Dimitrios G. Karpouzas

Microbial Degradation of Organophosphorus Xenobiotics: Metabolic Pathways and Molecular Basis

Organophosphorus (OP) xenobiotics are used worldwide as pesticides and petroleum additives. OP compounds share the major portion of the pesticide market globally. Owing to large-scale use of OP compounds, contaminations of soil and water systems have been reported from all parts of the world. OP compounds possess very high mammalian toxicity and therefore early detection and subsequent decontamination and detoxification of the polluted environment is essential. Additionally, about 200,000 tons of extremely toxic OP chemical warfare agents are required to be destroyed by 2007 under Chemical Warfare Convention (1993). Chemical and physical methods of decontamination are not only expensive and time-consuming, but also in most cases they do not provide a complete solution. These approaches convert compounds from toxic into less toxic states, which in some cases can accumulate in the environment and still be toxic to a range of organisms. Bioremediation provides a suitable way to remove contaminants from the environment as, in most of the cases, OP compounds are totally mineralized by the microorganisms. Most OP compounds are degraded by microorganisms in the environment as a source of phosphorus or carbon or both. Several soil bacteria have been isolated and characterized, which can degrade OP compounds in laboratory cultures and in the field. The biochemical and genetic basis of microbial degradation has received considerable attention. Several genes/enzymes, which provide microorganisms with the ability to degrade OP compounds, have been identified and characterized. Some of these genes and enzymes have been engineered for better efficacy. Bacteria capable of complete mineralization are constructed by transferring the complete degradation pathway for specific compounds to one bacterium. In the present article, we review microbial degradation and metabolic pathways for some OP compounds. The biochemical and molecular basis of OP degradation by microbes and the evolution and distribution of genes/enzymes are also reviewed. This article also examines applications and future use of OP-degrading microbes and enzymes for bioremediation, treatment of OP poisoning, and as biosensors.

Degradation and Adsorption of Pesticides in Compost-Based Biomixtures as Potential Substrates for Biobeds in Southern Europe

Biobeds have been used in northern Europe for minimizing point source contamination of water resources by pesticides. However, little is known regarding their use in southern Europe where edaphoclimatic conditions and agriculture practices significantly differ. A first step toward their adaptation in southern Europe is the use of low-cost and easily available substrates as biomixture components. This study investigated the possibility of replacing peat with agricultural composts in the biomixture. Five composts from local substrates including olive leaves, cotton crop residues, cotton seeds, spent mushroom substrate, and commercial sea wrack were mixed with topsoil and straw (1:1:2). Degradation of a mixture of pesticides (dimethoate, indoxacarb, buprofezin, terbuthylazine, metribuzin, metalaxyl-M, iprodione, azoxystrobin) at two dose rates was tested in the compost biomixtures (BX), in corresponding peat biomixtures (OBX), and in soil. Adsorption-desorption of selected pesticides were also studied. Pesticide residues were determined by gas chromatography with nitrogen-phosphorus detector, except indoxacarb, which was determined with a microelectron capture detector. Overall, BX degraded the studied pesticides at rates markedly higher than those observed in soil and OBX, in which the slowest degradation rates were evident. Overall, the olive leaf compost biomixture showed the highest degradation capacity. Adsorption studies showed that OBX and BX had higher adsorption affinity compared to soil. Desorption experiments revealed that pesticide adsorption in biomixtures was not entirely reversible. The results suggest that substitution of peat with local composts will lead to optimization of the biobed system for use in Mediterranean countries.

Factors influencing the ability of Pseudomonas putida epI to degrade ethoprophos in soil.

An ethoprophos-degrading Pseudomonas putida strain (epI) was used in laboratory-based experiments to inoculate soils containing residues of the nematicide. Inoculum densities as low as 104 cells g−1 were sufficient to degrade a fresh addition of ethoprophos within 16 days. P. putida epI was able to rapidly degrade aged as well as freshly applied ethoprophos residues. However, slower degradation of aged residues was observed in the later stages of incubation suggesting that a fraction of the pesticide residues had become inaccessible to the introduced bacteria. The effects of environmental and soil factors on the ability of P. putida epI to degrade ethoprophos were also studied. The bacterium was able to rapidly degrade ethoprophos at 20 and 35°C and at soil water potentials of −33 and −10 kPa. However, its degrading ability was significantly reduced but not completely inhibited at 5°C or at a soil water potential of −1500 kPa. The P. putida strain was active in soils with pH 6.8 and 8.3, but there was a complete loss of degrading ability in a soil with pH 5.4. Efficient degradation of ethoprophos by P. putida epI was observed in soils with organic matter contents varying from 0.3 to 8.5%. However, degradation was somewhat slower in the soil with the higher organic matter content, presumably due to increased pesticide adsorption and decreased bioavailability. The results indicate that the ethoprophos-degrading P. putida epI was efficient as a bioremediation agent in a range of environmental and soil conditions.

