Inoculation of soil with an Isoproturon degrading microbial community reduced the pool of "real non-extractable" Isoproturon residues.

During pesticides degradation, biogenic non-extractable residues ("apparent NER") may not share the same environmental fate and risks with the "real NER" that are bound to soil matrix. It is not clear how microbial community (MC) inoculation for pesticides degradation would influence the NER composition. To investigate degradation efficiency of pesticides Isoproturon (IPU) and NER composition following MC inoculation, clay particles harboring MC that contains the IPU degrading strain, Sphingomonas sp., were inoculated into soil receiving 14C-labeled IPU addition. Mineralization of IPU was greatly enhanced with MC inoculation that averagely 55.9% of the applied 14C-IPU was consumed up into 14CO2 during 46 days soil incubation. Isoproturon degradation was more thorough with MC than that in the control: much less amount of metabolic products (4.6% of applied IPU) and NER (35.4%) formed in MC treatment, while the percentages were respectively 30.3% for metabolites and 49.8% for NER in the control. Composition of NER shifted with MC inoculation, that relatively larger amount of IPU was incorporated into the biogenic "apparent NER" in comparison with "real NER". Besides its well-recognized role on enhancing mineralization, MC inoculation with clay particles benefits soil pesticides remediation in term of reducing "real NER" formation, which has been previously underestimated.

Effects of carbon amendment on in situ atrazine degradation and total microbial biomass.

This study elucidates the effects of carbon amendment on metabolic degradation of atrazine (6-chloro-N(2)-ethyl-N(4)-isopropyl-1,3,5-triazine-2,4-diamine) and total microbial biomass in soil. Degradation of (14)C-ring-labelled atrazine was monitored in laboratory incubations of soils supplemented with 0, 10, 100 and 1000 µg g(-1) sucrose concentrations. An experiment to determine the effect of carbon amendment on total microbial biomass and soil respiration was carried out with different concentrations of sucrose and non-labelled atrazine. The soils were incubated at a constant temperature and constant soil moisture at water potential of -15 kPa and a soil density of 1.3 g cm(-3). Mineralization of (14)C-ring-labelled atrazine was monitored continuously over a period of 59 d in the first experiment. The CO(2) production was monitored for 62 d in the second experiment and microbial biomass determined at the end of the incubation period. The addition of 1000 µg g(-1) sucrose reduced atrazine mineralization to 43.5% compared to 51.7% of the applied amount for the treatment without sucrose. The addition of 1000 µg g(-1) sucrose modified the transformation products to 1.08 µg g(-1) deisopropylatrazine (DIA), 0.32 µg g(-1) desethylatrazine (DEA) and 0.18 µg g(-1) deisopropyl-2-hydroxyatrazine (OH-DIA). Treatment without sucrose resulted in formation of 0.64 µg g(-1) hydroxyatrazine (HA), 0.28 µg g(-1) DIA and 0.20 µg g(-1) OH-DIA. Atrazine dealkylation was enhanced in treatments with 100 and 1000 µg g(-1) of sucrose added. HA metabolite was formed in the control (no sucrose) and in the presence of 10 µg g(-1) of sucrose, whereas DEA was only detected in treatment with 1000 µg g(-1) sucrose. Results indicate that total microbial biomass increased significantly (P < 0.001) with the addition of 1000 µg g(-1) sucrose.

Bioelectroventing: an electrochemical-assisted bioremediation strategy for cleaning-up atrazine-polluted soils.

