Microplate MPN-enumeration of monocyclic- and dicyclic-aromatic hydrocarbon degraders via substrate phase-partitioning

Abstract

The high aqueous solubility of monoaromatic and some diaromatic oil components may hinder classical growth-based MPN enumeration of bacterial mono- and di-aromatics degraders because these aromatics are toxic in high concentrations. We developed a microplate MPN method for the enumeration of toluene-, xylene-, naphthalene-, biphenyl- and benzothiophene-degraders on the basis of phase-partitioning of substrate between a biologically inert organic phase and an aqueous mineral salt medium. This way, it was possible to maintain non-toxic, aqueous concentrations in the microplate wells. Depletion of aqueous aromatics by growth of the degraders was prevented by the continuous solubilization of aromatics from the silicone phase. The method was validated by MPN enumerating degrader cultures both with phase-partitioned aromatics and with tryptic soy broth as carbon sources. The applicability of the method was demonstrated by MPN-enumerating mono- and di-aromatic degraders in soils of varying hydrocarbon pre-exposure.

Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate

“…Naphthalene was dissolved to Silicone oil AR20 (Sigma-Aldrich) to yield a concentration of 1% (w v -1 ) and added individually to each well (10 ll/well) as described by Johnsen and Henriksen (2009). Using silicone-water partition coefficient, Johnsen and Henriksen (2009) estimated the maximum aqueous concentration of naphthalene to be 18 mg l -1 in their system.…”

“…The first experiment was a method comparison between our method referred to as 'the vapour method' and the method of Johnsen and Henriksen (2009) referred to as 'the silicone oil method'. In the second experiment we investigated the effect of increasing concentration of oil contamination in soil on the number of naphthalene degraders detected with the vapour method.…”

“…Aromatic substrates are typically dissolved in a suitable volatile and added to each well individually Johnsen et al 2002;Kirk et al 2005). Johnsen and Henriksen (2009) mixed the aromatic substrates with inert mineral oil in order to reduce the toxicity of pure aromatics to the degrader community. Distinguishing growth-positive wells from negative wells appears to be tricky as there are several methods published for this purpose (reviewed by Johnsen 2010).…”

“…Distinguishing growth-positive wells from negative wells appears to be tricky as there are several methods published for this purpose (reviewed by Johnsen 2010). The most common way of detecting growth is to use respiration indicators (INT and WST-1) which change colour upon reduction by respiratory active cells (Haines et al 1996;Wrenn and Venosa 1996;Johnsen et al 2002;Johnsen and Henriksen 2009). The detection of growth by INT indicator is based on visual estimation where objectivity is hard to accomplish.…”

Fruit Quality of Tomato Affected by Single and Combined Bioeffectors in Organically System

“…From each sample three parallel 20 μl aliquots from each dilution were transferred to sterile polystyrene 96-well microplates. After incubation at 22°C for 1 week, the plates were tested for growth using the respiration indicator iodonitrotetrazolium violet (INT) (Sigma-Aldrich) (Johnsen and Henriksen, 2009 (Mukherjee, 2006;Vladar, 2008). Fruit quality measurements: The fruits (skins and fruit-pulp) were homogenized with a blender and homogenates were stored at -25°C until analysis.…”