**2. Experimentals**

Bioluminescent bacteria, *Photobacterium kishitanii*, collected from the skin of a cuttlefish and *Todarodes pacificus* (for sashimi), were purified and used. In a well-stirred solution, dissolved oxygen is in equilibrium with the atmospheric oxygen. This may not be the case with a bioluminescent bacterial suspension. As reported above, the luminescent reaction consumes oxygen to produce light. Simultaneously, production of the substrate FMNH2 requires energy that is produced by respiration. Karatani calculated the energy required to produce light and concluded that the bacterial bioluminescence is an extremely oxygen-consuming process. A bioluminescent bacterial suspension was, therefore, suspected to show a very low dissolved oxygen (DO) concentration. In this study, we began with the measurement of both DO and luminescent intensity through the period of oscillation.

As the luminescent reaction occurs inside the cell, the luminescent intensity is affected by the [DO] inside the cell rather than that in the suspension. Because the dynamic measurement of [DO] within a bacterium is considered to be difficult, we focused on any change in cell density during the oscillation period. The colour of bacterial bioluminescence is determined by the fluorescent protein (LumP) (Sato Y, Shimizu S, Ohtaki A, Noguchi K, Miyatake H, Dohmae N, Sasaki S, Odaka M, Yohda M., Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7 dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution., J Bacteriol. 2010 Jan;192(1):127-33.). We then, therefore, measured the spectral change in luminescence through the oscillation period.

luciferase (Raushel, F. M. & T. O. Baldwin; (1989), Lee, J., Y. Y. Wang and B. G. Gibson; (1991), Hastings, J. W. (1996), Shirazy, N. H., B. Ranjbar, S. Hosseinkhani, K. Khalifeh, A. R. Madvar and H. Naderi-Manesh (2007)). The reaction requires a flavin mononucleotide (FMNH2), a long-chain aliphatic aldehyde, and O2 to produce light (Balny, C. and J. W. Hastings (1975), Kurfurst, M., S. Ghisla and J. W. Hastings (1983), Tu, S. C., B. Lei, M. Liu, C.

FMNH2+RCOOH+O2--->FMN+RCOOH+H2O+hν This reaction is catalysed by bacterial luciferase (Karatani, H.; Izuta, T. & Hirayama, S. (2007)). This enzyme is synthesised by a process called quorum sensing, in which the synthesis occurs only after the cells recognise each other to be above a threshold in density. Two substrates, FMNH2 and RCHO (linear alkyl aldehyde), of the reaction are also synthesised in the cell. The substrate with the least amount is, therefore, the ratedetermining factor. The intensity of the bioluminescence has been reported primarily in connection with the oxygen concentration, but, theoretically, two other compounds might be candidates. Bacterial luminescence that has been used for environmental monitoring has been reviewed (Girott, S.; Ferri, E.N.; Fumo, M.G.; & Maiolini, E. (2008). Recently, an oscillation in luminescence from a well-stirred bacterial suspension was reported (Sato, Y. and S. Sasaki (2008)). Here, in this chapter, the relationship between the oxygen and

Bioluminescent bacteria, *Photobacterium kishitanii*, collected from the skin of a cuttlefish and *Todarodes pacificus* (for sashimi), were purified and used. In a well-stirred solution, dissolved oxygen is in equilibrium with the atmospheric oxygen. This may not be the case with a bioluminescent bacterial suspension. As reported above, the luminescent reaction consumes oxygen to produce light. Simultaneously, production of the substrate FMNH2 requires energy that is produced by respiration. Karatani calculated the energy required to produce light and concluded that the bacterial bioluminescence is an extremely oxygen-consuming process. A bioluminescent bacterial suspension was, therefore, suspected to show a very low dissolved oxygen (DO) concentration. In this study, we began with the measurement of both

As the luminescent reaction occurs inside the cell, the luminescent intensity is affected by the [DO] inside the cell rather than that in the suspension. Because the dynamic measurement of [DO] within a bacterium is considered to be difficult, we focused on any change in cell density during the oscillation period. The colour of bacterial bioluminescence is determined by the fluorescent protein (LumP) (Sato Y, Shimizu S, Ohtaki A, Noguchi K, Miyatake H, Dohmae N, Sasaki S, Odaka M, Yohda M., Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7 dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution., J Bacteriol. 2010 Jan;192(1):127-33.). We then, therefore, measured the

K. Tang and C. Jeffers (2000)).

oscillation mode was investigated.

**2. Experimentals** 

Changes in the luminescence spectra are also reported.

DO and luminescent intensity through the period of oscillation.

spectral change in luminescence through the oscillation period.

