Propylene glycol-specific dehydrogenases as functional biomarkers for monitoring biodegradation in sites contaminated by de-icing chemicals

The area under study at Gardermoen airport (Oslo, Norway) is a glacial contact formation with sand and gravels dominating near the ground surface. Due to the northern climate, every winter large amounts of de-icing chemicals, i.e. propylene glycol (PG) and potassium formate, are commonly used in the airport for the removal of snow and ice from airplanes and runways, respectively. Even though these contaminants are easily degradable by biotic or abiotic factors, they may still threaten groundwater, due to the system overloading. The present study, performed within the FPVII European research project: “SoilCAM - Soil Contamination: Advanced integrated characterisation and time-lapse Monitoring”, deals with the physic-chemical and microbiological characterization of the site and the development of suitable methods for monitoring PG biodegradation on site under reclamation. For this purpose, functional biomarkers were selected to evaluate their potential use in Real Time quantitative PCR (q-PCR) experiments directly on soil DNA. The soils are highly leached, with low biological and biochemical activities. Therefore, pollutant transfer to the groundwater occurs quickly and is virtually unhindered. Bacterial strains isolated from the soil were able to degrade PG in aerobic conditions at 4, 15 and 30 °C. The PG-degrading population was mainly composed by different species of Pseudomonas, as shown by denaturing gradient gel electrophoresis (DGGE) analysis on soil DNA. Gene probes for PG-specific alcohol dehydrogenase (ADH-IIG) detected the presence of such genes in the isolates. The deduced amino acid sequence of representative strains presented over 92% identities with PG-specific dehydrogenase-related proteins. ADH-IIG detected in soil DNA indicated that PG-degrading strains were present along the soil profile from 0 to 100 cm. The application of q-PCR analysis on DNA from soil mesocosm experiments will confirm the suitability of ADH-IIG as biomarker for monitoring PG biodegradation in soil systems

Soil-catalyzed polymerization of phenolics in polluted waters

Some biotic and abiotic soil components are able to catalyze phenol oxidation, producing water-insoluble polymers. In phenol-polluted water bodies, this phenomenon could be exploited to prevent phenol dispersion. The reaction kinetics of phenol polymerization catalyzed by soil samples drawn from unsaturated and aquifer layers was measured in slurry, aerated batch reactors. Catechol was used as a model phenol. The observed catalytic activity is essentially abiotic and can be attributed to inorganic soil components. The rate of phenol removal is first-order with respect to both catechol and soil concentration. Soil activity towards other phenolic compounds was tested, as well. Diphenols show the highest reactivity. Comparisons were performed with the enzymatic activity of phenol oxidases-containing mushroom tissues whose use has been envisaged in the treatment of phenol-polluted waters. The use of phenol oxidases can complement the intrinsic activity of soil for the removal of recalcitrant phenols

Biodegradation of aircraft deicing fluids in soil slurries

Aircraft de-icing fluids (ADF) containing propylene-glycol (PG) and other additives are used at Northern airport in winter. Potassium formate is also used for de-icing of runways and taxiways. Due to the mechanical removal of snow from the runways and to the drift of ADF from the aircrafts during take-off, the ADFs and formate are mixed with snow. The chemicals infiltrate the soil surface along the runways when the melting of snow begins in spring. It is important to ascertain that these chemicals do not contaminate groundwaters and surface waters. The unsaturated zone can act as a natural purification system, because PG and potassium formate are easily degraded by soil bacterial communities under aerobic conditions, whereas anaerobic degradation in groundwaters is considerably slower. Nevertheless, they still constitute a threat to the groundwater if biodegradation rates are not sufficient compared to infiltration velocities in the unsaturated zone. We present experimental data on the biodegradation of PG and formate under aerobic and anaerobic conditions. Soil samples from the Oslo International Airport at Gardermoen have been used as source of degrading microorganisms. Gardermoen Airport is built on Norway’s largest rain-fed aquifer and must operate on the condition that the groundwaters are not affected. All biodegradation tests were carried out in shaken flask reactors containing soil suspensions in PG or formate solutions. This is an extremely simplified system as compared to what actually occurs at the site, all phenomena related to mass transfer and to soil heterogeneities having been removed. In order to characterise and to quantify a complex reacting system such as that at hand, however, it is a universally acknowledged practice to measure the intrinsic kinetic parameters, once all the unnecessary complications have been removed. In order to afford the actual situation to be modelled in detail, the identification and measurement of the kinetic parameters is a necessary prerequisite. The rate and the extent of biodegradation has been found to be severely limited by many factors, such as temperature, biomass concentration, availability of additional nutrients (nitrogen and phosphorus sources) and of electron acceptors (oxygen, nitrate, sulphate, iron and manganese oxides)

Oxidative polymerisation of phenols by a phenol oxidase from green olives

The phenol oxidase activity of green olive drupae was characterised in view of possible applications for the de-phenolisation of polluted waters. For experimental convenience, activity measurements were performed on acetone powders and raw-extracts from drupae tissues. Phenols were oxidised in the presence of dissolved molecular oxygen. Oxidation products were o-quinones undergoing non-enzymatic polymerisation. Final products were insoluble polymers that could be easily removed from the reaction mixture by centrifugation. The kinetics of activity loss was analysed and a possible "suicide substrate" deactivation mechanism, analogous to that of mushroom tyrosinase, has been proposed. Substrate specificity was particularly high towards o-diphenols and p-methyl substituted phenols. For real applications, the observed, low operational stability discourages the use of purified and immobilised enzyme in lieu of minced tissues. Furthermore, the seasonal availability of olive suggests an on-site use for polyphenol removal from vegetation waters resulting from the milling process

