**3. Results and discussion**

The effects of the suspension volume on the oscillation mode are shown in Fig. 5. Remarkable oscillatory waves were observed in the case of 10, 20, and 30 mL but not in the case of 50 mL. It was noteworthy that, even with the largest volume, the 50 mL suspension showed the smallest luminescent intensity. This might be due to the shortage of the oxygen supply, as the fixed liquid-air interface area could allow a fixed amount of oxygen diffusion into the suspension. In the case of 50 mL, the distributed oxygen to each cell should be smaller than in the case of other volumes. As the case of 30 mL showed the most distinct oscillation, this volume was chosen for further experiments.

Two sensors with filters were placed at the same height so that the stirrer bar did not affect the measurement. All the optical setup, including the magnetic stirrer, was enclosed in the incubator.

The effects of the suspension volume on the oscillation mode are shown in Fig. 5. Remarkable oscillatory waves were observed in the case of 10, 20, and 30 mL but not in the case of 50 mL. It was noteworthy that, even with the largest volume, the 50 mL suspension showed the smallest luminescent intensity. This might be due to the shortage of the oxygen supply, as the fixed liquid-air interface area could allow a fixed amount of oxygen diffusion into the suspension. In the case of 50 mL, the distributed oxygen to each cell should be smaller than in the case of other volumes. As the case of 30 mL showed the most distinct

Fig. 4. System setup for the measurement of spectral change.

oscillation, this volume was chosen for further experiments.

**3. Results and discussion** 

Fig. 5. Suspension volume effect on the oscillation in bacterial bioluminescence.

First, oscillation in bioluminescence was observed only in the case with the oscillation broth (Sato, Y. & S. Sasaki (2008)). We were interested in the use of the common marine broth and tried to determine the broth dilution effect on the mode of oscillation (whether or not it oscillated) because, in our previous report, the oscillation was thought to be the result of a lack of nutrients. Therefore, even with the marine broth, the oscillation was observed (Fig. 6). In cases of no dilution, clearer peaks were observed than in the cases with dilution. In addition, the luminescence measurement was performed with a cap on the glass tube. This case also showed, though with a different mode, an oscillatory behaviour. Through measurement with two different broths, the effect of oxygen supply into the suspension on the mode change of oscillation was strongly indicated.

Oscillation in Bacterial Bioluminescence 175

(a)

(b)

The measurement conditions were the same as those reported in Fig. 5.

Fig. 6. Broth-dilution effect on the oscillation in bacterial bioluminescence.

Encouraged by the above results, we tried to see the stirring effect on the bioluminescence over a shorter period because, during measurements lasting more than a day (1,440 min), the cell density effect on the luminescence could not be ignored. We, therefore, used brightly glowing suspensions (5 – 19 hours after inoculation / 108-109 cells mL-1) and investigated the effect of stirring on the luminescence intensity. First, the dark suspension was stirred until the luminescence reached a stable intensity. The result is shown in Fig. 7 (a). The luminescence intensity was gradually increased. This might be due to the increase in the fluorescence activity of LumP. In other bioluminescent bacteria, *V. fischeri* Y1, a fluorescent protein changes the fluorescent activity in its redox states; i.e., when reduced, the fluorescence is lost, and, when oxidised, the original fluorescence is retrieved (Karatani, H.; Izuta, T.; & Hirayama, S. (2007)). LumP in P. kishitanii might have similar characteristics.

The measurement conditions were the same as those reported in Fig. 5.

Fig. 6. Broth-dilution effect on the oscillation in bacterial bioluminescence.

Encouraged by the above results, we tried to see the stirring effect on the bioluminescence over a shorter period because, during measurements lasting more than a day (1,440 min), the cell density effect on the luminescence could not be ignored. We, therefore, used brightly glowing suspensions (5 – 19 hours after inoculation / 108-109 cells mL-1) and investigated the effect of stirring on the luminescence intensity. First, the dark suspension was stirred until the luminescence reached a stable intensity. The result is shown in Fig. 7 (a). The luminescence intensity was gradually increased. This might be due to the increase in the fluorescence activity of LumP. In other bioluminescent bacteria, *V. fischeri* Y1, a fluorescent protein changes the fluorescent activity in its redox states; i.e., when reduced, the fluorescence is lost, and, when oxidised, the original fluorescence is retrieved (Karatani, H.; Izuta, T.; & Hirayama, S. (2007)). LumP in P. kishitanii might have similar characteristics.

