Taxonomy, purification and chemical characterization of four bioactive compounds from new Streptomyces sp. TN256 strain.

A new actinomycete strain designated TN256, producing antimicrobial activity against pathogenic bacteria and fungi, was isolated from a Tunisian Saharan soil. Morphological and chemical studies indicated that strain TN256 belonged to the genus Streptomyces. Analysis of the 16S rDNA sequence of strain TN256 showed a similarity level ranging between 99.79 and 97.8% within Streptomyces microflavus DSM 40331(T) and Streptomyces griseorubiginosus DSM 40469(T) respectively. The comparison of its physiological characteristics showed significant differences with the nearest species. Combined analysis of the 16 S rRNA gene sequences (FN687758), fatty acids profile, and results of physiological and biochemical tests indicated that there were genotypic and phenotypic differentiations of that isolate from other Streptomyces species neighbours. These date strongly suggest that strain TN256 represents a novel species with the type strain Streptomyces TN256 (=CTM50228(T)). Experimental validation by DNA-DNA hybridization would be required for conclusive confirmation. Four active products (1-4) were isolated from the culture broth of Streptomyces TN256 using various separation and purification steps and procedures. 1: N-[2-(1H-indol-3-yl)-2 oxo-ethyl] acetamide 'alkaloid' derivative; 2: di-(2-ethylhexyl) phthalate, a phthalate derivative; 3: 1-Nonadecene and 4: Cyclo (L: -Pro-L: -Tyr) a diketopiperazine 'DKP' derivative. The chemical structure of these four active compounds was established on the basis of spectroscopic studies NMR and by comparing with data from the literature. According to our biological studies, we showed in this work that the pure compounds (1-4) possess antibacterial and antifungal activities.

Assessment of the genotoxicity of quinolone and fluoroquinolones contaminated soil with the Vicia faba micronucleus test.

The genotoxicity of quinolone and fluroquinolones was assessed using the micronucleus (MN) test on Vicia faba roots by direct contact exposure to a solid matrix. Plants were exposed to quinolones (nalidixic acid) and fluoroquinolones (ciprofloxacin and enrofloxacin) alone or mixed with artificially contaminated soils. Four different concentrations of each of these antibiotics were tested (0.01, 0.1, 1 and 10 mg/Kg) for nalidixic acid and (0.005, 0.05, 0.5 and 5 mg/Kg) for ciprofloxacin and enrofloxacin. These antibiotics were also used in mixture. Exposure of Vicia faba plants to each antibiotic at the highest two concentrations showed significant MN induction. The lowest two concentrations had no significant genotoxic effect. The mixture of the three compounds induced a significant MN induction whatever the mixture tested, from 0.02 to 20 mg/Kg. The results indicated that a similar genotoxic effect was obtained with the mixture at 0.2 mg/Kg in comparison with each molecule alone at 5-10 mg/Kg. Data revealed a clear synergism of these molecules on Vicia faba genotoxicity.

Giemsa versus acridine orange staining in the fish micronucleus assay and validation for use in water quality monitoring.

This study concerns a comparative analysis of the acridine orange and Giemsa staining procedures for the fish erythrocyte micronucleus assay. The goal was to optimize the assay in the context of field water monitoring. Fish (Carassius carassius) were exposed to a reference genotoxic agent, cyclophosphamide monohydrate 5 mg l(-1) for 2, 4, and 6 days before testing. Slides from each individual were scored using the two procedures. The results show that the assay was more sensitive when acridine orange was used. When slides were Giemsa stained, the presence of ambiguous artefacts, leading to false positives and increasing random variance, reduced the contrast between exposed and control samples. Acridine Orange staining was then applied in the context of water quality monitoring. Fish were exposed for 4 days to water sampled in two hydrological contexts: basal flow and spring flood. The results show that exposure to spring flood water in an agricultural stream can induce mutagenicity.

Mutagenic impact on fish of runoff events in agricultural areas in south-west France.

When heavy rainfall follows herbicide application, the intense surface runoff causes stream water contamination. Aquatic organisms are then briefly exposed to a complex mixture of contaminants. The aim of the present study is to investigate the genotoxic impact of such events on fish. A model fish, the Crucian carp (Carassius carassius) was exposed in controlled conditions, for 4 days, to water sampled daily in the Save River (France). The watershed of this stream is representative of agricultural areas in south-west France. Three hydrological conditions were compared: basal flow, winter flood, and spring flood. Chemical analysis of the water samples confirmed the higher contamination of the spring flood water, mainly explained by a peak of metolachlor. Genotoxicity was evaluated by micronucleus (MN) test and comet assay in peripheral erythrocytes. A significant increase in DNA breakdowns compared to controls was detected by the comet assay for all conditions. Exposure to spring flood water resulted in the highest damage induction. Moreover, induced chromosomal damage was only detected in this condition. In addition, fish were exposed, for 4 days, to an experimental mixture of 5 herbicides representative of the spring flood water contamination. Fish exhibited moderate DNA damage induction and no significant chromosomal damage. The mutagenicity induced by field-collected water is then suspected to be the result of numerous interactions between contaminants themselves and environmental factors, stressing the use of realistic exposure conditions. The results revealed a mutagenic impact of water contamination during the spring flood, emphasizing the need to consider these transient events in water quality monitoring programs.