Use of the south Italy lysimeter station for soil remediation studies

Remediation of soil and groundwater contamination by organic chemicals has become an urgent problem. Besides of laboratory studies, real-scale studies are required for the validation of remediation techniques. Our scope is to assess the validity of the lysimeter as a tool for remediation program development. Soil lysimeter will be used as an experimental tool for the study of pollutant transport and degradation in both soil and groundwater at the field-scale, providing the necessary data for 1D-modeling. Variations undergone by the autochthonous microflora under the effect of contaminant addition will be monitored, as well. The soil lysimeter will aid the implementation of in situ bioremediation techniques, such as bioventing, air sparging, biostimulation, bioaugmentation, phytoremediation. All these general remarks have been taken into account in defining the layout of the AMRA multiple-lysimeter installation (built in Piana di Monte Verna, Caserta, Italy). It consists in eight units that operate individually and simultaneously. Programmable hydraulic systems are provided to simulate the fluctuations in water-table level, as well a complete set of sensors that enable the continuous monitoring of crucial parameters, including the soil sample weight, temperature, humidity, water tension. Sampling ports for gas, water, and soil off-line chemical analysis can be envisaged or on-line sensors can be used for some analytes. As of now, four units contain an uncontaminated agricultural soil (from a nearby field) and four an industrial soil (from the dismissed industrial site ILVA Bagnoli, Napoli). Two case studies have been selected to assess the validity of the lysimeter as a tool for remediation program development: 1) monitoring of a soil subject to periodic releases of olive mill waste water and 2) reactive transport of a crude oil spil

Recycle of olive mill wastewater for fertigation after treatment in soil slurry reactor

OMW are rich in organic and mineral compounds that could be usefully recycled as fertilizers. Their high polyphenol content, however, can affect soil health because of the related microbial- and phyto-toxicity. Therefore, a detoxification treatment should be desirable before using OMW for agricultural purposes. The aim of this work is to test the feasibility of a simple treatment of OMW in soil slurry reactors, with specific care to the effects of spreading the treated wastewater onto the soil

Catalytic and sorptive properties of NAPL-polluted soils

Catalytic activity towards phenol oxidation was characterized on sediment samples from a NAPL-polluted site in Thuringia, Germany. Catechol, a common metabolic intermediate in the pathways for aromatic biodegradation, was used as a model pollutant

Toxicity attenuation of olive mill wastewater in soil slurries

Olive mill wastewaters (OMW) are endowed with micro- and phyto-toxicity bound to the high polyphenol content. This work focuses on the potentiality of agricultural soil as a natural catalyst to promote polyphenol oxidation and polymerisation and thus detoxify OMW. We have shown that model polyphenols usually found in OMW are fully converted in soil slurries and products show no toxicity to the growth of a typical soil bacterium (B. cereus) and reduced phytotoxicity in germination tests (using the sensitive English cress seeds). Such preliminary data suggest the feasibility of an economically sustainable treatment of OMW in aerated soil slurries

A lysimeter station for contaminated soil studies

A lysimeter station has been set up by AMRA for the study of the reactive transport of contaminants in soil at the field-scale. It consists of eight lysimeters that can operate individually and simultaneously. Each lysimeter is a steel cylinder (diameter 1.14m, height 2m) filled with an undisturbed soil monolith, whose upper surface is directly exposed to the external environmental agents. A set of sensors enables the remote monitoring of overall lysimeter weight as well as of temperature, water potential and water content at several soil depths along the soil monolith. Water and gas samples can be obtained for chemical and biological analysis. A programmable hydraulic system is provided to simulate the fluctuations in water-table level. A gas-sparging system is already installed for bioventing applications

Phenol removal by enzymes: benefits and shortcomings

Comparisons have been performed of different phenol-oxidizing enzymes (phenol-oxidases, tyrosinases, laccases) from different sources. Specific reference has been made to substrate specificity, operational stability and contaminant immobilization efficiency, in order to assess suitability for environmental applications. Phenol-oxidase and tyrosinase enzymes, from plant sources, have been tested in batch experiments by using minced tissues, acetone powders and raw extracts. Substrate specificity is usually broad and absorption also takes place when tissues are used. Operational stability is low, due to product inactivation. Final reaction-products are insoluble polymers that can be removed by physical methods or immobilized into the humic matrix of the soil. As regards suitability for environmental applications, stability problems generally discourage the use of purified, immobilized enzymes. Minced tissues or raw extracts from plant tissues may be used successfully in wastewater at low phenol concentration without enzyme recovery, in view of the reasonable cost of the sources. As regards phenol-contaminated soil, the widespread presence of endogenous, mainly abiotic, activity makes the addition of plant enzymes unproductive for the most reactive contaminants such as di-phenols. Since batch experiments with soil slurries in the presence of variously substituted mono-phenols show exiguous natural activity, the use of plant extract or tissues with high phenol-oxidase activity is not completely ruled out. Comparison with literature data on wastewater and contaminated soil aerobic treatment shows that the rate of phenol removal by enzymes and natural abiotic catalysts can proceed faster than biodegradation, due to toxicity effects on microbial metabolism