(b)

Oscillation in Bacterial Bioluminescence 177

The stirrer was switched off at 0, 20, 40, 60, 80, and 100 s and on at 10, 30, 50, 70, 90, and 110 s. The

measurement was performed at 17C.

Data was recorded every ten minutes.

Fig. 9. Time courses of dissolved oxygen and luminescence.

Fig. 8. Effect of stirring on the bright suspension.

In (a), the stirrer was switched on at 200, 400, 600, 800, 1,000, and 1,200 min and off at 300, 500, 700, 900, and 1,100 min. In (b), the stirrer was switched on at 60 s and off at 180 s. The measurements in both (a) and (b) were performed at 17C. Photographs in (c) were taken at a 5 s interval. Luminescence from the suspension after the stirrer was switched on was measured for two minutes (Fig. 7 (b)). A local maximal luminescence was observed right after the stirring (ca. 60 s), and then, a gradual increase was observed. This characteristic might be related to the LumP fluorescence ability, but the photographs of the luminescence showed no significant colour change (Fig. 7 (c)).

Fig. 7. Time course of the luminescence from the dark suspension after repeated stirring (a), a typical luminescence curve showing two peaks of intensity (b), and interval photographs of luminescence from the suspension in experiment (b) (c).

The effect of stirring on the bright (originally well-stirred) suspension luminescence resulted in different outcomes (Fig. 8). The luminescence increased after switch-off and decreased after switch-on. This tendency is the opposite of the results in Fig. 7 (a). The reason for the decreasing tendency of luminescence under the stirred condition is difficult to explain as long as we regard the suspension to be homogeneous. As is reported later, the condition of the cells in the suspension seemed to be inhomogeneous.

The suspension DO characteristic during the oscillation is shown in Fig. 9. As is evident from the figure, the DO during the oscillation was approximately zero. This result was considered to be reasonable, since the origin of bioluminescence was an oxygen-quenching mechanism. One evolutionary purpose of bioluminescence is oxygen quenching (Rees, J.F (1998), Timmins, GS. (2001), Szpilewska, H., Czyz, A. & Wegrzyn, G. (2003)). In a wellstirred condition, oxygen in the atmosphere diffused into the suspension, but most of it was assumed to be consumed by both the luminescence reaction and respiration. *Vibrio fisheri* was reported to perform anaerobic respiration using a certain gene regulator (Septer, AN.; Bose, JL.; Dunn, AK. & Stabb, EV. (2010).). No such report was available for the *Photobacterium* species. As a result, there was no significant relationship between the suspension DO and oscillatory waves. From this result, we recognised the importance of considering the DO within rather than outside the cell.

(c)

In (a), the stirrer was switched on at 200, 400, 600, 800, 1,000, and 1,200 min and off at 300, 500, 700, 900, and 1,100 min. In (b), the stirrer was switched on at 60 s and off at 180 s. The measurements in both (a)

Fig. 7. Time course of the luminescence from the dark suspension after repeated stirring (a), a typical luminescence curve showing two peaks of intensity (b), and interval photographs

The effect of stirring on the bright (originally well-stirred) suspension luminescence resulted in different outcomes (Fig. 8). The luminescence increased after switch-off and decreased after switch-on. This tendency is the opposite of the results in Fig. 7 (a). The reason for the decreasing tendency of luminescence under the stirred condition is difficult to explain as long as we regard the suspension to be homogeneous. As is reported later, the condition of

The suspension DO characteristic during the oscillation is shown in Fig. 9. As is evident from the figure, the DO during the oscillation was approximately zero. This result was considered to be reasonable, since the origin of bioluminescence was an oxygen-quenching mechanism. One evolutionary purpose of bioluminescence is oxygen quenching (Rees, J.F (1998), Timmins, GS. (2001), Szpilewska, H., Czyz, A. & Wegrzyn, G. (2003)). In a wellstirred condition, oxygen in the atmosphere diffused into the suspension, but most of it was assumed to be consumed by both the luminescence reaction and respiration. *Vibrio fisheri* was reported to perform anaerobic respiration using a certain gene regulator (Septer, AN.; Bose, JL.; Dunn, AK. & Stabb, EV. (2010).). No such report was available for the *Photobacterium* species. As a result, there was no significant relationship between the suspension DO and oscillatory waves. From this result, we recognised the importance of

