A method to remove environmental inhibitors prior to the detection of waterborne enteric viruses by reverse transcription-polymerase chain reaction.

A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the reverse transcription-polymerase chain reaction (RT-PCR). Environmental inhibitors, concentrated along with viruses during water sample processing, are removed by the method through a series of steps that includes dialysis, solvent extraction, ultrafiltration and glass purification. The method was tested by spiking sodium phosphate with poliovirus type 1 with or without humic or fulvic acids and then measuring virus recovery by plaque assay and RT-PCR. Results of the study indicated that (i) 90% of the spiked virus could be recovered from samples at the end of the ultrafiltration step, (ii) virus was detected in the final eluate of samples containing as much as 0.5 mg of humic acid or 5.0 mg of fulvic acid, and (iii) as little as 0.06 plaque forming units (PFU) was detectable per RT-PCR reaction. These results indicate that the described purification method along with RT-PCR is a feasible approach for detecting waterborne human enteric viruses in the presence of interfering substances.

Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments.

A variety of gel filtration resins (Sephadex G200 and G150; Sepharose 6B, 4B and 2B; Bio-Gel P100, P200; and Toyopearl HW 55, HW 65, and HW 75) were evaluated for their efficacy in removing PCR-inhibitory substances from feedlot soil DNA crude extracts using gravity-flow disposable columns. Sepharose resins demonstrated the best properties for DNA purification when compared to other gel filtration resins, and Sepharose 2B was the most efficient purification resin based upon flow rate and the elution of DNA and humic acids from the columns. A method for purifying large solution volumes of DNA extract economically was also developed using low-cost disposable Disposaflex columns. Crude DNA extracts of cattle feedlot soil and aquifer sediment impacted by animal and human wastes were easily purified using the Disposaflex column method regardless of whether a gentle chemical lysis or a bead mill homogenization DNA extraction method was employed.

On-column concentration of bisphenol A with one-step removal of humic acids in water.

An efficient extraction method for bisphenol A from environmental water including contaminants was developed using surface selective localization of functional group, on a polymeric separation device. The polymer utilized in this study was prepared through a kind of molecular imprinting technique, namely fragment imprinting effect utilizing a pseudo-template molecule (p-tert.-butylphenol) instead of bisphenol A. The concentration of bisphenol A onto the polymer device prepared, up to 1000 times concentration from environmental water including contaminants (humic acids), was achieved very easily with interesting exclusion effect for humic acids. The results obtained in this study suggest that molecular imprinting with the pseudo-template molecule is quite an effective way for selective concentration of the diluted target molecule from other contaminants including similar functional group with the target molecule.

Discrete spacers for photometric characterization of humic acids separated by capillary isotachophoresis.

A group of twenty discrete spacers suitable for photometric characterization of humic acids (HAs) after their isotachophoretic (ITP) separation at pH 10 was found. The spacers, inorganic and organic acids and amino acids of suitable acid-base and migration properties exhibiting no light absorption in the UV region of the light spectrum, made possible to perform this characterization in a sensitive spike mode of the ITP analysis. Using this approach a complex mixture of humic constituents present in a test HA preparation was separated into 22 fractions migrating in the interzonal boundary layers formed by the zones of discrete spacers and 21 fractions mixed with the zones of the spacers. A photometric monitoring of the fractions in the ITP stack at a 405 nm detection wavelength provided an adequate selectivity and sensitivity into the characterization. Relative sizes of the detected fractions of the test HA preparation ranged from 0.2-0.3 to 27.5% (based on the response of the phototometric detector at 405 nm). The fractions representing ca. 0.2-0.3% of the total peak area could be still quantified when 800 ng of the test preparation was loaded onto the ITP column. A typical repeatability of the total area of the detection signal corresponding to humic constituents in the ITP stack was ca. 2.5%. Repeatabilities of the peak areas of the fractions of the humic constituents defined by the spacers ranged from 2 to 6% for the fractions representing 1% or more of the total area and from 8 to 12% for those representing less than 1%. No marks of aggregations of the humic constituents were detected and reproducible ITP profiles (fingerprints) of the studied humic preparation were achieved under the developed working conditions.

Separation methods in the chemistry of humic substances.

