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Microalgae have been proven efficient biological vectors for heavy metal uptake. In order to further study their biosorption potential, a strain of Desmodesmus pleiomorphus (L) was isolated from a strongly contaminated industrial site in Portugal. Under different initial Zn(2+) concentrations, metal removal by that strain reached a maximum of 360 mg Zn/g biomass after 7 days, at 30 mg Zn/l, after an initial rapid phase of uptake. Comparative studies were carried out using a strain of the same microalgal species that is commercially available (ACOI 561): when exposed to 30 mg Zn/l, it could remove only 81.8 mg Zn/g biomass. Biosorption experiments using inactivated biomass of the isolated strain reached a maximum Zn(2+) uptake of 103.7 mg/g. Metal removal at various initial pH values was studied as well; higher removal was obtained at pH 5.0. The microalga strain L, isolated from the contaminated site, exhibited a much higher removal capacity than the commercial strain, and the living biomass yielded higher levels of metal removal than its inactivated form.

Introduction: Tinctures are widely used liquid pharmaceutical preparations traditionally obtained by maceration of one or more medicinal plants in ethanol-water solutions. Such a process results in the extraction of virtually hundreds of structurally diverse compounds with different polarities. Owing to the large chemical diversity of the constituents present in the herbal tinctures, the analytical tools used for the quality control of tinctures are usually optimised only for the detection of single chemical entities or specific class of compounds., Objective: In order to overcome the major limitations of the current methods used for analysis of tinctures, a new methodological approach based on NMR spectroscopy and MS spectrometry has been tested with different commercial tinctures., Methodology: Diffusion-edited 1H-NMR (1D DOSY) and 1H-NMR with suppression of the ethanol and water signals have been applied here for the first time to the direct analysis of commercial herbal tinctures derived from Echinacea purpurea, Hypericum perforatum, Ginkgo biloba and Valeriana officinalis. The direct injection of the tinctures in the MS detector in order to obtain the corresponding metabolic profiles was also performed., Results: Using both NMR and MS methods it was possible, without evaporation or separation steps, to obtain a metabolic fingerprint able to distinguish between tinctures prepared with different plants. Batch-to-batch homogeneity, as well as degradation after the expiry date of a batch, was also investigated., Conclusion: The techniques proposed here represent fast and convenient direct analyses of medicinal herbal tinctures.

Industrial wastewater treatment comprises several processes to fulfill the discharge permits or to enable the reuse of wastewater. For tannery wastewater, constructed wetlands (CWs) may be an interesting treatment option. Two-stage series of horizontal subsurface flow CWs with Phragmites australis (UP series) and Typha latifolia (UT series) provided high removal of organics from tannery wastewater, up to 88% of biochemical oxygen demand (BOD(5)) (from an inlet of 420 to 1000 mg L(-1)) and 92% of chemical oxygen demand (COD) (from an inlet of 808 to 2449 mg L(-1)), and of other contaminants, such as nitrogen, operating at hydraulic retention times of 2, 5 and 7 days. No significant (P<0.05) differences in performance were found between both the series. Overall mass removals of up to 1294 kg COD ha(-1)d(-1) and 529 kg BOD(5)ha(-1)d(-1) were achieved for a loading ranging from 242 to 1925 kg COD ha(-1)d(-1) and from 126 to 900 kg BOD(5)ha(-1)d(-1). Plants were resilient to the conditions imposed, however P. australis exceeded T. latifolia in terms of propagation.

This study focused on the diversity of bacterial communities from two series of two-stage constructed wetlands (CWs) treating tannery wastewater, under different hydraulic conditions. Series were separately planted with Typha latifolia and Phragmites australis in expanded clay aggregates and operated for 31 months. The effect of plant species, hydraulic loading and unit stage on bacterial communities was addressed through bacterial enumeration and denaturating gradient gel electrophoresis (DGGE). Diverse and distinct bacterial communities were found in each system unit, which was related in part to the type of plant and stage position (first or second unit in the series). Numerical analysis of DGGE profiles showed high diversity in each unit with an even distribution of species. No clear relation was established between the sample collection time, hydraulic loading applied and the bacterial diversity. Isolates retrieved from plant roots and substrates of CWs were affiliated with gamma-Proteobacteria, Firmicutes, alpha-Proteobacteria, Sphingobacteria, Actinobacteria and Bacteroidetes. Both series were effective in removing organic matter from the inlet wastewater, however, based on batch degradation experiments it seems that biodegradation was limited by the recalcitrant properties of the wastewater.

This work investigates the potential of Rubus ulmifolius, indigenous to a metal contaminated site--Esteiro de Estarreja--for phytoremediation purposes. The site has a long history of metal contamination. The accumulation of lead (Pb), arsenic (As) and nickel (Ni) in different sections--roots, stems and leaves - of the plant was assessed and compared to the levels of those metals in the soil and in the available fraction. The distribution of metals throughout the area was quite heterogeneous, presenting levels of As, Pb and Ni of up to 3078, 1400 and 135 mg kg(-1), respectively, and the metal content in the sections of R. ulmifolius collected in the banks of the stream varied among sites of collection. Levels of metals were higher in the plant roots: As levels (mgkg(-1)) ranged from 277 to 1721 in the roots, 30 to 110 in the stems, and 60 to 265 in the leaves; Pb concentrations (mgkg(-1)) ranged from 248 to 1178 in the roots, 35 to 133 in the stems, and 25 to 149 in the leaves; and Ni (mgkg(-1)) ranged from 48 to 151 in the roots. Significant correlations were found between the total levels of Pb and As in the soil and the levels in the roots of the plant; further correlations between total and available levels in the soil and metals in other plant tissues were generally found as non-significant. According to the metal accumulation patterns of R. ulmifolius, this species seems to be valuable for application in phytostabilisation strategies.