Luminescence from the suspension after the stirrer was switched on was measured for two minutes (Fig. 7 (b)). A local maximal luminescence was observed right after the stirring (ca. 60 s), and then, a gradual increase was observed. This characteristic might be related to the LumP fluorescence ability,

but the photographs of the luminescence showed no significant colour change (Fig. 7 (c)).

and (b) were performed at 17C. Photographs in (c) were taken at a 5 s interval.

of luminescence from the suspension in experiment (b) (c).

the cells in the suspension seemed to be inhomogeneous.

considering the DO within rather than outside the cell.

The stirrer was switched off at 0, 20, 40, 60, 80, and 100 s and on at 10, 30, 50, 70, 90, and 110 s. The measurement was performed at 17C.

Fig. 8. Effect of stirring on the bright suspension.

Data was recorded every ten minutes.

Fig. 9. Time courses of dissolved oxygen and luminescence.

Oscillation in Bacterial Bioluminescence 179

The oscillation mode observed under the same suspension condition differed, as shown in the figures. These differences should be kept in mind for the following experiments. As reported above, the luminescence from LumP (peak wavelength: ca. 475 nm) was the main part of the observed light. The ratio of the luminescence at throughout the oscillation was estimated by the use of optical filters. The results are shown in Fig. 11 (a). A blue light with a spectral peak at 479 nm appeared ca. 1 h after a green light (521 nm) and quenched 4 h before that. This result indicated the change in the fluorescence ability at the beginning and at the end of the oscillation. When the luminescence intensity at 521 nm was plotted against that at 479 nm, the two showed a linear relationship (Fig. 11 (b)). This indicated that the

For the first time, we found an oscillation in bioluminescence intensity. The next step would be to identify the initial reason for the oscillation. Since a definitive answer is not yet available, we propose the hypothesis explained below. Bacterial luminescence spectral change has been reported (Eckstein, JW.; Cho, KW.; Colepicolo, P.; Ghisla, S.; Hastings, JW. & Wilson, T. (1990).; Karatani, H.; Matsumoto, S.; Miyata, K.; Yoshizawa, S.; Suhama, Y. & Hirayama, S. (2006).; Karatani, H.; Yoshizawa, S. & Hirayama, S. (2004).). Under the DO-rich condition, the LumP fluorescence capacity is high, and a blue light is evident, whereas, under a DO-poor condition, luciferin-luciferase luminescence (with a peak wavelength of 540 nm) occupies the main part, and a green light is evident. When the luminescence spectra measured with and without stirring were compared, a slight difference in the peak wavelength was observed (Fig. 12). This result agreed with the above-mentioned report.

(a)

LumP fluorescence ability was stable during the oscillation period.

The cell density was expressed by the optical density (OD) in the measurement. OD was measured as the decrease in near-infrared light measured at the sensor (Fig. 3). This OD probe light did not affect the bioluminescence measurement using solar cells. Four results of the simultaneous measurement of DO and luminescence are shown in Fig. 10 (a) – (d). We searched for the common characteristics between the DO and luminescent curves in the four cases and found that, after the luminescence peak, a plateau in the DO curve appeared. This might be due to the decrease in DO inside the cell after the luminescence that inhibited the respiration. Lack of oxygen might have suppressed the energy production by the respiration.

A 100 mL oscillation broth in a 500 mL Erlenmeyer flask was used for each measurement. Measurements were performed at room temperature (20-23ºC).

Fig. 10. Time courses of the luminescence and optical density in four experiments under the same condition.