Separation methods are widely used to isolate humic substances (HSs), to fractionate them before further investigation, and to obtain information about their structure and properties. Among the chromatographic methods, techniques based on a size-exclusion effect appear to be most useful, as they allow us to relate elution data to the molecular mass distribution of HSs. The limitations of this approach are discussed in this review. Gas chromatography with mass spectrometric detection is typically used to identify the products of pyrolysis or thermochemolysis of HSs; this technique is considered most important in the structural investigation of HSs. Electrophoretic methods (especially capillary zone electrophoresis) provide detailed characterization of HSs, but it is very difficult to relate the electrophoretic data to any specific subfraction, structure or properties of HSs. The electrophoretic patterns are often called "fingerprints" and can potentially be used for the identification and classification of HSs. This is limited, however, by the great diversity of the procedures employed and by the low degree of harmonization--no data on reproducibility and between-laboratory comparability are available. The same holds true, to a certain degree, for most methods utilized for the characterization of HSs. Separation methods play an important role in the examination of the interactions of HSs with heavy metals and other chemical pollutants. They allow us to determine binding constants and other data necessary to predict the mobility of chemical pollutants in the environment.

Evaluation of the permeation of peat substances through human skin in vitro.

Peat and various peat extracts have been successfully applied for a variety of clinical indications. Quite apart from the physico-thermal effects, new studies point towards the so-called "chemical effects" of peat containing substances. These effects include a stimulatory response of the spontaneous contractile activity (SCA) of smooth muscle (SM) tissue. The effects are, however, dependent on the possible permeability of pharmacologically active substances as naturally occurring ingredients of peat. Since peat is a mixture of various products it is necessary to examine the various peat types based upon their biological activity on SM tissue. In order to unequivocally prove the pharmacological activity of cutaneous peat treatment, in vitro permeation measurements of these actives across excised human skin can be used.HPLC analysis revealed that aqueous peat extracts contain up to 18 fractions of water-soluble compounds of fulvic and ulmic acids. These compounds have been found to have a stimulatory response on the contractile activity of SM tissue. In vitro diffusion studies showed that the permeability of these substances across human full thickness skin (thickness: 200 um(-1)) is highly selective and the resulting stimulatory activity is dependent on the permeated fraction. Especially, the HPLC fractions 7-11 and 14 are able to permeate human skin. Fractions 7-11 show a moderate stimulatory effect of SCA on SM for more than 90 min whereas fraction 14 shows the strongest stimulatory effect which was, however, suppressed after 87 min. These results show that the cutaneous therapy with peat treatment results in transcutaneaous permeation of biologically active fulvic and ulmic acid derivatives explaining the additional "chemical" effect of peat treatment in clinical practice.

Characterization of isolated fractions of dissolved organic matter from natural waters and a wastewater effluent.

Dissolved organic matter (DOM) was concentrated from natural waters and the effluent of a wastewater treatment plant using a portable reverse osmosis (RO) system. The humic acid (HA), fulvic acid (FA) and hydrophilic (HyI) fractions were isolated and purified by the XAD-8 resin combined with the cation exchange resin method. The FA fractions predominated in natural waters and accounted for 54-68% of the total amount of dissolved organic carbon (DOC), whereas the HA and HyI fractions constituted, respectively, 13-29 and 9-30% of the total DOC. The effluent of wastewater was almost devoid of HA and the HyI fraction exceeded FA. The elemental compositions of HA and FA were in the ranges typical for natural humic materials, but the HyI fractions did not exhibit humic character. 1H NMR spectra revealed that the HyI fractions were almost devoid of aromatic protons and the aliphatic region featured more sharp signals than HA and FA fractions, indicating that HyI fractions were consisted of more simple compounds and less complex mixtures. The aliphatic functional groups in these fractions of DOM samples followed the order HA < FA HyI. The rate of Cu complexation with the HyI fraction was faster than the rate with the HA or FA fraction of the Suwannee River DOM, implying that copper reacted with relatively weak ligands faster than with strong ligands.

Removal of organic matter from water by PAC/UF system.