Factors influencing the ability of Pseudomonas putida strains epI and II to degrade the organophosphate ethoprophos.

Two strains of Pseudomonas putida (epI and epII), isolated previously from ethoprophos‐treated soil, were able to degrade ethoprophos (10 mg l−1) in a mineral salts medium plus nitrogen (MSMN) in less than 50 h with a concurrent population growth. Addition of glucose or succinate to MSMN did not influence the degrading ability of Ps. putida epI, but increased the lag phase before rapid degradation commenced with Ps. putida epII. The degrading ability of the two isolates was lost when the pesticide provided the sole source of phosphorus. Degradation of ethoprophos was most rapid when bacterial cultures were incubated at 25 and 37 °C. Pseudomonas putida epI was capable of completely degrading ethoprophos at a slow rate at 5 °C, compared with Ps. putida epII which could not completely degrade ethoprophos at the same time. Pseudomonas putida epI was capable of degrading ethoprophos when only 60 cells ml−1 were used as initial inoculum. In contrast, Ps. putida epII was able to totally degrade ethoprophos when inoculum densities of 600 cells ml−1 or higher were used. In general, longer lag phases accompanied the lower inoculum levels. Both isolates rapidly degraded ethoprophos in MSMN at pHs ranging from 5·5 to 7·6, but not at pH 5 or below.

The Impact of Biofumigation and Chemical Fumigation Methods on the Structure and Function of the Soil Microbial Community

Biofumigation (BIOF) is carried out mainly by the incorporation of brassica plant parts into the soil, and this fumigation activity has been linked to their high glucosinolate (GSL) content. GSLs are hydrolyzed by the endogenous enzyme myrosinase to release isothiocyanates (ITCs). A microcosm study was conducted to investigate the effects induced on the soil microbial community by the incorporation of broccoli residues into soil either with (BM) or without (B) added myrosinase and of chemical fumigation, either as soil application of 2-phenylethyl ITC (PITC) or metham sodium (MS). Soil microbial activity was evaluated by measuring fluorescein diacetate hydrolysis and soil respiration. Effects on the structure of the total microbial community were assessed by phospholipid fatty acid analysis, while the impact on important fungal (ascomycetes (ASC)) and bacterial (ammonia-oxidizing bacteria (AOB)) guilds was evaluated by denaturating gradient gel electrophoresis (DGGE). Overall, B, and to a lesser extent BM, stimulated microbial activity and biomass. The diminished effect of BM compared to B was particularly evident in fungi and Gram-negative bacteria and was attributed to rapid ITC release following the myrosinase treatment. PITC did not have a significant effect, whereas an inhibitory effect was observed in the MS-treated soil. DGGE analysis showed that the ASC community was temporarily altered by BIOF treatments and more persistently by the MS treatment, while the structure of the AOB community was not affected by the treatments. Cloning of the ASC community showed that MS application had a deleterious effect on potential plant pathogens like Fusarium, Nectria, and Cladosporium compared to BIOF treatments which did not appear to inhibit them. Our findings indicate that BIOF induces changes on the structure and function of the soil microbial community that are mostly related to microbial substrate availability changes derived from the soil amendment with fresh organic materials.

On-farm biopurification systems for the depuration of pesticide wastewaters: recent biotechnological advances and future perspectives.

Point source contamination of natural water resources by pesticides constitutes a serious problem and on-farm biopurification systems (BPS) were introduced to resolve it. This paper reviews the processes and parameters controlling BPS depuration efficiency and reports on recent biotechnological advances which have been used for enhancing BPS performance. Biomixture composition and water management are the two factors which either individually or through their interactions control the depuration performance of BPS. Which process (biodegradation or adsorption) will dominate pesticides dissipation in BPS depends on biomixture composition and the physicochemical properties of the pesticides. Biotechnological interventions such as augmentation with pesticide-degrading microbes or pesticide-primed matrices have resulted in enhanced biodegradation performance of BPS. Despite all these advancement in BPS research, there are still several issues which should be resolved to facilitate their full implementation. Safe handling and disposal of the spent biomixture is a key practical issue which needs further research. The use of BPS for the depuration of wastewaters from post-farm activities such as postharvest treatment of fruits should be a priority research issue considering the lack of alternative treatment systems. However, the key point hampering optimization of BPS is the lack of fundamental knowledge on BPS microbiology. The use of advanced molecular and biochemical methods in BPS would shed light into this issue in the future.

Novel biomixtures based on local Mediterranean lignocellulosic materials: evaluation for use in biobed systems.