The cell density was expressed by the optical density (OD) in the measurement. OD was measured as the decrease in near-infrared light measured at the sensor (Fig. 3). This OD probe light did not affect the bioluminescence measurement using solar cells. Four results of the simultaneous measurement of DO and luminescence are shown in Fig. 10 (a) – (d). We searched for the common characteristics between the DO and luminescent curves in the four cases and found that, after the luminescence peak, a plateau in the DO curve appeared. This might be due to the decrease in DO inside the cell after the luminescence that inhibited the respiration. Lack of oxygen might have suppressed the energy production by the

(a) (b)

(c) (d)

Fig. 10. Time courses of the luminescence and optical density in four experiments under the

A 100 mL oscillation broth in a 500 mL Erlenmeyer flask was used for each measurement.

Measurements were performed at room temperature (20-23ºC).

same condition.

respiration.

The oscillation mode observed under the same suspension condition differed, as shown in the figures. These differences should be kept in mind for the following experiments. As reported above, the luminescence from LumP (peak wavelength: ca. 475 nm) was the main part of the observed light. The ratio of the luminescence at throughout the oscillation was estimated by the use of optical filters. The results are shown in Fig. 11 (a). A blue light with a spectral peak at 479 nm appeared ca. 1 h after a green light (521 nm) and quenched 4 h before that. This result indicated the change in the fluorescence ability at the beginning and at the end of the oscillation. When the luminescence intensity at 521 nm was plotted against that at 479 nm, the two showed a linear relationship (Fig. 11 (b)). This indicated that the LumP fluorescence ability was stable during the oscillation period.

For the first time, we found an oscillation in bioluminescence intensity. The next step would be to identify the initial reason for the oscillation. Since a definitive answer is not yet available, we propose the hypothesis explained below. Bacterial luminescence spectral change has been reported (Eckstein, JW.; Cho, KW.; Colepicolo, P.; Ghisla, S.; Hastings, JW. & Wilson, T. (1990).; Karatani, H.; Matsumoto, S.; Miyata, K.; Yoshizawa, S.; Suhama, Y. & Hirayama, S. (2006).; Karatani, H.; Yoshizawa, S. & Hirayama, S. (2004).). Under the DO-rich condition, the LumP fluorescence capacity is high, and a blue light is evident, whereas, under a DO-poor condition, luciferin-luciferase luminescence (with a peak wavelength of 540 nm) occupies the main part, and a green light is evident. When the luminescence spectra measured with and without stirring were compared, a slight difference in the peak wavelength was observed (Fig. 12). This result agreed with the above-mentioned report.

Oscillation in Bacterial Bioluminescence 181

The blue curve indicates the spectrum of luminescence at 479 nm, and the brown curve indicates that at

400 450 500 550 600 **wavelength / nm**

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

During cell cultivation, the variety of cell phases was assumed to increase with cell growth even when the inoculated cells had the same, synchronised cell phases. In the glowing suspension, the cell condition was assumed to be inhomogeneous. A photograph of the bioluminescent suspension after the stirrer was switched off is shown in Fig. 13. A slowly precipitating block of cells was glowing as brightly as the air-liquid interface part. At that moment, the DO in the middle of the suspension was zero. Unlike others, this block of cells

Fig. 12. Bioluminescence spectra with and without stirring (normalized).

emitted light even under the [DO]=0 condition.

521 nm.

0

0.2

0.4

0.6

0.8

1

1.2

(b)

An approximation line between the two luminescences is illustrated. The coefficient of determination (R2) was calculated to be 0.9564.

Fig. 11. Bioluminescence oscillation observed in two colours (a) and relationship between blue (479 nm) and green (521 nm) colours (b).

(b)

An approximation line between the two luminescences is illustrated. The coefficient of determination

Fig. 11. Bioluminescence oscillation observed in two colours (a) and relationship between

(R2) was calculated to be 0.9564.

blue (479 nm) and green (521 nm) colours (b).

Fig. 12. Bioluminescence spectra with and without stirring (normalized).

During cell cultivation, the variety of cell phases was assumed to increase with cell growth even when the inoculated cells had the same, synchronised cell phases. In the glowing suspension, the cell condition was assumed to be inhomogeneous. A photograph of the bioluminescent suspension after the stirrer was switched off is shown in Fig. 13. A slowly precipitating block of cells was glowing as brightly as the air-liquid interface part. At that moment, the DO in the middle of the suspension was zero. Unlike others, this block of cells emitted light even under the [DO]=0 condition.