The absence of suitable terminal electron acceptors (TEA) in soil might limit the oxidative metabolism of environmental microbial populations. Bioelectroventing is a bioelectrochemical strategy that aims to enhance the biodegradation of a pollutant in the environment by overcoming the electron acceptor limitation and maximizing metabolic oxidation. Microbial electroremediating cells (MERCs) are devices that can perform such a bioelectroventing. We also report an overall profile of the 14 C-ATR metabolites and 14 C mass balance in response to the different treatments. The objective of this work was to use MERC principles, under different configurations, to stimulate soil bacteria to achieve the complete biodegradation of the herbicide 14 C-atrazine (ATR) to 14 CO2 in soils. Our study concludes that using electrodes at a positive potential [+600 mV (versus Ag/AgCl)] ATR mineralization was enhanced by 20-fold when compared to natural attenuation in electrode-free controls. Furthermore, ecotoxicological analysis of the soil after the bioelectroventing treatment revealed an effective clean-up in < 20 days. The impact of electrodes on soil bioremediation suggests a promising future for this emerging environmental technology.

Large variation in glyphosate mineralization in 21 different agricultural soils explained by soil properties.

Glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) have frequently been detected in surface water and groundwaters. Since adequate glyphosate mineralization in soil may reduce its losses to environment, improved understanding of site specific factors underlying pesticide mineralization in soils is needed. The aim of this study was to investigate the relationship between soil properties and glyphosate mineralization. To establish a sound basis for resilient correlations, the study was conducted with a large number of 21 agricultural soils, differing in a variety of soil parameters, such as soil texture, soil organic matter content, pH, exchangeable ions etc. The mineralization experiments were carried out with 14C labelled glyphosate at a soil water tension of -15 kPa and at a soil density of 1.3 g cm-3 at 20 ±â€¯1 °C for an incubation period of 32 days. The results showed that the mineralization of glyphosate in different agricultural soils varied to a great extent, from 7 to 70% of the amount initially applied. Glyphosate mineralization started immediately after application, the highest mineralization rates were observed within the first 4 days in most of the 21 soils. Multiple regression analysis revealed exchangeable acidity (H+ and Al3+), exchangeable Ca2+ ions and ammonium lactate extractable K to be the key soil parameters governing glyphosate mineralization in the examined soils. A highly significant negative correlation between mineralized glyphosate and NaOH-extractable residues (NaOH-ER) in soils strongly suggests that NaOH-ER could be used as a simple and reliable parameter for evaluating the glyphosate mineralization capacity. The NaOH-ER were composed of glyphosate, unknown 14C-residues, and AMPA (12%-65%, 3%-34%, 0%-11% of applied 14C, respectively). Our results highlighted the influential role of soil exchangeable acidity, which should therefore be considered in pesticide risk assessments and management to limit efficiently the environmental transfers of glyphosate.

Isolation and characterization of 1,2,4-trichlorobenzene mineralizing Bordetella sp. and its bioremediation potential in soil.

A soil which has been polluted with chlorinated benzenes for more than 25 years was used for isolation of adapted microorganisms able to mineralize 1,2,4-trichlorobenzene (1,2,4-TCB). A microbial community was enriched from this soil and acclimated in liquid culture under aerobic conditions using 1,2,4-TCB as a sole available carbon source. From this community, two strains were isolated and identified by comparative sequence analysis of their 16S-rRNA coding genes as members of the genus Bordetella with Bordetella sp. QJ2-5 as the highest homological strain and with Bordetella petrii as the closest related described species. The 16S-rDNA of the two isolated strains showed a similarity of 100%. These strains were able to mineralize 1,2,4-TCB within two weeks to approximately 50% in liquid culture experiments. One of these strains was reinoculated to an agricultural soil with low native 1,2,4-TCB degradation capacity to investigate its bioremediation potential. The reinoculated strain kept its biodegradation capability: (14)C-labeled 1,2,4-TCB applied to this inoculated soil was mineralized to about 40% within one month of incubation. This indicates a possible application of the isolated Bordetella sp. for bioremediation of 1,2,4-TCB contaminated sites.

Enhanced degradation of isoproturon in an agricultural soil by a Sphingomonas sp. strain and a microbial consortium.

