R² = 0.999
10
789
y =-2.331 x + 46.659 R² = 0.999
40 35
8
30 25
6
20 4
15 10
2
5 0
0
5
10
15
20
0
25
Viable cell concentration [ln CFU ml-1]
0
4
8
12
16
20
Viable cell concentration [ln CFU ml-1]
FIG. 1. Relationship between the logarithmic viable cell concentrations and values of tΔ0.5 for E. coli strain K12 (A) and S. cerevisiae strain KA31a (B). Means and standard deviations were obtained for 9 experiments and 7 experiments for E. coli and for S. cerevisiae, respectively. The viable cell concentration shows the concentration of the cell suspension used as the inoculum for the microplate cultivation.
using the dry sheet media Compact Dry EC and YM (Nissui Pharmaceutical, Tokyo, Japan) for E. coli and S. cerevisiae, respectively. A serial dilution of culture broth of 102 to 108 CFU ml− 1 was prepared. A 15-μl sample of each diluted cell suspension was inoculated into 135 μl of cultivation media to give the initial cell concentration of 101 to 107 CFU ml− 1 in the wells of a 96-well microplate (Asahi Glass, Tokyo, Japan). The media for E. coli and S. cerevisiae were LB medium (Bacto tryptone, 10 g L− 1; Bacto yeast extract, 5 g L− 1; NaCl, 10 g L− 1; pH 7.2) and 2% YPD medium (Bacto yeast extract, 10 g L− 1; Bacto peptone, 20 g L− 1; glucose, 20 g L− 1), respectively. The microplate was set in the Bio Microplate Reader “HiTS” (High Throughput Screening) (Scinics, Tokyo, Japan), and cultivation was carried out. Before cultivation, the absorbance values at 660 nm of the cell suspensions, measured by HiTS, were 0.01 and 0.02 for 107 CFU ml− 1 of E. coli and 106 CFU ml− 1 of S. cerevisiae. Microplate cultivation was carried out with shaking at a speed of 10 (approximately 220 rpm) at 37°C for E. coli or at 30°C for S. cerevisiae. The
absorbance value at 660 nm in each well was monitored automatically at 30 min intervals. The absorbance values in each well during cultivation were used to generate the growth curve. The tΔ0.5 value, which is the cultivation time when the absorbance increased by 0.5 from the initial absorbance, was calculated using software (HiTS reader for Windows version 1.71-RK, Scinics). Using the growth curve, the viable cell concentration X [CFU ml− 1] can be described as a function of the cultivation time t as follows: X ¼ X 0 e½μ ðtl Þ
ð1Þ
where X0 [CFU ml− 1], μ [h− 1] and l [h] are the initial cell concentration, specific growth rate and lag time, respectively. A culture of a homogeneous population can show a uniform lag time. If the cell concentration at tΔ0.5 was defined as XΔ0.5, Eq. 1 can be written as follows: X Δ0:5 ¼ X 0 e½ μðt Δ0:5 lÞ
ð2Þ
Cell suspension Cap mat (silicone rubber)
Vacuum-packed polyethylene bag 8-well microplate strips
Colony counting High pressure treatment
Standard curve
Up to 80 samples Standards
(standards)
Blank
Viable cell concentration
Microplate cultivation
9
-1
(samples)
OD 660
10 CFU ml
Bio Microplate Reader
107CFU ml-1
Incubation time [h] Cultivation time
Viable cell concentration FIG. 2. Schematic diagram of the microplate method.
790
HASEGAWA ET AL.