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with 1(max) of 0.094 h(-1) and S (m) of 1,435 mg l(-1). Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.

A suspended-growth bioreactor (SGB) was operated for the treatment of a gaseous stream mimicking emissions generated at a leather industrial company. The main volatile organic compounds (VOCs) present in the gaseous stream consisted of 1-methoxy-2-propanol, 2,6-dimethyl-4-heptanone, 2-butoxyethanol, toluene and butylacetate. A microbial consortium able to degrade these VOCs was successfully enriched. A laboratory-scale SGB was established and operated for 210-d with an 8h cycle period and with shutdowns at weekends. Along this period, the SGB was exposed to organic loads (OL) between 6.5 and 2.3 x 10(2) g h(-1) m(-3). Most of the compounds were not detected at the outlet of the SGB. The highest total VOC removal efficiency (RE) (ca 99%) was observed when an OL of 1.6 x 10(2) g h(-1) m(-3) was fed to the SGB. The maximum total VOC elimination capacity (1.8 x 10(2) g h(-1) m(-3)) was achieved when the OL applied to the SGB was 2.3 x 10(2) g h(-1) m(-3). For all the operating conditions, the SGB showed high levels of degradation of toluene and butylacetate (RE approximately equal to 100%). This study also revealed that recirculation of the gaseous effluent improved the performance of the SGB. Overall, the SGB was shown to be robust, showing high performance after night and weekend shutdown periods.

Toxicity tests were performed to assess the effect of tannery wastewater with different treatment levels on two wetland plants, Phragmites australis and Typha latifolia, which are frequently used in constructed wetlands (CWs) for water treatment, and thus deepen the knowledge on their capacity to withstand the application of industrial wastewater. Trifolium pratense, a plant generally used as an indicator in toxicity tests, was included as a control. End points measured were germination percentage, shoot length, root elongation, and biomass growth of the plants. When tannery effluent, with a low treatment level, was supplied to the wetland plants germination occurred even at effluent concentrations of 100%, whereas germination of T. pratense was completely inhibited, almost invariably, at effluent concentration of 50%. Higher germination levels were achieved when the plants were exposed to effluent originating from the outlet of constructed wetland pilot units, allowing germination of all tested plants, indicating a significant decrease in its toxicity level. Experiments conducted with the same plants using different growing substrata as the germination matrix, namely expanded clay aggregates (Filtralite MR 3-8 and Filtralite NR 3-8) and two types of sand (fine gravel and standard sand) have shown that higher germination levels were achieved in standard sand and that P. australis was the plant species showing higher germination in all cases, reinforcing the robustness of this plant to environmental stress. The phytoextraction potential of P. australis, was evaluated by subjecting the plant to tannery wastewater supplemented with 50 and 150 mg Cr/L. After 6 weeks of exposure, levels up to 4825, 883, and 627 mg Cr/kg were found in the rhizome, shoot, and leaves, respectively, although phytotoxic signs in the plant were evident. This plant might not be considered a chromium hyperacumulator, but the potential to extract and accumulate this metal on its rhizomes is high.

The aim of this study was to investigate the performance of horizontal subsurface flow constructed wetlands planted with Typha latifolia treating tannery wastewater under long-term operation. Two expanded clay aggregates (Filtralite MR3-8-FMR and Filtralite NR3-8-FNR) and a fine gravel-FG were used as substrate for the constructed wetland units plus one unit with FMR was left as an unvegetated control. The systems were subject to three hydraulic loadings, 18, 8 and 6cmd(-1), and to periods of interruption in the feed. The relationship between the substrate, plant development and removal efficiency, especially of organic matter, was investigated. Organic loadings up to 1800kg BOD(5)ha(-1)d(-1) and 3849kg COD ha(-1)d(-1) were applied leading to mass removals of up to 652kg BOD(5)ha(-1)d(-1) and 1869kg COD ha(-1)d(-1), respectively. The three different substrates were adequate for the establishment of T. latifolia, although the clay aggregates allowed for higher plant propagation levels. The units with FNR and FMR achieved significantly higher COD and BOD5 removal when compared to the FG and to the unplanted units. The systems proved to be tolerant to high organic loadings and to interruptions in feed suggesting this technology as a viable option for the biological treatment of tannery wastewater.