Isoproturon (IPU) degradation in an agricultural soil inoculated with an isolated IPU-degrader strain (Sphingomonas sp. strain AK1, IS) or a microbial consortium (MC) harboring this strain, with or without carrier material, were investigated in soil microcosm experiments during 46 days. Effect of the carrier material and inoculation size on IPU-degradation efficacy of the inoculants were studied. Mineralization, extractable residues and non-extractable residues of 14C-labeled IPU were analyzed. The low IPU mineralization in untreated soil (7.0%) was enhanced to different extents by inoculation of IS (17.4%-46.0%) or MC (58.9%-67.5%). Concentrations of IPU residues in soils amended with MC (0.002-0.095 µg g dry soil-1) were significantly lower than in soils amended with IS (0.02-0.67 µg g dry soil-1) and approximately 10 times lower than in the uninoculated soil (0.06-0.80 µg g dry soil-1). Less extractable residues and non-extractable residues were detected in soil with higher IPU mineralization. Inoculation size (as indicated by the volume of liquid cultures or by the number of carrier particles) determined the IPU-removal efficacy of IS in soil, but this effect was less pronounced for MC. The low sorption of IPU to soil and the decreasing IPU-mineralizing rates suggested incapability of IS to establish the IPU-mineralizing function in the soil. The thorough removal of IPU and persistent IPU-mineralizing activity of soil inoculated with MC indicated a high persistence of IPU-metabolic trait. Our results showed that microbial consortia might be more efficient than single degrader strains to enhance clean-up of organic chemicals in soil.

Influence of a heavy rainfall event on the leaching of [14C]isoproturon and its degradation products in outdoor lysimeters.

In four different agricultural soils the long-term leaching behaviour of [14C]isoproturon was studied in outdoor lysimeters (2 m length, 1 m2 surface area). The herbicide was applied in spring 1997 and spring 2001. At the end of the first 4-year-investigation period between 0.13% and 0.31% of the applied radioactivity was leached. Isoproturon or known metabolites could not be detected in the leachate. However, shortly after the second application isoproturon and its degradation products 2-hydroxy-isoproturon and monodemethyl-isoproturon were leached via preferential flow in one of the lysimeters (Mollic gleysol) in concentrations of 4.5 microg L-1, 3.1 microg L-1 and 0.9 microg L-1, respectively, thus considerably exceeding the EU threshold limit of 0.1 microg L-1 for ground and drinking water. The results indicate that in soils where mass flow transfer dominates, leaching of isoproturon to groundwater is of low probability whereas in highly structured soils which have the tendency to form macropores, isoproturon can be transported via preferential flow to the groundwater.

Predicting isoproturon long-term mineralization from short-term experiment: Can this be a suitable approach?

A worldwide used pesticide - isoproturon (IPU) - was selected to test whether short-term experiments can be used to predict long-term mineralization of IPU in soil. IPU-mineralization was measured for 39 and 265 days in four different agricultural soils with a low mineralization dynamic. Additionally, in one soil IPU dissipation, formation and dissipation of metabolites, formation of non-extractable residues (NER) and (14)C-microbial biomass from (14)C-IPU were monitored for 39 and 265 days. The data from short-term and long-term experiments were used for model fitting. The long-term dynamics of IPU mineralization were considerably overestimated by the short-term experiments in two soils with neutral pH, while in two other soils with low pH and lower mineralization, the long-term mineralization of IPU could be sufficiently predicted. Additional investigations in one of the soils with neutral pH showed that dissipation of IPU and metabolites could be correctly predicted by the short-term experiment. However, the formation of NER and (14)C-microbial biomass were remarkably overestimated by the short-term experiment. Further, it could be shown that the released NER and (14)C-microbial biomass were the main contributors of (14)CO2 formation at later incubation stages. Taken together, our results indicate that in soils with neutral pH short-term experiments were inadequate to predict the long-term mineralization of IPU.

Stimulating soil microorganisms for mineralizing the herbicide isoproturon by means of microbial electroremediating cells.

