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High-throughput monitoring of bacterial growth at elevated hydrostatic pressure on a BMG LABTECH microplate reader

S. Lucas Black (1, 2), F. Bruce Ward (1), Rosalind J. Allen (2) (1) Institute of Cell Biology, (2) School and Physics and Astronomy, University of Edinburgh 03/2009

Introduction

Microorganisms display an astonishing ability to survive and proliferate under extreme environmental conditions, including high and low temperatures, high acidity or alkalinity, high salt concentrations, and high hydrostatic pressure. The physiological and biochemical origins of these capabilities are in many cases poorly understood. Technologically, extremophiles have the potential to provide new products for molecular biology and other biotech areas.


Our work focuses on the deep sea bacterium Photobacterium profundum SS9. This γ- proteobacterium is a close relative of Vibrio cholerae. P. profundum SS9 was isolated in 1984 from the Sulu Sea at a depth of 2.5 km, and is a piezophile: it grows better at elevated hydrostatic pressure than at atmospheric pressure.  P. profundum SS9 can grow at pressures ranging from 0.1 MPa to 90 MPa, with an optimal growth pressure  of 28 MPa. Like most marine organisms, P. profundum SS9 has a requirement for NaCl. It has been observed that the physiological effects of increased hydrostatic pressure are similar to those of osmotic pressure (increased salt). For example, P. profundum SS9  produces a similar range of intracellular osmolytes in response to both salt and hydrostatic pressure. The aim of our work is to understand better this intriguing similarity.

An established method to measure microbial growth is to measure absorbance at 600 nm. Absorbance and light scattering increase with increasing number of particles, in this case the number of microbes.

 

In this application note, the growth of the deep sea bacterium Photobacterium profundum SS9 was analyzed with regard to hydrostatic and osmotic pressure.

 

Monitoring the OD600 using a BMG LABTECH microplate reader, it was revealed that hydrostatic pressure has a marked effect on the range of NaCl concentration at which P. profundum SS9 is able to grow. When the hydrostatic pressure was increased from 0.1 MPa to 28 MPa, growth of P. profundum SS9 was largely unaffected at intermediate salt concentrations (200-400 mM NaCl), but growth was strongly inhibited at higher NaCl concentration, compared to the results at 0.1 MPa. The optimum NaCl concentration range was 300-400 mM for both hydrostatic pressures tested.

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