Assessment of average exposure to organochlorine pesticides in southern Togo from water, maize (Zea mays) and cowpea (Vigna unguiculata).

Drinking water, cowpea and maize grains were sampled in some potentially exposed agro-ecological areas in Togo and analysed for their contamination by some common organochlorine pesticides. A total of 19 organochlorine pesticides were investigated in ten subsamples of maize, ten subsamples of cowpea and nine subsamples of drinking water. Analytical methods included solvent extraction of the pesticide residues and their subsequent quantification using gas chromatography-mass spectrometry (GC/MS). Estimated daily intakes (EDIs) of pesticides were also determined. Pesticides residues in drinking water (0.04-0.40 microg l(-1)) were higher than the maximum residue limit (MRL) (0.03 microg l(-1)) set by the World Health Organization (WHO). Dieldrin, endrin, heptachlor epoxide and endosulfan levels (13.16-98.79 microg kg(-1)) in cowpea grains exceeded MRLs applied in France (10-50 microg kg(-1)). Contaminants' levels in maize grains (0.53-65.70 microg kg(-1)) were below the MRLs (20-100 microg kg(-1)) set by the Food and Agriculture Organization (FAO) and the WHO. EDIs of the tested pesticides ranged from 0.02% to 162.07% of the acceptable daily intakes (ADIs). Population exposure levels of dieldrin and heptachlor epoxide were higher than the FAO/WHO standards. A comprehensive national monitoring programme on organochlorine pesticides should be undertaken to include such other relevant sources like meat, fish, eggs and milk.

Evaluation of a TiO2 photocatalysis treatment on nitrophenols and nitramines contaminated plant wastewaters by solid-phase extraction coupled with ESI HPLC-MS.

Nitration reactions of aromatic compounds are commonly involved in different industrial processes for pharmaceutical, pesticide or military uses. For many years, most of the manufacturing sites used lagooning systems to treat their process effluents. In view of a photocatalytic degradation assay, the wastewater of a lagoon was investigated by using HPLC coupled with mass spectrometry. The wastewater was highly concentrated in RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and two herbicides Dinoterb (2-tert-butyl-4,6-dinitrophenol) and Dinoseb (2-sec-butyl-4,6-dinitrophenol). First of all, an analytical method using solid-phase extraction (SPE) combined with HPLC ESI MS/MS was put in work for identification and titration of RDX, HMX and the two dinitrophenols in a complex natural matrix. Then, the UV/TiO2 treatment was investigated for pollutants removal. Dinitrophenolic compounds were significantly degraded after a 8-h-exposition of the wastewater/TiO2 suspension, whereas RDX and HMX were poorly affected.

Qualitative and quantitative evolution of polyphenolic compounds during composting of an olive-mill waste-wheat straw mixture.

Olive-mill wastes represent a significant environmental problem in Mediterranean areas due to their important production during a short period of time. Their high polyphenol, lipid and organic acid concentrations turn them into phytotoxic wastes. This work examined the evolution of polyphenolic compounds during the composting of an olive-mill waste-wheat straw mixture by using quantitative (Folin-Ciocalteu) and qualitative (High Pressure Liquid Chromatography, HPLC) analyses. The polyphenol content decreased strongly by 93% while the significant correlations obtained between polyphenol content and different humification indices and lignin losses suggested that polyphenols contribute to the synthesis of humic substances. In addition, the significant correlation found between germination index, polyphenols and humification indices must be related to the progressive substrate detoxification as humification process progressed, leading to a non-phytotoxic and partially humified end-product. Nine polyphenolic compounds were identified by HPLC, most of which had disappeared by the end of composting, when only traces of hydroxytyrosol, caffeic and syringic acids and oleuropein were detected.

Fate of polycyclic aromatic hydrocarbons during composting of activated sewage sludge with green waste.