The absence of suitable terminal electron acceptors (TEA) in soil might limit the oxidative metabolism of environmental microbial populations. Microbial electroremediating cells (MERCs) consist in a variety of bioelectrochemical devices that aim to overcome electron acceptor limitation and maximize metabolic oxidation with the purpose of enhancing the biodegradation of a pollutant in the environment. The objective of this work was to use MERCs principles for stimulating soil bacteria to achieve the complete biodegradation of the herbicide (14) C-isoproturon (IPU) to (14) CO(2) in soils. Our study concludes that using electrodes at a positive potential [+600 mV (versus Ag/AgCl)] enhanced the mineralization by 20-fold respect the electrode-free control. We also report an overall profile of the (14) C-IPU metabolites and a (14) C mass balance in response to the different treatments. The remarkable impact of electrodes on the microbial activity of natural communities suggests a promising future for this emerging environmental technology that we propose to name bioelectroventing.

Stimulation of reductive dechlorination of hexachlorobenzene in soil by inducing the native microbial activity.

The reductive dechlorination and behaviour of (14)C-hexachlorobenzene (HCB) was investigated in an arable soil. The activity of the native anaerobic microbial communities could be induced by saturating the soil with water. Under these conditions high rates of dechlorination were observed. After 20 weeks of incubation only 1% of the applied 14C-HCB could be detected in the fraction of extractable residues. Additional organic substances, like wheat straw and lucerne straw, however considerably delayed and reduced the dechlorination process in the soil. The decline of HCB was not only caused by dechlorination but also by the formation of non-extractable residues, whereby their amounts varied with time depending on the experimental conditions. Several dechlorination products were detected, indicating the following main HCB transformation pathway: HCB --> PCB --> 1,2,3,5-TeCB --> 1,3,5-TCB --> 1,3-DCB, with 1,3,5-TCB as main intermediate dechlorination product. The other TeCB-, TCB- and DCB-isomers were also detected in low amounts, showing the presence of more than one dechlorination pathway. Since the methane production rates were lowest when the dechlorination rates were highest, it can be assumed that methanogenic bacteria were not involved in the dechlorination process of HCB. The established 14C-mass balances show, that with increasing dechlorination and incubation times, the 14C-recoveries decreased.

Microsomal detoxification enzymes in yam bean [Pachyrhizus erosus (L.) urban].

Cytochrome P450s and glutathione-S-transferases (GSTs) constitute two of the largest groups of enzyme families that are responsible for detoxification of exogenous molecules in plants. Their activities differ from plant to plant with respect to metabolism and substrate specificity which is one of the reasons for herbicide selectivity. In the tuber forming yam bean, the legume Pachyrhizus erosus, their activities at the microsomal level were investigated to determine the detoxification status of the plant. The breakdown of the herbicide isoproturon (IPU) to two distinct metabolites, 1-OH-IPU and monodesmethyl-IPU, was demonstrated. GST activity was determined with model substrates, but also by the catalysed formation of the fluorescent glutathione bimane conjugate. This study demonstrates for the first time microsomal detoxification activity in Pachyrhizus and the fluorescence image description of microsomal GST catalysed reaction in a legume.

Short-term bioavailability of carbon in soil organic matter fractions of different particle sizes and densities in grassland ecosystems.