The composition of biomixtures strongly affect the efficacy of biobeds. Typically, biomixture consists of peat (or compost), straw (STR) and topsoil (1:2:1 by volume). Straw guarantees a continuous supply of nutrients and high microbial activity. However, in south Europe other lignocellulosic materials including sunflower crop residues (SFR), olive leaves, grape stalks (GS), orange peels, corn cobs (CC) and spent mushroom substrate (SMS) are also readily available at no cost. Their potential utilization in biomixtures instead of STR was tested in pesticide degradation and adsorption studies. The microbial activity in these biomixtures was also assessed. The GS-biomixture was the most efficient in pesticide degradation, while CC- and SFR-biomixtures showed comparable degrading efficacy with the STR-biomixture. The SMS-biomixture was also highly efficient in degrading the pesticide mixture with degradation rates being correlated with the proportion of SMS in the biomixture. Microbial respiration was positively correlated with the degradation rates of metalaxyl, azoxystrobin and chlorpyrifos, compared to phenoloxidase which showed no correlation. Biomixtures containing alternative lignocellulosic materials showed a higher adsorption affinity for terbuthylazine and metribuzin compared to the STR-biomixture. We provide first evidence that STR can be substituted in biomixtures by other lignocellulosic materials which are readily available in south Europe.

Impact of Nitrogen and Sulfur Fertilization on the Composition of Glucosinolates in Relation to Sulfur Assimilation in Different Plant Organs of Broccoli

Broccoli (Brassica oleracea var. italica) is one of the most important winter season vegetables and a rich source of chemoprotective molecules, including glucosinolates (GSL). The aim of this study was to investigate the impact of nitrogen (N) and sulfur (S) fertilization on GSL concentration and composition in different parts of broccoli plants. A greenhouse experiment was performed, with four different treatments of sulfur (10, 30, 70, and 150 kg/ha) and three treatments of nitrogen (50, 250, and 600 kg/ha). GSL concentrations and plant growth responded to the N supply, but this was not observed above the 250 kg N/ha dose. On the contrary, plant growth did not respond to the S supply, whereas GSL concentrations showed a sharp response to the whole range of S applications (from 10 to 150 kg/ha). Glucosinolate composition was altered differentially in the examined plant parts. Aliphatic GSL were more abundant in the florets and leaves, whereas indolyl GSLs were dominant in roots, in which aromatic GSL were also observed. High nitrogen fertilization had a higher impact on indolyl compared to aliphatic GSLs concentration. More importantly, a high concentration of aliphatic GSL, >2.4 micromol/g dry weight (dw), and high S assimilation into aliphatic GSL were consistently observed in the florets compared to other broccoli parts, indicating adaptable processes for nitrogen and sulfur regarding synthesis and transport of aliphatic GSL for these organs.

Degradation and adsorption of terbuthylazine and chlorpyrifos in biobed biomixtures from composted cotton crop residues

BACKGROUND Biobeds have been well studied in northern Europe, whereas little is known regarding their use in southern Europe. The degradation and adsorption of terbuthylazine (TA) and chlorpyrifos (CP) were studied in three different biomixtures composed of composted cotton crop residues, soil and straw in various proportions, and also in sterilised and non-sterilised soil. RESULTS Compost biomixtures degraded the less hydrophobic TA at a faster rate than soil, while the opposite was evident for the more hydrophobic CP. These results were attributed to the rapid abiotic hydrolysis of CP in the alkaline soil (pH 8.5) compared with the lower pH of the compost (6.6), but also to the increasing adsorption (K(d) = 746 mL g(-1)) and reduced bioavailability of CP in the biomixtures compared with soil (K(d) = 17 mL g(-1)), as verified by the adsorption studies. CONCLUSIONS Compost had a dual but contrasting effect on degradation that depended on the chemical nature of the pesticide studied: a positive effect towards TA owing to increasing biodegradation and a negative effect towards CP owing to increasing adsorption. Overall, composted cotton crop residues could be potentially used in local biobed systems in Greece, as they promoted the degradation of hydrophilic pesticides and the adsorption of hydrophobic pesticides.

Emerging technologies in bioremediation: constraints and opportunities

Intensive industrialisation, inadequate disposal, large-scale manufacturing activities and leaks of organic compounds have resulted in long-term persistent sources of contamination of soil and groundwater. This is a major environmental, policy and health issue because of adverse effects of contaminants on humans and ecosystems. Current technologies for remediation of contaminated sites include chemical and physical remediation, incineration and bioremediation. With recent advancements, bioremediation offers an environmentally friendly, economically viable and socially acceptable option to remove contaminants from the environment. Three main approaches of bioremediation include use of microbes, plants and enzymatic remediation. All three approaches have been used with some success but are limited by various confounding factors. In this paper, we provide a brief overview on the approaches, their limitations and highlights emerging technologies that have potential to revolutionise the enzymatic and plant-based bioremediation approaches.