Two up-flow fixed-bed reactors (UFBRs), inoculated with activated sludge and operated for 162 days, were fed 1mmolL(-1)d(-1) with two model halogenated compounds, 2-fluorobenzoate (2-FB) and dichloromethane (DCM). Expanded clay (EC) and granular activated carbon (GAC) were used as biofilm carrier. EC did not have any adsorption capacity for both model compounds tested, whereas GAC could adsorb 1.3mmolg(-1) GAC for 2-FB and 4.5mmolg(-1) GAC for DCM. Both pollutants were degraded in both reactors under simultaneous feeding. However, biodegradation in the EC reactor was more pronounced, and re-inoculation of the GAC reactor was required to initiate 2-FB degradation. Imposing sequential alternating pollutant (SAP) feeding caused starvation periods in the EC reactor, requiring time-consuming recovery of 2-FB biodegradation after resuming its feeding, whereas DCM degradation recovered significantly faster. The SAP feeding did not affect performance in the GAC reactor as biodegradation of both pollutants was continuously observed during SAP feeding, indicating the absence of true starvation.

Photopolymerized sol-gel monolithic columns for use in capillary electrochromatography were prepared in 125 microm i.d. polyacrylate-coated fused-silica capillaries. The polyacrylate-coating, unlike the polyimide one, is transparent to the radiation used (approximately 370 nm), and thus, no coating removal is necessary. This is a very important particularity since intrinsic capillary column characteristics, such as flexibility and mechanical resistance, are unchanged. A mixture containing metacryloxypropyltrimethoxysilane (MPTMS) as the polymeric precursor, hydrochloric acid as the catalyst, toluene as the porogen and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (Irgacure 819) as the photoinitiator was irradiated at 370 nm for 20 min inside the capillaries to prepare the columns through sol-gel approach. The versatility and viability of the use of polyacrilate as a new capillary external coating were shown through preparation of two columns under different conditions, which were tested in electrochromatography for separation of standard mixture containing thiourea (marker compound), propylbenzene, phenanthrene and pyrene.

A detailed classification of a novel bacterial strain, designated F11(T), capable of degrading fluorobenzene as a sole carbon and energy source, was performed by using a polyphasic approach. This Gram-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium was isolated from a sediment sample collected from an industrially contaminated site in northern Portugal. The predominant whole-cell fatty acids were C(19 : 0) cyclo omega8c, C(16 : 0), C(18 : 1)omega7c, C(18 : 0), C(18 : 0) 3-OH and C(16 : 0) 3-OH. The G+C content of the DNA was 62.9 mol% and the major respiratory quinone was ubiquinone 10 (UQ-10). 16S rRNA gene sequence analysis revealed that strain F11(T) was a member of the class Alphaproteobacteria and was phylogenetically related to the genus Labrys, having sequence similarities of 95.6 and 93.1 % to the type strains of Labrys monachus and Labrys methylaminiphilus, respectively. DNA-DNA hybridization experiments revealed levels of relatedness of <70 % between strain F11(T) and the type strains of L. monachus and L. methylaminiphilus (38.6 and 34.1 %, respectively), justifying the classification of strain F11(T) as representing a novel species of the genus Labrys. The name Labrys portucalensis sp. nov. is proposed for this organism. The type strain is F11(T) (=LMG 23412(T)=DSM 17916(T)).

Two up-flow fixed bed reactors (UFBR) were operated for 8 months treating a model synthetic wastewater containing 2-fluorobenzoate (2-FB) and dichloromethane (DCM). The stability of the reactors under dynamic conditions, that is, sequentially alternating pollutants (SAP), shock loads, and starvation periods was assessed. Two support materials were used: expanded clay (EC) that does not adsorb 2-FB or DCM, and granular-activated carbon (GAC) that adsorbs 180 mg g(-1) of 2-FB and 390 mg g(-1) of DCM. The reactors were inoculated with a 2-FB-degrading strain (FB2) and a DCM degrader (TM1). 2-FB was fed at organic loads ranging from 0 to 800 mg L(-1) d(-1), while DCM was fed at 0-250 mg L(-1) d(-1). 2-FB or DCM were never detected at the outlet of the GAC reactor, while in the EC reactor outlet small amounts were observed. Nevertheless, the highest biological elimination capacity was observed in the EC reactor (over 700 mg L(-1) d(-1) of 2-FB). DGGE analysis revealed a fairly stable bacterial community with the largest shifts occurring during starvation periods and changes in feed composition. Several bacterial strains isolated from the reactors showed capacity for 2-FB degradation, while only strain TM1 degraded DCM., (Copyright 2007 Wiley Periodicals, Inc.)

Zn accumulation in Solanum nigrum grown in naturally contaminated soil in the presence of different types of organic amendments was assessed. Under the same conditions, the response of the plant to inoculation with two different isolates of arbuscular mycorrhizal fungi (AMF) (Glomus claroideum and Glomus intraradices) was also evaluated. S. nigrum grown in the non-amended soil always presented higher Zn accumulation in the tissues, with the addition of amendments inducing reductions of up to 80 and 40%, for manure and compost, respectively, and enhancing plant biomass yields. The establishment of S. nigrum in the Zn contaminated soil combined with the application of amendments led to a 70-80% reduction in the amount of Zn leached through the soil. The use of S. nigrum in combination with manure appeared as an effective method for reducing the effects of soil contamination, diminishing Zn transfer to other environmental compartments via percolation.