The quality, stability and availability of organic carbon (OC) in soil organic matter (SOM) can vary widely between differently managed ecosystems. Several approaches have been developed for isolating SOM fractions to examine their ecological roles, but links between the bioavailability of the OC of size-density fractions and soil microbial communities have not been previously explored. Thus, in the presented laboratory study we investigated the potential bioavailability of OC and the structure of associated microbial communities in different particle-size and density fractions of SOM. For this we used samples from four grassland ecosystems with contrasting management intensity regimes and two soil types: a Haplic Cambisol and a typical Chernozem. A combined size-density fractionation protocol was applied to separate clay-associated SOM fractions (CF1, <1 µm; CF2, 1-2 µm) from light SOM fractions (LF1, <1.8 g cm(-3); LF2, 1.8-2.0 g cm(-3)). These fractions were used as carbon sources in a respiration experiment to determine their potential bioavailability. Measured CO2-release was used as an index of substrate accessibility and linked to the soil microbial community structure, as determined by phospholipid fatty acids (PLFA) analysis. Several key factors controlling decomposition processes, and thus the potential bioavailability of OC, were identified: management intensity and the plant community composition of the grasslands (both of which affect the chemical composition and turnover of OC) and specific properties of individual SOM fractions. The PLFA patterns highlighted differences in the composition of microbial communities associated with the examined grasslands, and SOM fractions, providing the first broad insights into their active microbial communities. From observed interactions between abiotic and biotic factors affecting the decomposition of SOM fractions we demonstrate that increasing management intensity could enhance the potential bioavailability of OC, not only in the active and intermediate SOM pools, but also in the passive pool.

Soil remediation with a microbial community established on a carrier: strong hints for microbial communication during 1,2,4-Trichlorobenzene degradation.

The objective of the present study was to get more insight into the mechanisms that govern the high mineralization potential of a microbial community attached on a carrier material, as we found in an earlier study (Wang et al., 2010). A 1,2,4-Trichlorobenzene (1,2,4-TCB) degrading microbial community - attached (MCCP) and non-attached (MCLM) on clay particles - was inoculated into a simplified mineral medium system. Signaling molecules (AHLs), cell growth and 1,2,4-TCB mineralization were measured at different sampling points. The production of AHLs in the MCCP system increased continuously with increasing key degrader (Bordetella sp.) cell growth and a positive correlation was observed between the production of AHLs and 1,2,4-TCB mineralization. In the MCLM system, however, 1,2,4-TCB mineralization was lower than in the MCCP system; the AHLs production per Bordetella cell was higher than in MCCP and there was no correlation between AHLs and mineralization. Moreover, in the MCCP system less different AHLs were produced than in the MCLM system. These results indicate that a microbial community attached on a carrier material has an advantage over a non-attached community: it produces signaling molecules with much less energy and effort to achieve a well-directed cell-to-cell communication resulting in a high and effective mineralization.

Effect of fluctuating soil humidity on in situ bioavailability and degradation of atrazine.

This study elucidates the effect of fluctuating soil moisture on the co-metabolic degradation of atrazine (6-chloro-N(2)-ethyl-N(4)-isopropyl-1,3,5-triazine-2,4-diamine) in soil. Degradation experiments with (14)C-ring-labelled atrazine were carried out at (i) constant (CH) and (ii) fluctuating soil humidity (FH). Temperature was kept constant in all experiments. Experiments under constant soil moisture conditions were conducted at a water potential of -15 kPa and the sets which were run under fluctuating soil moisture conditions were subjected to eight drying-rewetting cycles where they were dried to a water potential of around -200 kPa and rewetted to -15 kPa. Mineralization was monitored continuously over a period of 56d. Every two weeks the pesticide residues in soil pore water (PW), the methanol-extractable pesticide residues, the non-extractable residues (NER), and the total cell counts were determined. In the soil with FH conditions, mineralization of atrazine as well as the formation of the intermediate product deisopropyl-2-hydroxyatrazine was increased compared to the soil with constant humidity. In general, we found a significant correlation between the formation of this metabolite and atrazine mineralization. The cell counts were not different in the two experimental variants. These results indicate that the microbial activity was not a limiting factor but the mineralization of atrazine was essentially controlled by the bioavailability of the parent compound and the degradation product deisopropyl-2-hydroxyatrazine.

Single application of sewage sludge--impact on the quality of an alluvial agricultural soil.