J. BIOSCI. BIOENG.,
This equation gives the following equation: t Δ0:5 ¼ −1=μ lnX 0 þ 1=μ lnX Δ0:5 þ l
ð3Þ
From Eq. 3, tΔ0.5 and ln X0 should be negatively linearly proportional. Actually, the tΔ0.5 was negatively linearly proportional to the logarithmic concentration of the diluted cell suspension, which was used as the inoculum for the microplate cultivation (Fig. 1). The specific growth rates μ of E. coli and S. cerevisiae were calculated from the slopes of the curves, 1.82 h− 1 and 0.43 h− 1, respectively. These values were equivalent to the μ values calculated from the growth curves based on Eq. 1, 1.38 ± 0.05 h− 1 and 0.46 ± 0.04 h− 1 for E. coli and S. cerevisiae, respectively. Thus, the curve shown in Fig. 1 can be used as a standard curve for enumeration of the cell suspension, which is applied to microplate cultivation. The new experimental method for analyzing HP microbial inactivation kinetics (the microplate method) constructed in this study is summarized in Fig. 2. The HP treatment of the cells was carried out using 8-well microplate strips (8-well stripwell, Corning, NY, USA). The culture broths of E. coli and S. cerevisiae were prepared by aerobic cultivation in LB medium for 24 h at 37°C and in 2% YPD medium for 48 h at 30°C, respectively. A 20-ml sample of each culture broth was mixed with 180 ml of 0.145 M NaCl solution. A 360-μl sample of the cell suspension was introduced into each well of the microplate strip. A silicon capmat (Capmats 96-well round silicone rubber, Whatman, NJ, USA) was cut into strips and was carefully put on the microplate strip to exclude air from the wells. The microplate strip with the capmat was soaked with distilled water, vacuum-packed in a polyethylene bag and subjected to the HP treatment using an HP apparatus (WIP, Kobe Steel, Kobe, Japan) at ambient temperature. For E. coli, HP treatments at 200, 250, 300 and 350 MPa for 0 to 360 s were carried out. For S. cerevisiae, HP treatments at 200, 225 and 250 MPa for 0 to 360 s were carried out. After the HP treatment, 15 μl of pressurized cell suspension was mixed with 135 μl of medium (LB medium for E. coli or 2% YPD medium for S. cerevisiae) in a new
0 -1 -2 -3 -4 -5 -6 -7 -8 -9 -10
N ¼ N 0 ⋅e–kt
B
0
100
200
300
400
0 -1 -2 -3 -4 -5 -6 -7 -8 -9 -10
0
Time [s]
C
0
y = 0.009 x - 5.866 R² = 0.981
400
0
microplate method conventional method
-3
y = 0.033 x - 11.643 R² = 0.979
-4 -5 y = 0.041 x - 13.776 R² = 0.995
-6
-6 250
300
-2
-4 -5
200
-1
y = 0.008x - 5.396 R² = 0.936
-3
-7 150
D
ln k
ln k
-2
100
Time [s]
microplate method conventional method
-1
ð4Þ
where N is the viable cell concentration [CFU ml− 1] at time t [s], N0 is the viable cell concentration [CFU ml− 1] at the time 0 [s] and k is the inactivation rate constant [s− 1]. The HP inactivation curves were compared with those obtained by the conventional method (dotted lines in Figs. 3A and 3B) (5,7,8), which was based on colony counting after HP treatment of the cell suspension in a polyethylene bag. For both strains, N/N0 values obtained via the microplate method at all pressure levels showed no significant difference from those obtained via the conventional method. Takano and Tsuchido demonstrated that the cell damage by heat treatment on E. coli showed larger values of the survival ratio determined by colony counting, compared with those determined by the growth delay method (11). This discrepancy was explained by the increase of the lag time in the heated cells, depending on the length of treatment. In contrast, they also reported that the heat treatment on Bacillus subtilis strain 168 showed no significant differences in the survival ratios, which were calculated by the colony-counting method and the growth delay method. Strain 168
ln (N/N0)
ln (N/N0)
A
96-well microplate. The microplate cultivation was carried out to obtain the tΔ0.5 values. At the same time, the cell suspensions without HP treatment, serial diluted with 0.145 M NaCl solution, were applied for microplate cultivation. The tΔ0.5 values obtained and the viable cell concentration of the cell suspensions by colony counting using the dry sheet media were used for generating the standard curve. Using the standard curve, the viable cell concentration of each pressurized cell suspension was calculated from the tΔ0.5 value. For both strains, the logarithmic values of the survival ratio N/N0, which is the viable cell concentration after HP treatment (N) divided by the viable cell concentration after HP treatment for 0 s (N0), were negatively proportional with the pressuring time (solid lines in Figs. 3A and 3B), indicating that the inactivation of both strains followed first-order kinetics, as expressed in the following equation:
350
Pressure [MPa]
450
-7 150
200
250
300
Pressure [MPa]
FIG. 3. Survival curves (A, B) and pressure dependency of the pressure inactivation rate constants (C, D) of E. coli strain K12 (A, C) and S. cerevisiae strain KA31a (B, D) for highpressure treatment assayed using the microplate method (closed symbols with solid lines) or the conventional method (open symbols with dotted lines). The pressure levels in panels A and B were 200 (diamonds), 225 (inverted triangles), 250 (squares), 300 (triangles) and 350 MPa (circles). Means and standard deviations were obtained for at least 3 experiments. Linear curves are shown based on Eq. 4 (A, B) and Eq. 5 (C, D).