Oscillation in Bacterial Bioluminescence 183

(a)

(b)

In (a), the relative bright cell density was calculated as 0.05\* (relative luminescence), whereas the relative dark cell density was calculated as {OD-0.05\*(relative luminescence)}. In (b), data at 2650 - 2850

Fig. 14. Time courses of bright and dark cells (a) and relative dark cell density plotted

min were chosen.

against the relative bright cell density (b).

The image was photographed using a digital still camera (GR Digital 3, Ricoh Company, Ltd.) with exposure time of 1/20 s, ISO 1600, f/1.9. The raw image was modified to enhance the contrast using image software (ImageJ).

Fig. 13. Image of brightly glowing cell block precipitating in the suspension.

The results in Fig. 10 indicated the possibility that the luminescence affected the cell growth; i.e., an increase in luminescence caused oxygen deficiency and inhibited the respiration needed for cell growth. Cell growth was assumed to be expressed by the time derivative of the optical density. We, therefore, plotted the time courses of relative luminescence and the time derivative of OD in the same time scale (Fig. 14 (a)). The result shown in Fig. 10 (c) was used because it showed five obvious peaks in the relative luminescence curve. As is clear in Fig. 14 (a), the peaks and valleys in the luminescence curve coincided with those in the time derivative of the optical density. We then plotted the derivative against the relative luminescence (Fig. 14 (b)). The obtained curve showed that the two parameters were in the relationship with a negative Pearson product-moment correlation coefficient.

The image was photographed using a digital still camera (GR Digital 3, Ricoh Company, Ltd.) with exposure time of 1/20 s, ISO 1600, f/1.9. The raw image was modified to enhance the contrast using

The results in Fig. 10 indicated the possibility that the luminescence affected the cell growth; i.e., an increase in luminescence caused oxygen deficiency and inhibited the respiration needed for cell growth. Cell growth was assumed to be expressed by the time derivative of the optical density. We, therefore, plotted the time courses of relative luminescence and the time derivative of OD in the same time scale (Fig. 14 (a)). The result shown in Fig. 10 (c) was used because it showed five obvious peaks in the relative luminescence curve. As is clear in Fig. 14 (a), the peaks and valleys in the luminescence curve coincided with those in the time derivative of the optical density. We then plotted the derivative against the relative luminescence (Fig. 14 (b)). The obtained curve showed that the two parameters were in the

Fig. 13. Image of brightly glowing cell block precipitating in the suspension.

relationship with a negative Pearson product-moment correlation coefficient.

image software (ImageJ).

In (a), the relative bright cell density was calculated as 0.05\* (relative luminescence), whereas the relative dark cell density was calculated as {OD-0.05\*(relative luminescence)}. In (b), data at 2650 - 2850 min were chosen.

Fig. 14. Time courses of bright and dark cells (a) and relative dark cell density plotted against the relative bright cell density (b).

The type of model that could describe such oscillatory behaviour should be identified. One of the best-known models is the one proposed by Alfred Lotka and, later, by Vito Volterra (Mounier, J.; Monnet, C.; Vallaeys, T.; Arditi, R.; Sarthou, AS.; Helias, A. & Irlinger, F. (2008).; Varon, M. & Zeigler, BP. (1978).; Tsuchiya, HM.; Drake, JF.; Jost, JL. & Fredrickson, AG. (1972).) This model is often used to characterise predator-prey interactions. If we were to adjust the bacterial bioluminescence in the model, the following might be examples:

> 2 2 *broth bright cell bright cell bright cell darkcell darkcell darkcell deadcell* (1)

Oscillation in Bacterial Bioluminescence 185

S.; Okamoto, T. & Fujii T. (2009)). The evaluation of surface-adsorbed cells was thought to be an effective way for this purpose. The characteristics of the luminescence from ca. 1.0 X 106 cells adsorbed on a glass surface are shown in Fig. 16. Irradiation of the cells was performed using a near-UV light (UV lamp—long wavelength, # 166-0500EDU, BIO RAD). The irradiation has the potential to cause a change in the redox state of FMN or other materials that produce an increase in luminescence. Bacterial bioluminescence from the electromagnetic viewpoint has been studied (Pooley DT. (2011)). Investigation of this luminescence from physico-chemical as well as biochemical viewpoints would be needed to

The initial values were [X[=1,000 and [Y]=100, with constants k1=0.009, k2=0.06, and kd=0.0001. The integration time was set at 1, and the calculation was performed using Microsoft Excel 2007 running on

Fig. 15. Solution of Equation (2) using a numerical calculation (Runge-Kutta method)

a personal computer.

explain the entire image of bacterial bioluminescence.