The effect of two different chelating agents [EDTA and EDDS (S,S-ethylenediaminedissucinic acid)] on Zn tissue accumulation in Solanum nigrum L. grown in a naturally contaminated soil was assessed. Under those conditions, the response of the plant to the inoculation with two different isolates of arbuscular mycorrhizal fungi (AMF)--Glomus claroideum and Glomus intraradices--was also studied. Plants grown in the local contaminated soil (Zn levels of 433mg kg(-1)) accumulated up to 1191mg kg(-1) of Zn in the roots, 3747mg kg(-1) in the stems and 3409mg kg(-1) in the leaves. S. nigrum plants grown in the same soil spiked with extra Zn (Zn levels of 964mg kg(-1)) accumulated up to 4735, 8267 and 7948mg Zn kg(-1) in the leaves, stems and roots, respectively. The addition of EDTA promoted an increase in the concentration of Zn accumulated by S. nigrum of up to 231% in the leaves, 93% in the stems and 81% in the roots, while EDDS application enhanced the accumulation in leaves, stems and roots up to 140, 124 and 104%, respectively. In the stems, the presence of Zn was predominantly detected in the cortex collenchyma cells, the starch sheath and the internal phloem and xylem parenchyma, and the addition of chelating agents did not seem to have an effect on the localisation of accumulation sites. The devise of a chelate-enhanced phytoextraction strategy, using chelating agents and AMF, is discussed.

Bacterial biofilters used in marine recirculation aquaculture systems need improvements to enhance nitrogen removal efficiency. Relatively little is known about biofilter autochthonous population structure and function. The present study was aimed at isolating and characterizing an autochthonous denitrifying bacterium from a marine biofilter installed at a recirculation aquaculture system. Colonization of four different media in a marine fish farm was followed by isolation of various denitrifying strains and molecular classification of the most promising one, strain T2, as a novel member of the Pseudomonas fluorescens cluster. This strain exhibits high metabolic versatility regarding N and C source utilization and environmental conditions for growth. It removed nitrate through aerobic assimilatory metabolism at a specific rate of 116.2 mg NO(3)-N g dw(-1) h(-1). Dissimilatory NO(3)-N removal was observed under oxic conditions at a limited rate, where transient NO(2)-N formed represented 22% (0.17 mg L(-1)) of the maximum transient NO(2)-N observed under anoxic conditions. Dissimilatory NO(3)-N removal under anoxic conditions occurred at a specific rate of 53.5 mg NO(3)-N g dw(-1) h(-1). The isolated denitrifying strain was able to colonize different materials, such as granular activated carbon (GAC), Filtralite and Bioflow plastic rings, which allow the development of a prototype bioreactor for strain characterization under dynamic conditions and mimicking fish-farm operating conditions.

The adsorption of a recalcitrant fluoroaromatic compound, fluorobenzene (FB), onto granular activated carbon (GAC) was evaluated. The respective isotherm was obtained and the Langmuir, Freundlich and Redlich-Peterson models were fitted to the experimental data, with the Redlich-Peterson model giving the best fitting. Freundlich model also provided a good fit but the Langmuir model could not adequately fit the experimental data, especially at high FB concentrations. Maximal adsorption capacity of FB onto GAC was found to be 388mg of FB per gram of GAC. The reversibility of the adsorption of FB onto GAC was investigated, both in the absence and presence of microorganisms. Abiotic desorption of FB occurred to a small extent (between 3% and 22%, for amounts of FB initially adsorbed to the GAC between 37 and 388mgg(-1)), and bioregeneration of GAC was shown to occur when the matrix was exposed to a FB degrading culture, with 58-80% of the adsorbed FB being biodegraded. A residual amount of FB showed not to be bioavailable, suggesting that part of the adsorbed FB may be irreversibly bound. The fraction of the non-bioavailable FB increased at higher amounts of adsorbed FB, from 19% to 33%. The results indicate that the GAC employed in this study has a good capacity to adsorb FB and that bioregeneration of this matrix is a feasible process.

The levels of zinc accumulated by roots, stems, and leaves of two plant species, Rubus ulmifolius and Phragmites australis, indigenous to the banks of a stream in a Portuguese contaminated site were investigated in field conditions. R. ulmifolius, a plant for which studies on phytoremediation potential are scarce, dominated on the right side of the stream, while P. australis proliferated on the other bank. Heterogeneous Zn concentrations were found along the banks of the stream. Zn accumulation in both species occurred mainly in the roots, with poor translocation to the aboveground sections. R. ulmifolius presented Zn levels in the roots ranging from 142 to 563 mg kg(-1), in the stems from 35 to 110 mg kg(-1), and in the leaves from 45 to 91 mg kg(-1), vs. average soil total Zn concentrations varying from 526 to 957 mg kg(-1). P. australis showed Zn concentrations in the roots from 39 to 130 mg kg(-1), in the stems from 31 to 63 mg kg(-1), and in the leaves from 37 to 83 mg kg(-1), for the lower average soil total Zn levels of 138 to 452 mg kg(-1) found on the banks where they proliferated. Positive correlations were found between the soil total, available and extractable Zn fractions, and metal accumulation in the roots and leaves of R. ulmifolius and in the roots and stems of P. australis. The use of R. ulmifolius and P. australis for phytoextraction purposes does not appear as an effective method of metal removing, but these native metal tolerant plant species may be used to reduce the effects of soil contamination, avoiding further Zn transfer to other environmental compartments.