The effects of sewage sludge on soil quality with regard to its nutrient and heavy metal content, microbial community structure and ability to maintain specific soil function (degradation of herbicide glyphosate) were investigated in a three months study using an alluvial soil (Eutric Fluvisol). Dehydrated sewage sludge significantly increased soil organic matter (up to 20.6% of initial content), total and available forms of N (up to 33% and 220% of initial amount, respectively), as well as total and plant available forms of P (up to 11% and 170% of initial amount, respectively) and K (up to 70% and 47% of initial amount, respectively) in the upper 2 cm soil layer. The increase of organic matter was most prominent 3d after the application of sewage sludge, after 3 months it was no longer significant. Contents of nutrients kept to be significantly higher in the sewage sludge treated soil till the end of experiment. Contents of some heavy metals (Zn, Cu, Pb) increased as well. The highest increase was found for Zn (up to 53% of initial amount), however it was strongly bound to soil particles and its total content was kept below the maximum permissible limit for agricultural soil. Based on molecular fingerprinting of bacterial 16S rRNA gene and fungal ITS fragment on 3rd day and 3rd month after sewage sludge amendment, significant short term effects on bacterial and fungal communities were shown due to the sewage sludge. The effects were more pronounced and more long-term for bacterial than fungal communities. The mineralization of (14)C-glyphosate in the sewage sludge soil was 55.6% higher than in the control which can be linked to (i) a higher glyphosate bioavailability in sewage sludge soil, which was triggered by the pre-sorption of phosphate originating from the sewage sludge and/or (ii) beneficial alterations of the sewage sludge to the physical-chemical characteristics of the soil.

Atrazine and terbuthylazine mineralization by an Arthrobacter sp. isolated from a sugarcane-cultivated soil in Kenya.

A tropical soil from a Kenyan sugarcane-cultivated field showed a very high capability to mineralize (14)C-ring-labeled atrazine. In laboratory experiments this soil mineralized about 90% of the applied atrazine within 98 d. The atrazine-degrading microbial community was enriched in liquid cultures containing atrazine as the sole N source and 100 mgL(-1) glucose as additional C source. From the enrichment culture a bacterial strain was isolated and identified by comparative sequence analysis of the 16S-rDNA as member of the genus Arthrobacter. The enriched mixed culture as well as the isolated strain, designated as Arthrobacter sp. strain GZK-1, could grow on atrazine and terbuthylazine as sole N-sources; Arthrobacter sp. GZK-1 mineralized (14)C-ring-labeled atrazine up to 88% to (14)CO(2) and (14)C-ring-labeled terbuthylazine up to 65% to (14)CO(2) in a liquid culture within 14 d. The enriched microbial consortium as well as the isolated strain could be a potential solution for the remediation of s-triazine polluted agricultural soils.

Quantifying the effect of soil moisture on the aerobic microbial mineralization of selected pesticides in different soils.

A standardized quantitative approach was developed to reliably elucidate the effect of increasing soil moisture on pesticide mineralization. The mineralization of three aerobically degradable and chemically different 14C-labeled pesticides (isoproturon, benazolin-ethyl, and glyphosate) was studied under controlled conditions in the laboratory at an identical soil density of 1.3 g cm(-3). The agricultural soils used are characterized by (i) large variations in soil texture (sand content 4-88%) and organic matter content (0.97-2.70% org. C), (ii) fairly diverse soil-water retention curves, and (iii) differing pH values. We quantified the effect of soil moisture on mineralization of pesticides and found that (i) at soil water potential < or = -20 MPa minimal pesticide mineralization occurred; (ii) a linear correlation (P < 0.0001) exists between increasing soil moisture (within a soil water potential range of -20 and -0.015 MPa), and increased relative pesticide mineralization; (iii) optimum pesticide mineralization was obtained at a soil water potential of -0.015 MPa, and (iv) when soil moisture approximated water holding capacity, pesticide mineralization was considerably reduced. As both selected pesticides and soils varied to a large degree, we propose that the correlation observed in this study may be also valid in the case of aerobic degradation of other native and artificial organic compounds in soils.