The level and fate of 16 polycyclic aromatic hydrocarbons (PAHs), targeted by the US Environmental Protection Agency (USEPA), has been studied over 90 days of composting of activated sludge with green waste, under a semi-arid climate. The total PAH calculated from the sum of the amounts of the 16 PAHs in the initial mixture of activated sludge and green waste, was lower than accepted European Union cut-off limits by about 0.48mgkg(-1). The treatment by composting led to a decrease of all PAHs mainly in the stabilization phase, but some differences could be observed between PAHs with three or fewer aromatic rings (N< or = 3) and those with four or more (N> or = 4). The former (except phenanthrene) exhibited a continuous decrease, while the latter PAHs with N of four or more and phenanthrene showed increases in the intermediate stages (30-60 days). This indicates the high potential sorption mainly of PAH with high molecular weight (> or = N4) plus phenanthrene, their tight adsorption makes them inaccessible for microbial attack. The high molecular weight PAHs showed a greater reduction of their bioavailability than those of low molecular weight. Naphthalene, with the lowest molecular weight, showed the smallest decrease (about 67.8%) compared to other PAHs of higher molecular weight (decrease reaching 100%). This is in agreement with the fact that the adsorption is less reversible with increased numbers of fused aromatic rings or an increase of their hydrophobicity.

Microbial community dynamics during composting of sewage sludge and straw studied through phospholipid and neutral lipid analysis.

The composting process involves a succession of different communities of microorganisms that decompose the initial material, transforming it into a stable final product. In this work, the levels of phospholipid fatty acid (PLFA), neutral lipid fatty acid (NLFA) and sterol were monitored in compost versus time, as indicators of the activity of various microorganisms (Gram-positive or Gram-negative bacteria, fungi, etc.). During composting, the PLFA and NLFA from Gram-negative bacteria and eukaryotes (2-OH 10; 3-OH 12; 2-OH 14; 13:0; 16:1; 18:1 trans) as well as some sterols of plant origin (e.g. monostearin sterols) decreased until the end of composting. In contrast, the branched fatty acids with iso- and anteiso-forms (i-15:0; a-15:0; i-16; i-17) increased mainly in the thermophilic phase, but decreased right after. The PLFA 18:2 (6; 9), which is used as an index of the occurrence of some fungi, rose strongly at the beginning of composting, but fell after peak heating. In contrast, the other main sterol indicative of fungi, ergosterol, decreased at the beginning of the thermophilic phase, but increased strongly by the end of composting. Accordingly, cluster and PCA analysis separated the PLFA of Gram-negative bacteria and eukaryotic cells from those of Gram-positive bacteria and long-chain fatty acids. The fungal PLFA considered, 18:2 (9, 12), was clustered more closely to iso- and anteiso-branched PLFAs. Stigmasterol, squalene and cholesterol occurred in the lower right part of the loading plot and were clustered more closely to iso-, anteiso-branched PLFAs and 18:2 w 6,9 suggesting their relationship to microbial activities. We also observed the tendency of resistance of fatty acid PLFAs and NLFAs of long chain (19:0 (cis-9); 20:0) and some recalcitrant sterols, e.g. sitosterol, at the end of composting. The presence of high levels of the latter in the final stage indicates their contribution to the structural stability of organic matter fractions. These recalcitrant components were more clustered and occurred in the lower right part of the loading plot.

13C NMR study of the effect of aerobic treatment of olive mill wastewater (OMW) on its lipid-free content.

Olive mill wastewater was treated by an aerobic bio-process at different values of pH (with or without addition of lime), for 45 days on a laboratory scale, to evaluate the reduction of the organic load. The lipid content showed an appreciable change in relation to the applied treatment both for total lipids and for the different fractions (neutral lipids, monoglycerides and phospholipids). 13C NMR spectroscopy was performed on initial and final samples both raw and after lipid extraction. The main spectral differences were observed in the C-alkyl region (0-50 ppm), in the C O-alkyl/N-alkyl region (50-110 ppm), and in the C-carboxylic (160-200 ppm) region, providing information on the alterations occurring in the different biochemical entities composing this complex biomatrix (e.g. lipids and carbohydrates) according to the treatment.

Phospholipid fatty acid analysis to monitor the co-composting process of olive oil mill wastes and organic household refuse.

The co-composting of olive oil mill wastes and household refuse was followed for 5 months. During the thermophilic phase of composting, the aerobic heterotrophic bacteria (AHB) count, showed a significant rise with a slight regression of fungal biomass. In the same way, phospholipid fatty acids PLFAs common in bacteria, showed a significant increase of hydroxyl and branched PLFAs. The evaluation of the ratio of octadecenoic PLFAs to stearic acid (C18:1omega/C18:0) revealed a significant reduction while a significant rise in the length of aliphatic chains evaluated by the stearic acid to palmitic acid ratio (C18:0/C16:0) was noted during the stabilization phase. The follow-up of PLFAs, indicates the degree of biodegradation that occurs during composting, it can be regarded an indicator of the stability and maturity of the end product.

Chemical and spectroscopic analysis of olive mill waste water during a biological treatment.