Wastewaters from leather processing are very complex and lead to water pollution if discharged untreated, especially due to its high organic loading. In this study the survival of different plant species in subsurface horizontal flow constructed wetlands receiving tannery wastewater was investigated. Five pilot units were vegetated with Canna indica, Typha latifolia, Phragmites australis, Stenotaphrum secundatum and Iris pseudacorus, and a sixth unit was left as an unvegetated control. The treatment performance of the systems under two different hydraulic loading rates, 3 and 6 cmd(-1), was assessed. COD was reduced by 41-73% for an inlet organic loading varying between 332 and 1602 kgha(-1)d(-1) and BOD(5) was reduced by 41-58% for an inlet organic loading varying between 218 and 780 kgha(-1)d(-1). Nutrient removal occurred to lower extents. Phragmites australis and Typha latifolia were the only plants that were able to establish successfully. Despite the high removal of organic content from the influent wastewater, during 17 months of operation, no significant differences in performance were observed between units.

Zn tissue accumulation in Solanum nigrum grown in a non-contaminated and a naturally contaminated Zn matrix and the effect of inoculation with different arbuscular mycorrhizal fungi (AMF) on metal uptake were assessed. S. nigrum grown in the contaminated soil always presented higher Zn accumulation in the tissues, accumulating up to 1622 mg Zn kg(-1). The presence of both Glomus claroideum and Glomus intraradices enhanced the uptake and accumulation of Zn by S. nigrum (up to 83 and 49% higher Zn accumulation, respectively). The main deposits of the metal were found in the intercellular spaces and in the cell walls of the root tissues, as revealed by autometallography, with the inoculation with different AMF species causing no differences in the location of Zn accumulation. These findings indicate that S. nigrum inoculated with selected heavy metal tolerant AMF presents extracting and accumulating capacities, constituting a potentially suitable remediation method for Zn polluted soils.

Alkoxy-amino-bis(phenolate) yttrium amide and alkoxide complexes promote the ring-opening polymerization of (3S,6S)-dimethyl-2,5-morpholinedione at 60-100 degrees C via a coordination-insertion polymerization mechanism.

Solanum nigrum was found to proliferate in sediments with high levels of metal pollution. The effect of Zn on plant growth and tissue metal accumulation was assessed. The response of the plant to the inoculation with four different isolates of arbuscular mycorrhizal fungi (AMF) (Glomus sp. BEG140, Glomus claroideum, Glomus mosseae and Glomus intraradices) was studied. While the isolates of AMF did not have a significant (P<0.05) influence on mycorrhizal colonisation, increasing Zn concentration to high levels (500 and 1000 mgkg(-1)) induced significant (P<0.05) decrease of the AMF colonisation. In general, the presence of AMF did not affect the growth and biomass of S. nigrum individuals. However, the level of metal in the matrix affected S. nigrum growth; plants grown at 100 mgkg(-1) had significantly (P<0.05) lower leaf, stem, root and total biomass than control ones and plants growing at 500 and 1000 mgkg(-1) had the significantly (P<0.05) lowest biomass. Plants inoculated with the AMF G. claroideum and G. intraradices presented significantly (P<0.05) higher Zn accumulation in all plant tissues. In general, the stem tissues had the higher Zn content while the leaves registered the lowest values, which indicate a high translocation of the metal. AMF inoculation had no significant (P<0.05) influence on the metal translocation within the plant. This study suggests that inoculation with the AMF G. claroideum or G. intraradices, can enhance the Zn accumulation in the tissues of S. nigrum, not affecting the plant translocation capacities.

The aerobic metabolism of fluorobenzene by Rhizobiales sp. strain F11 was investigated. Liquid chromatography-mass spectrometry analysis showed that 4-fluorocatechol and catechol were formed as intermediates during fluorobenzene degradation by cell suspensions. Both these compounds, unlike 3-fluorocatechol, supported growth and oxygen uptake. Cells grown on fluorobenzene contained enzymes for the ortho pathway but not for meta ring cleavage of catechols. The results suggest that fluorobenzene is predominantly degraded via 4-fluorocatechol with subsequent ortho cleavage and also partially via catechol.

An up-flow fixed bed reactor (UFBR) was established to investigate the biodegradation of fluorobenzene (FB) under a number of operating conditions, which included variation in the concentration of FB in the feed stream (up to 180 mg l(-1)) and temporary suspension of feeding. Degradation of FB was followed for a period of 8 months under a continuous flow regime. During the operation of the UFBR, FB was never detected in the reactor effluent, being biodegraded by the microbial biofilm or adsorbed to the granular activated carbon (GAC). Biodegradation of FB was observed from the beginning of the reactor operation, and overall, it accounted for 50% of the total amount fed to the bioreactor. High organic loads of FB (210-260 mg d(-1) dm(-3)) were found to affect the biological removal efficiency, possibly due to an inhibitory effect caused by the higher FB concentrations fed to the bioreactor (149-179 mg l(-1)). When FB feeding was suspended for 1 month, biodegradation continued, indicating that the adsorbed FB became bioavailable. Biofilm bacterial dynamics were followed throughout the UFBR operation by denaturing gradient gel electrophoresis and plate-counting techniques, showing that a quite stable community was found in the bioreactor, and this was mainly attributed to the high selective pressure exerted by the presence of FB.

