Assay Microorganisms generate electrons from catabolic reactions, these can be disrupted by toxicants

1. SciTOXTM unit quantifies reduced mediator MED- KFC(II) H + Toxicants inhibit catabolism MED+ + - e KFC(III) e - Non - toxic substrate promotes catabolism + H Bacteria SciTOXTM - a low cost toxicity analyser for environmental applicationsN Glithero1, J Hay1, N Pasco1, D Patterson2, R Wattinger2 and R Weld11 Lincoln Ventures Ltd, PO Box 133, Christchurch, NEW ZEALAND2 Int2egy NZ Ltd www.integy-llc.com Assay • Microorganisms generate electrons from catabolic reactions, these can be disrupted by toxicants • Electrons donated by microorganisms reduce ferricyanide mediator to ferrocyanide • Toxic inhibition of microorganisms slows electron production and reduces catabolic activity depending on dosage • Mean generated from three replicates for each toxicant concentration to quantify ferrocyanide production and displayed as ratio of activity compared to zero-toxicant control • The EC50 is defined as the concentration of toxicant that provokes a response from the microorganisms halfway between the baseline and maximum response. Graphs and tabled EC50 values generated by non-linear Hillslope analysis (Sigma Plot 10) Aim To address an unmet need for technology capable of rapidly assessing toxic effects on microorganisms. A miniaturised method with rapid analysis based on limiting current microelectrode amperometry is presented Background Features • Control via touch screen • SciTOXTM unit bioreactors facilitate incubation (heating and agitation) of toxic sample and microorganisms • 15 minute incubation with optional simultaneous replicates followed by 20 second analysis • Direct toxicity assessment using microelectrode amperometry • Toxicant dose dependent response quantifies toxicity • Tolerant of variable sources of microorganisms - a wastewater treatment plant can use bacteria from its own effluent Applications • Measuring impact of contaminants and toxicants in aqueous environments • Rapid quantification of toxicity Applied trials Toxicants • Phenol • 2,4-dichlorophenol (2,4-DCP) • 3,5-dichlorophenol (3,5-DCP) • Acetone Microorganisms • Activated sludge flora from wastewater treatment plant • Escherichia coli • Bacillus subtilis Experimental Method • Microorganisms sourced from both laboratory cultures and wastewater treatment plants and adjusted to desired concentrations • Toxicants diluted to produce dose dependent response curve, zero toxicant control included • Primary incubation: equal volumes of toxicant and microorganisms incubated together 5 mins at 25°C with mild agitation for mixing • Secondary incubation: mediator (potassium ferricyanide) added and incubation continued for 10 mins Potassium ferricyanide reduced to potassium ferrocyanide by microorganisms Analysis • Ferrocyanide quantified immediately upon completion of secondary incubation using microelectrode amperometry • Incubated sample with toxicant added compared to incubated sample having no toxicant • Microorganism activity calculated from difference between the two Results Conclusions • The SciTOXTM unit produces usable toxicity measurements within 16 minutes • Quantification of ferrocyanide is reproducible with <5% variance between toxicity test replicates • A 1mm ø Au reference/auxillary electrode and 50µm ø Pt working electrode are capable of facilitating chronoamperometric measurements while microorganisms are present in analyte • A potential step of 200mV is adequate to drive the reactions as both electrodes exhibit the same equilibrium potential vs. Ag/AgCl electrode • This technology can accommodate a range of microorganisms, based on our trials: E. coli, B. subtilis and activated sludge (comprising dozens of bacteria species depending on sludge age and waste source) • Activated sludge microorganisms more resistant to toxicants than laboratory microorganism cultures with 3,5-DCP maximum inhibition at 0.55 activity for activated sludge and 0.25 for E. coli. For 2,4-DCP, maximum inhibition at 0.65 activity for activated sludge and activity still declining at 100mg/L for both E. coli and B. subtilis • Despite differences in the degree of signal attenuation between microorganism sources, the EC50 values to 2,4-DCP and 3,5-DCP remained similar Research funded by New Zealand Foundation for Research, Science and Technology Acknowledgements EC50 values for 2,4-DCP and E. coli with varying exposure times EC50 values for toxicants with target microorganisms Int2egy NZ Ltd.