In these reactions, we regarded that


If we write

A: broth, X: bright cell, Y: dark cell, P: dead cell, then the above equations can be written as

$$\begin{array}{c} \begin{array}{l} A+X \xrightarrow{k\_1} \xrightarrow{k\_1} \end{array} \begin{array}{l} \begin{array}{l} X \\ \end{array} \\ Y \xrightarrow{k\_d} \xrightarrow{k\_d} P \end{array} \end{array} \tag{2}$$

We consider A, the broth, to be infinite and not to decrease through the oscillation reaction (however, in an experiment, it does). As X and Y are the function of the time t, we can write two equations, such as,

$$\begin{aligned} \frac{dX}{dt} &= k\_1[A][X] - k\_2[X][Y] \\ \frac{dY}{dt} &= k\_2[X][Y] - k\_d[Y] \end{aligned} \tag{3}$$

These are the typical equations that appear in the model. We have a numerical solution of the two equations, i.e., the time course of X and Y through the simulation using a common spreadsheet software that runs on a personal computer.

Model (1) is not proved to interpret what is going on in the oscillation, but we can approach the real image of the oscillatory reaction. By changing the parameters k1, k2, and kd, we will have curves that look like what we observe, and we should then determine the values for the three parameters and evaluate their suitability from a biochemical viewpoint.

As reported in relation to Fig. 13, luminescence from the suspension with a volume of several tens – hundreds of mL might contain luminescence from cells of different conditions. Future investigation of cells with similar conditions is indicated, therefore, to be necessary. The relationship between the bacterial motility and luminescence was investigated (Sasaki,

The type of model that could describe such oscillatory behaviour should be identified. One of the best-known models is the one proposed by Alfred Lotka and, later, by Vito Volterra (Mounier, J.; Monnet, C.; Vallaeys, T.; Arditi, R.; Sarthou, AS.; Helias, A. & Irlinger, F. (2008).; Varon, M. & Zeigler, BP. (1978).; Tsuchiya, HM.; Drake, JF.; Jost, JL. & Fredrickson, AG. (1972).) This model is often used to characterise predator-prey interactions. If we were to adjust the bacterial bioluminescence in the model, the following might be examples:

> *broth bright cell bright cell bright cell darkcell darkcell*

2. one bright cell becomes a dark cell as a result of interaction with a dark cell (both cells

A: broth, X: bright cell, Y: dark cell, P: dead cell, then the above equations can be written as

*d*

*k*

*Y P*

1 2

*k k*

*A X X XY Y*

We consider A, the broth, to be infinite and not to decrease through the oscillation reaction (however, in an experiment, it does). As X and Y are the function of the time t, we can write

1 2

These are the typical equations that appear in the model. We have a numerical solution of the two equations, i.e., the time course of X and Y through the simulation using a common

Model (1) is not proved to interpret what is going on in the oscillation, but we can approach the real image of the oscillatory reaction. By changing the parameters k1, k2, and kd, we will have curves that look like what we observe, and we should then determine the values for

As reported in relation to Fig. 13, luminescence from the suspension with a volume of several tens – hundreds of mL might contain luminescence from cells of different conditions. Future investigation of cells with similar conditions is indicated, therefore, to be necessary. The relationship between the bacterial motility and luminescence was investigated (Sasaki,

*dX kAX kXY*

*dY kXY kY*

[ ][ ] [ ][ ]

[ ][ ] [ ] *<sup>d</sup>*

2

the three parameters and evaluate their suitability from a biochemical viewpoint.

*dt*

*dt*

spreadsheet software that runs on a personal computer.

 

2 2

*darkcell deadcell*

1. one bright cell divides into two bright cells with the supply of infinite broth;

consume oxygen as a result of respiration and become dark ones);

In these reactions, we regarded that

3. a dark cell becomes a dead cell.