Obesity constitutes a serious health problem. Inflammation, which has recently been shown to follow adipocyte death, is at the basis of a series of pathogenic complications of obesity. Here we demonstrate, through modelling using the finite element method, that the bigger the adipocyte, the more fragile it becomes to rupture when submitted to common physical forces. This indicates that adipocyte size is an important determinant of cell death. Interventions to prevent adipocyte hypertrophy may, therefore, help to reduce the risk associated with obesity.

The culturable bacteria colonizing the rhizosphere of plants growing in the area of discharge of a tannery effluent were characterized. Relative proportions of aerobic, denitrifying, and sulfate-reducing bacteria were determined in the rhizosphere of Typha latifolia, Canna indica, and Phragmites australis. Aerobic bacteria were observed to be the most abundant group in the rhizosphere, and plant type did not seem to influence the abundance of the bacterial types analyzed. To isolate bacteria able to degrade polyphenols used in the tannery industry, enrichments were conducted under different conditions. Bacterial cultures were enriched with individual polyphenols (tannins Tara, Quebracho, or Mimosa) or with an undefined mixture of tannins present in the tannery effluent as carbon source. Cultures enriched with the effluent or Tara tannin were able to degrade tannic acid. Six bacterial isolates purified from these mixed cultures were able to use tannic acid as a sole carbon source in axenic culture. On the basis of 16S ribosomal DNA sequence analysis, these isolates were closely related to organisms belonging to the taxa Serratia, Stenotrophomonas maltophilia, Klebsiella oxytoca, Herbaspirillum chlorophenolicum, and Pseudomonas putida.

The presence of actinorhizas and arbuscular mycorrhizas may reduce plant stresses caused by adverse soil conditions. A greenhouse experiment was conducted using a sediment with a high pH, resulting from the disposal of waste originated at an acetylene and polyvinylchloride factory, in which Black alder (Alnus glutinosa) seedlings were inoculated either with Glomus intraradices BEG163 (originally isolated from the same sediment), Frankia spp. or both symbionts. After a 6-month growth period, plants inoculated with both symbionts had significantly greater leaf area, shoot height and total biomass when compared with the uninoculated control, the Frankia spp. and the G. intraradices treatments alone. In dual inoculated plants the N and P leaf content was significantly increased. A defoliation experiment was performed to evaluate the stress recovery of A. glutinosa and plants inoculated with both symbionts had a faster leaf regrowth and produced greater numbers of leaves. The dual inoculation resulted in greater numbers of and larger root nodules than when inoculated with Frankia spp. alone. The length and NADH diaphorase activity of the extraradical mycelium of G. intraradices was also significantly greater when dual inoculation was performed. The inoculation with Frankia spp. alone was shown to improve A. glutinosa growth, whereas G. intraradices alone had no positive effect under these environmental conditions. However, when the two symbionts were inoculated together a synergistic effect was observed resulting in a greater benefit for the plants and for both symbionts. The relevance of these findings for the phytorestoration of anthropogenic stressed sediments with high pH is discussed.

A pure bacterial strain capable of aerobic biodegradation of fluorobenzene (FB) as the sole carbon and energy source was isolated by selective enrichment from sediments collected from a polluted site. 16S rRNA and fatty acid analyses support that strain F11 belongs to a novel genus within the alpha-2 subgroup of the Proteobacteria, possibly within a new clade related to the order Rhizobiales. In batch cultures, growth of strain F11 on FB led to stoichiometric release of fluoride ion. Maximum experimental growth rate of 0.04 h-1 was obtained at FB concentration of 0.4 mM. Growth kinetics were described by the Luong model. An inhibitory effect with increasing FB concentrations was observed, with no growth occurring at concentrations higher than 3.9 mM. Strain F11 was shown to be able to use a range of other organic compounds, including other fluorinated compounds such as 2-fluorobenzoate, 4-fluorobenzoate and 4-fluorophenol. To our knowledge, this is the first time biodegradation of FB, as the sole carbon and energy source, by a pure bacterium has been reported.

An aquatic community was recovered from a waste discharge container fed with several aromatic pollutants. After 3 months of selective enrichment with p-chlorophenol and p-nitrophenol, two microalgae species, Chlorella vulgaris and Coenochloris pyrenoidosa, were recovered from the microbial consortium. As an axenic culture, this microalgae consortium was able to remove p-chlorophenol under different photo-regimes. Cultures grown under a 24h light regime were capable of biodegrading 50mg l(-1) of p-chlorophenol within 5 days. Addition of zeolite, an adsorbing material, did not improve the p-chlorophenol removal. However, when p-chlorophenol at 150mgl(-1) was fed to the culture supplemented with zeolite, the growth rate of the consortium improved, but the lag phase was longer (16 against 14 days in the absence of zeolite).

Epifluorescence microscope methods, namely BacLight, direct epifluorescence filter technique and Rhodamine 123, consistently underestimated plate bacterial counts in a 4-chlorophenol degrading consortium. Cells capable of passing through 0.2 microm filters, referred as 'ultramicrocells', were found. Although cell counts were higher when traditional methods were used, BacLight and direct epifluorescence filter technique were convenient techniques for the systematic monitoring of bacteria involved in biodegradation processes, as results were consistent and available within a short time.

