Biosensors and Bioelectronics 22 (2007) 1251–1259 Use of protoplasts from paired heterogenic bacterial species to detect tin contaminants: Prospects for biosensor development Douglas Mountfort a,∗ , Olivier Laczka b , Claire Debarnot b , Audrey Bonnin c , Neil Pasco d , Gareth Lloyd-Jones e a Cawthron Institute, Private Bag 2, Nelson, New Zealand b ESIL, Luminy Case 925, 13288 Marseille, Cedex 09, France c ENSIL, Limoges, France d Lincoln Technology, P.O. Box133, Lincoln, Christchurch 8152, New Zealand e Landcare Research, P.O. Box 69, Lincoln, New Zealand Received 16 March 2006; accepted 3 May 2006 Available online 3 July 2006 Abstract Two different bacteria gave different respiratory responses to the test analytes, tributyl tin (TBT) and cadmium as expressed by positive sigmoid responses by Halomonas sp. (slope, +1.71 [TBT]; +1.76 [Cd]) and negative sigmoid responses by Bacillus pumilis (slope, -1.06 [TBT]; -0.59 [Cd]). The EC 50 values determined from Hill plots for the response of Halomonas sp. to the TBT and Cd were 1 and 8.5 mM, respectively, which were lower by a factor of 10 than the corresponding values for B. pumilis. With protoplasts of B. pumilis there was a major shift in the signal from sigmoid negative to positive with TBT (+1.35) but not Cd (-0.5), while the signals with the remaining protoplast–analyte combinations remained unchanged. For all four protoplast–analyte combinations the EC 50 values were in the order of 10–100-fold lower than those for their whole cell counterparts. When other analytes were tested the protoplasts gave a similar response to tin as for TBT, but detected copper and 2,4-dichlorophenol with similar signal profiles to Cd and with lower sensitivity. The difference in signal and higher sensitivity of the two species protoplast system towards TBT/tin compared to the other analytes tested, suggests that it may feasible to develop this approach for the detection of tin residues. © 2006 Elsevier B.V. All rights reserved. Keywords: Microbial; Biosensors; Respiration; Response; Tin residues 1. Introduction Tin and its residues are recognised as one of the major metal pollutants in estuaries and bays and particularly in areas where heavy industry has been long been established, or where there are shipping ports, it constitutes a serious threat to marine and estuarine ecosystems (Eisler, 1989; King et al., 1989). The bioci- dal effects of tin have lead to its use in organic formulations (e.g. tributyltin) as an anti-foulant on ships to prevent fouling on hulls by marine organisms. However because of its adverse effect on non-target species, such as shellfish, the use of TBT as an anti- foulant on ships will be prohibited under International Maritime Organization convention by 2008. Tin and its organic deriva- tives are also used in many agricultural and horticultural spray ∗ Corresponding author. Tel.: +64 3 5482319; fax: +64 3 5469464. E-mail address: douglas.mountfort@cawthron.org.nz (D. Mountfort). formulations and their use, combined with tin residues in waste streams poses a risk to life in lakes and streams adjacent to treated areas as well as a health threat through contamination of potable waters (Bundy et al., 1997). Although conventional analytical techniques have been developed to detect organo-tins such as TBT, no technique is yet available that is able to measure total tin residues using a field device. A method developed to detect tin in all its formulations on site would have value in detecting toxic degradation products in marine and freshwater systems from for- mulations applied in sprays over many years, anti-foulants, as well as residues from heavy metal processing industries. This paper explores the application of micro-organisms in developing a biosensor to detect total tin with the view of its application in measuring the effectiveness of remediation programmes. Recent reviews about the development of microbial biosen- sors for monitoring environmental pollutants (D’Souza, 2001; Keane et al., 2002; Belkin, 2003) identify two general biosen- sor strategies; the first is to measure broad properties such as 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.05.010