Satoshi Sasaki

*Tokyo University of Technology Japan* 

#### **1. Introduction**

Live cells show various dynamic characteristics, such as cell division or material production. When we consider that a cell is a chemical reactor that contains an enzyme in its structure, the rates of chemical reaction catalysed by them depend on the cell density. As the amount of enzyme within the cell differs according to the rate of expression of a specific gene, the rate of the reaction also depends on the condition of the cell. In short, chemical reactions caused by cells are nonlinearly related to the cell density; the reaction rate is not proportional to the cell density. This is one remarkable aspect of live cells. In the field of chemical analysis, bacterial cell behaviour is often used. For example, changes in respiration caused by chemical compounds that inhibit the respiratory chain (such as KCN) can be quantified, theoretically, by measuring the changes in the dissolved oxygen concentration.

Biomaterial-based devices have been reported, such as biochips or biosensors. These are not truly "bio" because they use an enzyme or antibody outside of the cell. Microbial sensors (Melidis, P.; Georgiou, D.(2002).; Kang. KH.; Jang. JK.; Pham. TH.; Moon. H.; Chang. IS. & Kim, BH. (2003).; Moon, H.; Chang, IS.; Kang, KH.; Jang, JK. & Kim, BH. (2004). ; Chang, IS.; Moon, H.; Jang, JK. & Kim, BH. (2005).; Kogure, H.; Kawasaki, S.; Nakajima, K.; Sakai, N.; Futase, K.; Inatsu, Y.; Bari, ML.; Isshiki, K. & Kawamoto, S. (2005).; Vaiopoulou, E.; Melidis, P.; Kampragou, E. & Aivasidis, A. (2004).; Yano, Y.; Numata, M.; Hachiya, H.; Ito, S.; Masadome, T.; Ohkubo, S.; Asano, Y. & Imato, T. (2001).; Kim, M.; Hyun, MS.; Gadd, GM.; Kim, GT.; Lee, SJ. & Kim, HJ. (2009). Davila, D.; Esquivel, JP.; Sabate, N. & Mas, J. (2011).), known as the analysis of devices that use live microbial cells as molecular-recognition material, are the only exception. This sensor, however, is based on a shift from one equilibrium to another. For example, a respiration inhibition-based microbial sensor measures a certain toxic compound because the dissolved oxygen concentration near the cells increases when the toxic compound exists. The main reason for the use of microorganisms is that they are more cost-effective than purified enzymes or antibodies. The dynamics of the bacterial cells are not at all used. The nonlinearity of cell behaviour has recently been studied (Wu, BM.; Subbarao, KV. & Qin, QM. (2008).; Kenkre, V. M.; &, Kumar, N. (2008).; Dobrescu, R. & Purcarea, VI. (2011)). A suitable bacterium model is, therefore, needed to start a fundamental study on the nonlinearity of the cell. In our group studies, bioluminescence characteristics have been identified (Sasaki S., Mori Y., Ogawa M., Funatsuka S.,(2010)). Bioluminescent bacteria are those that emit light autonomously without the need of excitation light. The bioluminescence reaction is catalysed by bacterial

Oscillation in Bacterial Bioluminescence 169

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,

**2.1 Relationship between the bacterial bioluminescence and dissolved oxygen** 

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

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

**concentration in a bacterial suspension** 

Inc.) was placed into the bacterial suspension (Fig. 2).

measurement.

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

#### 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 oscillation mode was investigated.

Changes in the luminescence spectra are also reported.