If we write

two equations, such as,

2 2

(1)

(2)

(3)

S.; Okamoto, T. & Fujii T. (2009)). The evaluation of surface-adsorbed cells was thought to be an effective way for this purpose. The characteristics of the luminescence from ca. 1.0 X 106 cells adsorbed on a glass surface are shown in Fig. 16. Irradiation of the cells was performed using a near-UV light (UV lamp—long wavelength, # 166-0500EDU, BIO RAD). The irradiation has the potential to cause a change in the redox state of FMN or other materials that produce an increase in luminescence. Bacterial bioluminescence from the electromagnetic viewpoint has been studied (Pooley DT. (2011)). Investigation of this luminescence from physico-chemical as well as biochemical viewpoints would be needed to explain the entire image of bacterial bioluminescence.

The initial values were [X[=1,000 and [Y]=100, with constants k1=0.009, k2=0.06, and kd=0.0001. The integration time was set at 1, and the calculation was performed using Microsoft Excel 2007 running on a personal computer.

Fig. 15. Solution of Equation (2) using a numerical calculation (Runge-Kutta method)

Oscillation in Bacterial Bioluminescence 187

The author thanks Dr. Hajime Karatani of the Kyoto Institute of Technology for his participation in discussions and Shoji Yamada, Kenshin Tamura, Shingo Kuriyama, Mika

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**5. Acknowledgments** 

1615-7591

0006-2960

pp. 2426-2430, ISSN 0956-5663

4, (February 1990), pp. 1466-1470, ISSN 0027-8424

Vol. 173, No. 1, (July 1995), pp. 5-11, ISSN 0378-1119

No. 5, (January 2007), pp. 566-570, ISSN 1474-905X

(February 2008), pp. 2-29, ISSN 0003-2670

82-90, ISSN 1844-122x

**6. References** 

Oil was used to prevent the bacterial environment from drying. Glass with an amino group modification (MAS coated glass slides, Matsunami Glass Ind., Ltd.) was used for the adsorption. The glass was soaked in a marine-broth-based bacterial suspension overnight. A measurement was performed using a luminescence meter (GENE LIGHT GL-200S, Microtec Nichion).

Fig. 16. Effect of irradiation to the luminescence from cells adsorbed on a glass surface.

## **4. Conclusion**

Oscillation in the bacterial bioluminescence mode is strongly dependent on the amount of oxygen supply to the solution. There is no clear relationship between the DO concentration and luminescence intensity, perhaps due to the consumption of oxygen by both the luminescence and respiration. The oscillation occurred at a very low DO concentration, and, when the time course of cell density was plotted with the same timescale as the luminescence intensity, the cell growth rate seemed to decrease after the strong luminescence. The fluorescence ability of LumP seemed constant during the oscillation period, but, at the beginning and at the end, it seemed to decrease. The characterisation of luminescence from a smaller number of cells would be necessary for further investigation of oscillation, considering that the suspension is a mixture of cell groups with a variety of cell phases.

## **5. Acknowledgments**

The author thanks Dr. Hajime Karatani of the Kyoto Institute of Technology for his participation in discussions and Shoji Yamada, Kenshin Tamura, Shingo Kuriyama, Mika Mochizuki, and Hajime Kimoto for their assistance with the experiments.

#### **6. References**

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**4. Conclusion** 

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Fig. 16. Effect of irradiation to the luminescence from cells adsorbed on a glass surface.

Oscillation in the bacterial bioluminescence mode is strongly dependent on the amount of oxygen supply to the solution. There is no clear relationship between the DO concentration and luminescence intensity, perhaps due to the consumption of oxygen by both the luminescence and respiration. The oscillation occurred at a very low DO concentration, and, when the time course of cell density was plotted with the same timescale as the luminescence intensity, the cell growth rate seemed to decrease after the strong luminescence. The fluorescence ability of LumP seemed constant during the oscillation period, but, at the beginning and at the end, it seemed to decrease. The characterisation of luminescence from a smaller number of cells would be necessary for further investigation of oscillation, considering that the suspension is a mixture of cell groups with a variety of cell


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