The treatment of olive mill waste water was studied on the laboratory scale. Physico-chemical analyses showed the final products had a mean pH of 5.4 without neutralisation and 5.7 when lime was added to the process. Raising the pH by adding lime had a positive outcome on the degradation of phenols, whose levels were reduced by over 76%. The lime also changed the structure of the organic matter, as seen in the infra-red spectra. Combining the FT-IR and 13C NMR data showed that with addition of lime, the density of aliphatic groups decreased to the benefit of aromatic groups, indicating that polymerisation of the organic matter occurred during the bioprocess. Under our experimental conditions, the biotransformation of olive mill waste water appears to favour the stabilisation of the organic matter through mechanisms analogous to those that lead to the formation of humus in the soil.

Assessment of the genotoxicity of olive mill waste water (OMWW) with the Vicia faba micronucleus test.

The present study concerns the genotoxicity of olive mill waste water (OMWW) generated in mills producing olive oil in Morocco. The Vicia faba micronucleus test was used to evaluate the genotoxicity of OMWW and the six major phenolic compounds identified by HPLC in this effluent. Five dilutions of OMWW were tested: 0.1, 1, 5, 10 and 20%. Maleic hydrazide was used as a positive control. The results showed that OMWW was genotoxic at 10% dilution. In order to investigate the components involved in this genotoxicity, the six major phenols present in this effluent, oleuropein, gallic acid, 4-hydroxyphenyl acetic acid, caffeic acid, paracoumaric acid and veratric acid, were studied at concentrations corresponding to the genotoxic concentration of the OMWW itself. Two phenols, gallic acid and oleuropein induced a significant increase in micronucleus frequency in Vicia faba; the four other phenols had no significant genotoxic effect. These results suggest that under the experimental conditions of our assay, OMWW genotoxicity was associated with gallic acid and oleuropein.

Agronomic and environmental impacts of a single application of heat-dried sludge on an Alfisol.

A field experiment was conducted on Alfisols in South-West France to assess the agronomic and environmental impacts of a single application of heat-dried sludge pellets at 11.1 Mg dry matter ha-1. The sludge pellets, with a moisture level of 9.5%, were spread on an irrigated crop of maize (Zea mays L.). This treatment was compared with inorganic fertilization (urea and diammonium phosphate mixed with KCl). Soil properties, yield and the composition of maize and the quality of drained water were monitored over 1 year to detect any changes resulting from sludge application. Amongst several determined soil properties, only two were significantly modified by the sludge application: The nitric nitrogen stock of the soil was higher in the inorganic fertilized plot, whereas Olsen-P soil content was higher in the sludge-amended plot. Agronomic recovery rates of N and P added by sludge were high: For the first crop following application, total amounts of N and P supplied by the sludge had the same efficiency as approximately 45% of the N and P amounts supplied by inorganic fertilizer. This ratio was 7% for the N uptake by the second maize crop. The quality and quantity of maize were equally good with both types of fertilization. During the 2 years following sludge spreading, N leaching remained as low in the sludged plot as in the inorganically fertilized one. The Cu, Zn, Cr, Cd, Pb and Ni composition of the drainage water was affected by neither of the types of amendment. From the heavy-metal contents of the soil, water and maize monitored over 1 year in the field experiment and from literature data for cow manure and atmospheric emissions, a theoretical balance between crop soil heavy-metal input and output over one century was drawn up. The long-term impact of cow manure on Zn, Ni and Cr in soil is higher than that of the studied heat-dried sludge. Obviously, sludge tended to cause a strong increase in soil Cu storage, valued for these soils, which are otherwise very Cu deficient.

Fate of polycyclic aromatic hydrocarbons during composting of lagooning sewage sludge.

The fate of 16 polycyclic aromatic hydrocarbons (PAHs), targeted by the USEPA agency, has been investigated during composting of lagooning sludge. Composting shows efficient decrease of the content and the bioavailability of each PAH. Biodegradation and sorption are suggested as the main mechanisms contributing to this decrease. During the stabilization phase of composting, extensive microbial degradation of PAHs, mainly those with a low number of aromatic rings, was achieved following development of intense thermophilic communities. However, partial sorption of PAH to non-accessible sites temporarily limits the mobility mainly of PAHs with a high number of aromatic rings plus acenaphthene and acenaphthylene, and allows them to escape microbial attack. During the maturation phase, the development of a mesophilic population could play an important role in the degradation of the remaining PAH. During this phase of composting, PAH sequestration and binding of their oxidative metabolites within new-formed humic substances might also explain PAH decrease at the end of composting. The tendency of change of content or bioavailability of various PAH compounds during composting is found to be strongly related to the number of their aromatic rings, their molecular weight and structure.