VOL. 113, 2012
TECHNICAL NOTE
was suggested not to increase in the lag time by the heat treatment. Our results suggested that no apparent damage causing an increase in the lag time for E. coli and S. cerevisiae occurred as the result of the HP treatment tested. Thus, we did not consider the effect of the HP treatment on the lag time in this study. Koseki and Yamamoto reported recovery of the injured cells by preservation of the cells under low temperature after HP treatment (13). Because the pressurized cell suspensions were immediately applied for microplate cultivation in this study, we did not consider the alteration of the viable cell concentration caused by the cell recovery. For both strains at all pressure levels tested, the k values calculated from the slope of the survival curves obtained by the microplate method were equivalent with those obtained by the conventional method. For E. coli, the k values for the microplate and conventional methods were 0.017 ± 0.003 and 0.021 ± 0.005 s− 1 (200 MPa), 0.031 ± 0.001 and 0.040 ± 0.012 s− 1 (250 MPa), 0.042 ± 0.003 and 0.046 ± 0.006 s− 1 (300 MPa), and 0.068 ± 0.011 and 0.075 ± 0.014 s− 1 (350 MPa), respectively. For S. cerevisiae, the k values for the microplate and conventional methods were 0.006 ± 0.002 and 0.004 ± 0.001 s− 1 (200 MPa), 0.016 ± 0.003 and 0.010 ± 0.001 s− 1 (225 MPa), and 0.030 ± 0.004 and 0.033 ± 0.008 s− 1 (250 MPa), respectively. The pressure dependencies of the k values of E. coli and S. cerevisiae were then analyzed. For both strains, the ln k values obtained for the microplate and conventional methods were positively proportional to the pressure levels (Figs. 3C and 3D). These results indicated that the HP inactivation reaction follows the basic equation for pressure dependency of the reaction velocity (14): k ¼ k 0 eðP ΔV
=RTÞ
ð5Þ
where k0 is a pre-exponential factor [s− 1], P is pressure [MPa], ΔV* is the activation volume [cm 3 mol − 1 ], R is the gas constant [J K− 1 mol− 1], and T is the absolute temperature [K]. The ΔV* values for E. coli, as determined by the microplate and conventional methods, were −25.0 ±2.1 and −20.1 ±4.4 cm3 mol− 1, respectively. The ΔV* values for S. cerevisiae, as determined by the microplate and conventional methods, were − 88.3 ± 1.3 and − 96.5 ± 17.5 cm3 mol− 1, respectively. The kinetics parameters obtained by the microplate method were equivalent with those values obtained by the microplate method. These results indicated apparent reproducibility of the HP inactivation behaviors analyzed by the microplate method and the conventional method. The microplate method was proven to be an alternative for the conventional method based on colony counting for a kinetics analysis of the pressure inactivation of microorganisms. In this study, we demonstrated how the HP treatment depends on the inactivation kinetics of E. coli and S. cerevisiae using the newly developed microplate method and compared the microplate method with the conventional colony-counting method. One of the critical characteristics of the new method is the high-throughput property. If one determines the viable cell concentration of a cell suspension using the conventional colony-counting method, serial dilutions of 101 to 108 usually need to be prepared. At least four dilutions from the serial dilution are used for agar plate cultivation to obtain the appropriate number of colonies formed on the medium plates. These steps are quite time consuming in the conventional method. In contrast, the microplate method does not require this serial dilution. Each cell suspension after the HP treatment was inoculated into a well of a 96-well microplate without dilution. Of the 96 wells, 16 wells are usually occupied by dilutions of cells without HP treatment for the standard curve, and the remaining 80 wells can be used for the treated cells. The viable cell concentration of 80 cell suspensions can thus be determined by only one cultivation. This method proved a remarkable high-throughput property, compared with the conventional method of colony counting.
