Solid-Phase Contact Assay That Uses alux-MarkedNitrosomonas europaeaReporter Strain To Estimate Toxicity of Bioavailable Linear Alkylbenzene Sulfonate in Soil

Abstract

Information about in situ toxicity of the bioavailable pools of adsorptive soil pollutants is a prerequisite for proper ecological risk assessment in contaminated soils. Such toxicity data may be obtained by assays allowing for direct exposure of introduced test microorganisms to the toxicants, as they appear in solid solution equilibria in the natural soil. We describe a novel sensitive solid-phase contact assay for in situ toxicity testing of soil pollutants based on a recombinant bioluminescent reporter strain of Nitrosomonas europaea. A slurry of the reporter strain and soil sample was shaken for 1 h, after which bioluminescence was measured either directly (soil slurry protocol) or in the supernatant obtained after centrifugation (soil extract protocol). The assay was validated for both protocols by using linear alkylbenzene sulfonate (LAS) as a toxic and adsorptive model compound in the soil samples. Interestingly, LAS showed the same toxicity to the reporter strain with either soil incubation (both protocols) or pure culture, suggesting that adsorbed LAS pools contributed to the observed toxicity. The solid-phase contact assay that used the reporter strain of lux-marked N. europaea was slightly more sensitive for the detection of LAS toxicity in soil than activity-based assays targeting indigenous nitrifiers and much more sensitive than assays targeting indigenous heterotrophic microbes. We conclude that the new solid-phase contact assay, which is based on direct interaction of the test microorganisms with bioavailable pools of the toxicants in soil, provides a most sensitive and relevant method for evaluating the in situ toxicity and assessing the risks of soil contaminants.

Light dazzles from the black box: whole-cell biosensors are ready to inform on fundamental soil biological processes

“…Differently, the measurement of biosensor responses to specific analyte in soil may allow an online monitoring of the elemental bioavailability by monitoring the induction of specific promoters, allowing a better assessment of bioaccumulation and biomagnification P. putida RB1353/RB1351 (pUTK9) Bioluminescence BTEX [19] P. putida RB1401 (pTOL) Bioluminescence BTEX [20] P. putida TVA8 (pUTK214) Bioluminescence BTEX [6] Stenotrophomonas sp ENV307 (pUTK60) Bioluminescence Alkylsulphonates [73] R. eutropha JMP134 (pUTK220) Green fluorescence PCB [49] Burkholderia sp (pUCD607) Bioluminescence PCB [15] Pseudomonas putida F1 (pUT mini-Tn5 luxCDABE) Bioluminescence PCB [138] N. europaea ATCC 19718(pHLUX20) Bioluminescence Alkylsulphonates [11] P. fluorescens A506 (pTS) Green fluorescence BTEX [120] Heavy metals and metalloids…”

“…A large number of biosensors is also available for detection of either natural organic substances or organic pollutants, potentially very useful for characterization of risks posed by contaminated soils. Alkanes and derivatives can be detected using biosensors developed by inserting the alk regulon of Pseudomonas oleovorans, with the luxAB genes from V. harveyi, and the constructed plasmid transformed in E. coli DH5R which becomes bioluminescent specifically when exposed to linear alkanes, whereas biosensors for linear alkylsulphonates based on Nitrosomonas europaea [11] or Stenotrophomonas and Ralstonia strains [73] have been constructed by luxAB gene fusion with catabolic genes. Biosensors responding to bioavailable benzene, toluene and xylene (BTEX) in soil have been constructed by inserting the luxCDBAE or luc genes (lucFF is the firefly Photinus pyralis luciferase gene) in different plasmidial genes involved in the catabolic pathways of such compounds, using P. putida strains [6,20] or E. coli [140].…”

Wastewater Toxicity Assessment by Whole Cell Biosensor

“…An intriguing development is a lux-marked ammonia-oxidising bacterium, Nitrosomonas europaea, to estimate toxicity in soils [250]. Bioluminescence output in this case should be limited strictly by the amount of reducing power generated from ammonia oxidation, and the tight coupling of the two should mean sensitive and rapid responses.…”

Effect of linear alkylbenzene sulfonates on the growth of aerobic heterotrophic cultivable bacteria isolated from an agricultural soil

“…These LAS concentrations are in the range of those that may be discharged in agricultural soils after the addition of sludge as a fertilizer ). Nevertheless, some functional groups of bacteria, such as the ammonia-oxidizers, show a higher sensitivity to the presence of the surfactant, and concentrations ranging between 8 and 10 mg kg -1 LAS are enough to induce a negative impact on Nitrosomonas and Nitrosospira strains (Elsgaard et al 2001a, b;Brandt et al 2001Brandt et al , 2002Brandt et al , 2003. The main biological effect of LAS is the disruption of biomembranes and the denaturation of proteins.…”

“…In microorganisms, LAS adsorption produces a depolarization of cell membranes, and consequently decreases the absorption of nutrients and modifies the release of substances from cell metabolism (Jensen 1999). Although a vast literature is available on the effects of LAS on the ecology and physiology of the soil microbial communities (Jensen 1999;Schwuger and Bartnik 1980;Elsgaard et al 2001a, b;Brandt et al 2002Brandt et al , 2003, few studies have isolated bacteria species or bacteria consortia capable to biodegrade the surfactant, and little is known about the degradation processes and the microbial community involved (Khleifat 2006).…”