This study presents the operation of a biotrickling filter when treating a monochlorobenzene (MCB) contaminated gaseous emission. Treatment dynamics were characterised by exposing the reactor to various MCB Organic Loads (OL). The use of different growth support materials, namely limestone, sand, ceramic and PVC pall-rings, was investigated. Limestone led to dogging of the reactor due to the accumulation of surface precipitates, but PVC pall-rings allowed for a uniform biofilm growth. The biotrickling filter presented maximum removal efficiency (RE, 95%) under OL regimes of 10 g m(-3)-reactor h(-1). Treatment inhibition was observed when the reactor was exposed to OL of 45 g m(-3)-reactor h(-1), with RE reaching a minimum value (8%) and elimination capacity of 8 g m(-3)-reactor h(-1). The first half of the reactor height was the predominant section for MCB biodegradation and increasing the mineral medium recirculation rate was beneficial for the overall treatment.

Microbial cultures able to degrade xenobiotic compounds are the key element for biological treatment of waste effluents and are obtained from enrichment processes. In this study, two common enrichment methods, suspension batch and immobilized continuous, were compared. The main selection factor was the presence of 1,3-dichloro-2-propanol (1,3-DCP) as the single carbon source. Both methods have successfully enriched microbial consortia able to degrade 1,3-DCP. When tested in batch culture, the degradation rates of 1,3-DCP by the two consortia were different, with the consortia obtained by batch enrichment presenting slightly higher rates. A preliminary morphological and biochemical analysis of the predominant colonial types present in each degrading consortia revealed the presence of different constituting strains. Three bacterial isolates capable of degrading 1,3-DCP as single strains were obtained from the batch enrichments. These strains were classified by 16S rRNA analysis as belonging to the Rhizobiaceae group. Degradation rates of 1,3-DCP were lower when single species were used, reaching 45 mg 1(-1) d(-1), as compared to 74 mg 1(-1) d(-1) of the consortia enriched on the batch method. Mutualistic interactions may explain the better performance of the enriched consortia.

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the beta-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter(-1) and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.

Phytoremediation technologies based on the combined action of plants and the microbial communities that they support within the rhizosphere hold promise in the remediation of land and waterways contaminated with hydrocarbons but they have not yet been adopted in large-scale remediation strategies. In this review plant and microbial degradative capacities, viewed as a continuum, have been dissected in order to identify where bottle-necks and limitations exist. Phenols, anilines and polyaromatic hydrocarbons (PAHs) were selected as the target classes of molecule for consideration, in part because of their common patterns of distribution, but also because of the urgent need to develop techniques to overcome their toxicity to human health. Depending on the chemical and physical properties of the pollutant, the emerging picture suggests that plants will draw pollutants including PAHs into the plant rhizosphere to varying extents via the transpiration stream. Mycorrhizosphere-bacteria and -fungi may play a crucial role in establishing plants in degraded ecosystems. Within the rhizosphere, microbial degradative activities prevail in order to extract energy and carbon skeletons from the pollutants for microbial cell growth. There has been little systematic analysis of the changing dynamics of pollutant degradation within the rhizosphere; however, the importance of plants in supplying oxygen and nutrients to the rhizosphere via fine roots, and of the beneficial effect of microorganisms on plant root growth is stressed. In addition to their role in supporting rhizospheric degradative activities, plants may possess a limited capacity to transport some of the more mobile pollutants into roots and shoots via fine roots. In those situations where uptake does occur (i.e. only limited microbial activity in the rhizosphere) there is good evidence that the pollutant may be metabolised. However, plant uptake is frequently associated with the inhibition of plant growth and an increasing tendency to oxidant stress. Pollutant tolerance seems to correlate with the ability to deposit large quantities of pollutant metabolites in the 'bound' residue fraction of plant cell walls compared to the vacuole. In this regard, particular attention is paid to the activities of peroxidases, laccases, cytochromes P450, glucosyltransferases and ABC transporters. However, despite the seemingly large diversity of these proteins, direct proof of their participation in the metabolism of industrial aromatic pollutants is surprisingly scarce and little is known about their control in the overall metabolic scheme. Little is known about the bioavailability of bound metabolites; however, there may be a need to prevent their movement into wildlife food chains. In this regard, the application to harvested plants of composting techniques based on the degradative capacity of white-rot fungi merits attention.

Using a continuous enrichment technique, a bacterial consortium capable of degrading 4-chlorophenol (4-CP) was obtained from the rhizosphere of Phragmites australis. A granular activated carbon (GAC) biofilm reactor was established using this consortium, and the degradation of 4-CP was investigated under continuous flow operation using a feed of 20-50 mg l(-1) with a hydraulic residence time of 17 min over a 6-month period. Chloride liberation occurred throughout the operation, and the reactor had 4-CP removal efficiencies of 69-100%. Periods of lower performance were attributed to clogging of the column with biomass and the formation of channels. Subsequently, the immobilized biofilm was subjected to a starvation period of 5 months, after which its degradative capacity was still maintained. The microbial consortium was characterized during the continuous flow experiment and dynamic population changes were observed throughout. One isolate recovered from the biofilm was shown to be capable of degrading 4-CP as a sole carbon and energy source.