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In addition, using an 8-well microplate strip as the pressure vessel, 8-cell suspensions with different strains and/or under different conditions of coexisting materials can be separately applied for HP treatment at the same time. This is also one of the high-throughput properties of the microplate method. In our previous study using the conventional method, the pressure inactivation kinetics analysis on E. coli were carried out with 6 different concentrations of NaCl solution (0.07 to 0.30 M) at 3 pressure levels (250 to 350 MPa) for 4 to 5 pressuring times (0 to 180 s) (5). Using the microplate method, duplicated data can be obtained by only 3 cultivations. In conclusion, we used microplates as the pressure vessels and cultivation vessels for the development of a new high-throughput microbial pressure inactivation kinetics analysis system (HT-PIKAS). This new system would facilitate an accelerated accumulation of useful knowledge on the HP-dependent inactivation of microorganisms. The accumulation of such knowledge could reveal the mechanism of the HP inactivation of microorganisms and could lead to better application of HP technology to sterilization and fermentation control. This work was supported by the program Niigata Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the Japan Science and Technology Agency and by the Salt Science Research Foundation. We thank Dr. Yoichi Noda (The University of Tokyo) for kindly providing the S. cerevisiae strain KA31a.
References 1. Michiels, C., Barlett, D. H., and Aertsen, A.: High-pressure microbiology. ASM Press, Washington, D.C. (2008). 2. Patterson, M. F. and Linton, M.: Factors affecting inactivation of food-borne bacteria by high pressure, p. 181–193, in: Michiels, C., Barltlett, D. H., and Aertsen, A. (Eds.), High-pressure microbiology. ASM Press, Washington, D.C. (2008). 3. Simpson, R. K. and Gilmour, A.: The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems, J. Appl. Microbiol., 83, 181–188 (1997). 4. Hauben, K. J. A., Bernaerts, K., and Michiels, C. W.: Protective effect of calcium on inactivation of Escherichia coli by high hydrostatic pressure, J. Appl. Microbiol., 85, 678–684 (1998). 5. Ueno, S., Shigematsu, T., Hasegawa, T., Higashi, J., Anzai, M., Hayashi, M., and Fujii, T.: Kinetic analysis of E. coli inactivation by high hydrostatic pressure with salts, J. Food Sci., 76, M47‐53 (2011). 6. Joo, W. L., Dong, S. C., Keum, T. H., and Hyun, J. P.: Effects of CO2 absorbent and high-pressure treatment on the shelf-life of packaged Kimchi products, Int. J. Food Sci. Technol., 38, 519–524 (2003). 7. Shigematsu, T., Nasuhara, Y., Nagai, G., Nomura, K., Ikarashi, K., Hirayama, M., Hayashi, M., Ueno, S., and Fujii, T.: Isolation and characterization of barosensitive mutants of Saccharomyces cerevisiae obtained by UV mutagenesis, J. Food Sci., 75, M509–M514 (2010). 8. Shigematsu, T., Nomura, K., Nasuhara, Y., Ikarashi, K., Nagai, G., Hirayama, M., Hayashi, M., Ueno, S., and Fujii, T.: Thermosensitivity of a barosensitive Saccharomyces cerevisiae mutant obtained by UV mutagenesis, High Pressure Res., 30, 524–529 (2010). 9. Shigematsu, T., Hayashi, M., Kikuchi, I., Ueno, S., Masaki, H., and Fujii, T.: A culture-dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment, FEMS Microbiol. Lett., 293, 240–247 (2009). 10. Horáková, K., Greifová, M., Seemannová, Z., Gondová, B., and Wyatt, G. M.: A comparison of the traditional method of counting viable cells and a quick microplate method for monitoring the growth characteristics of Listeria monocytogenes, Lett. Appl. Microbiol., 38, 181–184 (2004). 11. Abe, F. and Minegishi, H.: Global screening of genes essential for growth in highpressure and cold environments: searching for basic adaptive strategies using a yeast deletion library, Genetics, 178, 851–872 (2008). 12. Takano, M. and Tsuchido, T.: Availability of growth delay analysis for the evaluation of total injury of stressed bacterial populations, J. Ferment. Technol., 60, 189–198 (1982). 13. Koseki, S. and Yamamoto, K.: Recovery of Escherichia coli ATCC 25922 in phosphate buffered saline after treatment with high hydrostatic pressure, Int. J. Food Microbiol., 110, 108–111 (2006). 14. Laidler, K. H.: The influence of pressure on the rates of biological reactions, Arch. Biochem., 30, 226–236 (1951).