Pollution monitoring using Photobacterium phosphoreum

Detection of marine contaminants is a very important factor in pollution management in which biological indicators can play a big part. Bioluminescence in microbes can provide a rapid visible indicator of this contamination making it a cheap and easily reproducible method of detection. This is possible as the production of luciferase coding for bioluminescence in marine bacteria can be switched off or suppressed in response to environmental pollution. Photobacterium phosphoreum is a bacterium regularly used to detect marine pollution in commercial systems, normally done by recording bioluminescence in discontinuous culture. However, this study looks at the importance of maintaining a continuous culture and immobilising the bacterium in order to measure light output using fibre optics.

The first aim of the experiment was to evaluate bioluminescence retention in immobilized cells. This was done by comparing immobilized suspensions of the culture in NaCl solution and alginate to a control of mobilized culture on Difco agar. These samples were all then stored at 4, -20 and -80ᵒC using glycerol as a cryoprotectant, recording bioluminescence weekly using a luminescent spectrophotometer allowing luciferase activity to be expressed as relative light units (RLU). This showed that luminescence is directly proportional to viable cell number in the culture making it an appropriate measure of the toxic effect of various chemicals. However there was limitation with the detection limits of instrumentation used meaning outside a 10³-10⁶ cells per ml range the relationship was non-linear and harder to detect. At 4ᵒC the NaCl solutions only retained their bioluminescence for 2 weeks, compared to the alginate cultures which retained light emission for up to 4 weeks without significant decline. At -20ᵒC and -80ᵒC the cultures in alginate also retained their bioluminescence for a significant amount of time. At -20ᵒC there was no improvement of light retention compared to 4ᵒC, however at -80ᵒC there was significant improvement in light retention. This is thought to be due to the fact that P. phosphoreum is a temperature sensitive bacterium, preferring ambient to low temperatures. At these lower temperatures it goes into a state of cryogenic protection, which protects the cells and their metabolic function, including the process of bioluminescence.

Other immobilization matrices were also tested by having their bioluminescence measured every hour whilst in storage, these included agarose, low melting point agarose and polyacrylsmide. Agarose and low melting point agarose lost significant bioluminescence almost immediately suggesting the gelling materials may have had a harmful effect on the bacteria. Whilst polyacrylsmide lost all bioluminescence straight away making it an unsuitable medium for this method.

The solidifying agents strontium chloride and calcium chloride were also tested for use as immobilizing the cell cultures. Calcium chloride was shown to be an insufficient solidifying agent, whilst Strontium chloride performed well, allowing it to be used in subsequent experiments involving the testing of toxic substances.

The second aim of the experiment was to put this method into practice and determine the effects of reference toxic chemicals including; Pb(NO₃)₂, NaAsO₂, NiCl₂, CdCl₂, HgCI₂, SDS and pentachlorophenol, on the bioluminescence of P. phosphoreum. Each chemical was tested using the optimised immobilization method, using alginate-glycerol media stroed at -80ᵒC. This involved using cultures in a flow- through cell method to achieve a semi-continuous culture, allowing for conditions to be controlled, including oxygen supply, pH and the concentrations of reference chemicals in the system.

 It was found that P. phosphoreum was still sensitive to all reference chemicals. However, when compared to mobile cultures the toxic effects were seen to be decreased as there was less contact between the toxic chemicals and the cells in cultures where the cells could not move around. Immobilization is thought to stabilize the biological activity in the culture which aids the use of this technique as a biosensor or pollution monitoring probe.

This experiment provides a great qualitative method for testing toxic chemicals on P. phosphoreum which allows for rapid and fairly reliable detection of chemicals in the environment. There are several factors such as the detection limits of instruments and the slight difference in sensitivity between mobilized and immobilized cells, which need to be accounted for when using this method. However, this is a step forward in monitoring and detecting marine pollution and its effects in the environment.

Chun U.H., Simonov N., Chen Y., and Britz M.L. (1996) Continuous pollution monitoring using Photobacterium phosphoreum. Resources, Conservation and Recycling, 18, 25–4.