A bacterial consortium that can degrade chloro- and nitrophenols has been isolated from the rhizosphere of Phragmitis communis. Degradation of 4-chlorophenol (4-CP) by a consortium attached to granular activated carbon (GAC) in a biofilm reactor was evaluated during both open and closed modes of operation. During the operation of the biofilm reactor, 4-CP was not detected in the column effluent, being either adsorbed to the GAC or biodegraded by the consortium. When 4-CP at 100 mg l-1 was fed to the column in open mode operation (20 mg g-1 GAC total supply), up to 27% was immediately available for biodegradation, the rest being adsorbed to the GAC. Biodegradation continued after the system was returned to closed mode operation, indicating that GAC bound 4-CP became available to the consortium. Biofilm batch cultures supplied with 10-216 mg 4-CP g-1 GAC suggested that a residual fraction of GAC-bound 4-CP was biologically unavailable. The consortium was able to metabolise 4-CP after perturbations by the addition of chromium (Cr VI) at 1-5 mg l-1 and nitrate at concentrations up to 400 mg l-1. The development of the biofilm structure was analysed by scanning electron microscopy and confocal laser scanning microscopy (CLSM) techniques. CLSM revealed a heterogeneous structure with a network of channels throughout the biofilm, partially occupied by microbial exopolymer structures.

The culture environment exerts a major effect on the glycosylation pattern of recombinant human interferon-gamma (IFN-gamma) produced by Chinese-hamster ovary (CHO) cells. The recombinant IFN-gamma is heterogeneous and consists of a mixture of fully (2N), partially (1N) and non-glycosylated (0N) glycoforms, and throughout batch cultures there is a decline in the proportion of fully glycosylated IFN-gamma. Glucose and glutamine, nutrients that are depleted early in such cultures, were prima facie candidates for causing such a shift in glycoform profile. Batch feeding of these nutrients did not prevent the decline in 2N glycoform, but the glycosylation pattern of IFN-gamma was affected by the initial glutamine concentration in the culture. Under different serum-free environments the extent of IFN-gamma glycosylation was affected by (1) the concentration of BSA, (2) the quality of BSA, (3) the lipid composition of the culture medium and (4) the presence of surfactants. Moreover, the inclusion of serum in cultures caused changes in the molecular masses of the major glycoforms, that was indicative of cleavage of the core polypeptide. The results reported emphasize the necessity of considering the effects of culture media on product quality as well as on product quantity during process optimization.

The role of bovine serum albumin in mammalian cell cultures and the possibility of its substitution by other components in a serum-free medium has been investigated. In this study, BSA was shown to be important for growth and product formation in CHO cells expressing recombinant human interferon-γ. There were indications that its stimulating growth effect was dependent on the source of BSA used and probably was related to the purification procedure used for the production of the desired albumin fraction. Cell growth did not occur in the absence of BSA but at low concentration (1 mg ml(-1)) it was stimulated by the addition of a combination of a commercial lipid mixture plus Pluronic F68. However, under the latter conditions IFN-γ production was adversely effected. The importance of individual lipid components was investigated using a statistical approach based on a Plackett-Burman design. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN-γ production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of low BSA medium, cell growth was similar to that at high BSA concentration (5 mg ml(-1)) but the IFN-γ concentration was significantly reduced (ca. 45%).

The importance of serum-free medium components on the growth of Chinese hamster ovary (CHO) cells and production of recombinant human interferon(IFN)-gamma was investigated. The complexity of the medium led to the adoption of a statistical optimization approach based on a Plackett-Burman design. From this analysis a set of nutritional components was identified as important for cell growth and recombinant protein production. Glycine was identified as an important determinant of specific growth rate, whereas for cell production bovine serum albumin (BSA), phenylalanine and tyrosine were also identified as important. BSA, sodium pyruvate, glutamate, methionine, proline, histidine, hydroxyproline, tyrosine and phenylalanine were shown to be important for IFN-gamma production. Other medium components, such as insulin, arginine, aspartate and serine produced an inhibitory effect on both cell growth and IFN-gamma production. The effect of the stimulatory nutrients as a whole group was tested by increasing their concentration in the medium. A significant improvement in specific cell growth rate, cell production and IFN-gamma production (up to 45%) was achieved on both shake-flask and fermentor cultures. An increase in the medium concentration of the negative variables had only a small inhibitory effect (approximately 10%) on the same parameters. Analysis of the effects of the group of stimulatory amino acids and BSA on CHO cell growth showed that the effect of the former was independent of BSA.

C-banding studies were performed on cultured peripheral blood lymphocytes from 100 patients with tumors of the nervous system and 30 controls. The classification of Patil and Lubs and the heterochromatic index (HI) of Neeley were used to evaluate the heteromorphism. No overall significant differences were found in the frequency of C-band variants when the findings in both tumoral populations and controls were compared, but we did find an excess of 9qh inversions in some groups of tumor patients. An increase in the amount of heterochromatin in patients with pituitary adenomas and high malignancy grade astrocytomas was noted.