#### **2.1 Relationship between the bacterial bioluminescence and dissolved oxygen concentration in a bacterial suspension**

Photobacterium belongs to a family of Gram-negative, facultatively aerobic bacteria (Urbanczyk, H.; Ast, JC. & Dunlap, PV. (2011)). We started by measuring the oxygen effect on bioluminescence. The intensity of the bioluminescence was measured using a self-made luminescence detector (five commercially available solar cells were connected in series) or optical power meter (Model 3664, Hioki E.E. Co.). The output voltage generated by both devices was measured and recorded with an A/D converting logger (NR 250, Keyence Co.). An oscillation broth (Yeast extract 2.5 g L-1, Bacto peptone 5 g L-1, and NaCl 30 g L-1) or marine broth (DifcoTM marine broth 2216, Becton, Dickinson, and Company) was prepared and filtrated using a 0.22 µm filter (Nalgene disposable filter unit, Thermo Fisher Scientific, Inc.). A glass cell with an inner diameter of 31 mm was placed over a magnetic stirrer. The schematic illustration of the measurement system is shown in Fig. 1. All the equipment was placed in an incubator (VS401, Versos Co., Ltd.) adjusted at 17C with 10, 20, 30, and 50 mL of oscillation broth to determine the effects of the air-liquid interface area/volume. In addition, the dilution effect of the marine broth on the oscillation mode was investigated by diluting the broth 1.5 and 3 times. For the simultaneous measurement of luminescence and dissolved oxygen concentration, an optical fibre-based DO sensor (FOXY R, Ocean Optics, Inc.) was placed into the bacterial suspension (Fig. 2).

An aluminium foil cap was placed loosely on the glass tube to prevent contamination during the measurement.

Fig. 1. Schematic illustration of the bioluminescence intensity measurement.

Oscillation in Bacterial Bioluminescence 171

A 500 mL Erlenmeyer flask with 100 mL oscillation broth was shaken at 100 rpm. Solar cells were attached on the flask surface. All the optical setup was enclosed in a self-made dark box.

**2.3 Spectral change in the bacterial bioluminescence during oscillation** 

were placed to cover the sensor windows of the optical power meter (Fig. 3).

density.

Fig. 3. Experimental setup for the simultaneous measurement of the luminescence and cell

Two optical filters that transmit wavelengths of 479 and 521 nm (Optical Coatings Japan)

An optical fibre sensor tip was placed vertically in the middle of the bacterial suspension. An aluminium foil cap was placed loosely on the glass tube to prevent contamination during the measurement.

Fig. 2. Schematic illustration of the system for the simultaneous measurement of the luminescence intensity and dissolved oxygen concentration.

#### **2.2 Simultaneous measurement of the luminescence and cell density during oscillation**

Continuous measurement of the optical density (OD) of the bacterial suspension was performed using an OD meter (ODBox-A, TAITEC Co.). A 500 mL Erlenmeyer flask with 100 mL of bacterial suspension was set over a rotary shaker (NR-2, TAITEC Co.), and, on the surface of the flask, five solar cells connected in a series were attached (Fig. 3). The generated voltage was measured and recorded by the same logger as reported in 2.1. All the equipment was placed in a self-made dark box, and measurements were performed at room temperature ranging from 20 to 23C.

An optical fibre sensor tip was placed vertically in the middle of the bacterial suspension. An aluminium foil cap was placed loosely on the glass tube to prevent contamination during the

Fig. 2. Schematic illustration of the system for the simultaneous measurement of the

**2.2 Simultaneous measurement of the luminescence and cell density during** 

Continuous measurement of the optical density (OD) of the bacterial suspension was performed using an OD meter (ODBox-A, TAITEC Co.). A 500 mL Erlenmeyer flask with 100 mL of bacterial suspension was set over a rotary shaker (NR-2, TAITEC Co.), and, on the surface of the flask, five solar cells connected in a series were attached (Fig. 3). The generated voltage was measured and recorded by the same logger as reported in 2.1. All the equipment was placed in a self-made dark box, and measurements were performed at room

luminescence intensity and dissolved oxygen concentration.

measurement.

**oscillation** 

temperature ranging from 20 to 23C.

A 500 mL Erlenmeyer flask with 100 mL oscillation broth was shaken at 100 rpm. Solar cells were attached on the flask surface. All the optical setup was enclosed in a self-made dark box.

Fig. 3. Experimental setup for the simultaneous measurement of the luminescence and cell density.

#### **2.3 Spectral change in the bacterial bioluminescence during oscillation**

Two optical filters that transmit wavelengths of 479 and 521 nm (Optical Coatings Japan) were placed to cover the sensor windows of the optical power meter (Fig. 3).

Oscillation in Bacterial Bioluminescence 173

*P. kishitanii* was inoculated into oscillation broth with each volume in a 22 mm diameter glass tube. The

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

suspension was stirred using a magnetic stirrer. The temperature was maintained at 17C. Fig. 5. Suspension volume effect on the oscillation in bacterial bioluminescence.

the mode change of oscillation was strongly indicated.

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.

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