The laboratory-scale ultrafiltration (UF) experiments were conducted to determine the effect of the presence of powdered activated carbon (PAC) on the UF process performance, in terms of flux decline and the possibilities of membranes cleaning during backwashing. Poly(vinylidene fluoride) membranes formed by the phase inversion technique were used in the UF experiments. A model solution was prepared as a mixture of humic acids (HA) and phenol in concentration of 10 and 1 mg l(-1), respectively. Commercial powdered activated carbons CWZ 11 and CWZ 30 (Gryfskand Sp. z o. o., Hajnówka, Poland) were used as the adsorbents. PAC dosage was in the range of 10-100 mg PAC l(-1). The process was carried out in the cross-flow system. It was found that PAC addition to the distilled water leads to a small drop in the permeate flux, regardless of PAC dose and its type. Although PAC particles are too large to block the membrane pores inside, they deposit on the membrane surface and partially can plug the surface pores. The experimental results demonstrate that the backwashing process applied in combined PAC/UF system was especially effective when PAC dosages were <20mg PAC l(-1). However, a similar permeate flux was maintained for all carbon dosages used and reached the value of about 1 m3 m(-2) d(-1). Moreover, no further drop in the permeate flux for PAC addition to the solution containing foulants (HA) was observed. Effectiveness of the removal of HA and phenol from the model solutions was also investigated. In the PAC/UF system HA were removed in about 90%, whereas the complete removal of phenol was achieved for PAC dosage equal to 100 mg l(-1).

An ultrasound assisted extraction of the available humic substance from marine sediments.

In this paper an ultrasound assisted procedure for the extraction of the bio-available fraction of humic substance in marine sediments is described. The proposed method is based on a preliminary 24 h 8 M HCl treatment already proposed in a previous paper [M. Mecozzi, E. Pietrantonio, M. Amici, Fres. Environ. Bull. 7 (1998) 605], followed by consecutive extractions by 0.5 M NaOH coupling to an ultrasonic treatment. The main advantages of the ultrasound procedure are the reduced times of extraction which take 30 min in contrast to the 24 h required by shaking method and the possibility to perform also the quantitative estimation of the extractable fraction of humic substance present in marine samples.

Description of characteristics of humic substances from different waste materials.

Humic acids (HAs) extracted from different organic wastes have been characterised by chemical methods. The chemical properties of HAs showed differences depending on the source from which they were obtained. The C content in HAs from organic wastes (41.1-63.2%) fluctuated around the C value in soil HA with the exception of composted bark and tobacco dust. Compared with soil HA, the N contents of HAs from sewage sludge and brewery sludge were found much higher than the others. E4:E6 ratios for HAs in organic wastes were generally greater than that for soil HA, which indicated a low degree of condensation and humification. The carboxyl and phenolic-OH group contents ranged 0.51-2.23 and 11.1-20.7 meq g(-1), respectively. High values of carboxyl and phenolic-OH contents indicated that these materials were still within early stages of humification.

Evaluation of the transformation of organic matter to humic substances in compost by coupling sec-page.

Humic acids (HAs) from soil and compost at the beginning (S0) and at the end of the stabilization process after 130 days (S130) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EMs) have been obtained from all samples and the HAFs weight content has been studied. A high degree of similarity in HAFs weight content between soil HA and a stabilized compost HAs130 has been observed. Such data seem to be reliable for monitoring the evolution of the compost organic matter to humic substances for their agricultural uses.

Oxidation of chlorophenols in soil at natural pH by catalyzed hydrogen peroxide: the effect of soil organic matter.

This investigation reports on the effects of soil organic matter (SOM) during the oxidation of chlorophenols with Fe2+-catalyzed H2O2 (Fenton oxidation) system. The soil pH was 7.1 and was not altered. Sorption experiments of soil pre-treated under various oxidation conditions were performed. Concentrations of organic matter in the liquid phase and soil before and after oxidation were analyzed. The results were correlated to the observation in batch Fenton oxidation tests. They showed that the oxidation of chlorophenols at natural soil pH depended on the dose of H2O2 and Fe2+. The soil organic content did not vary significantly after various Fenton treatments, while the sorption of chlorophenols was 10-25% less by the oxidation. The concentration of chlorophenols in the liquid phase exhibited a "decrease and rebound" phenomenon in the batch Fenton oxidation tests. It appeared that the oxidation of SOM resulted in the release of sorbed chlorophenols which were then oxidized by the excess H2O2. An "oxidation-desorption-oxidation" scheme was proposed to describe one of the interaction mechanisms among the oxidant, SOM, and chlorophenols during oxidation.

Photoinductive efficiency of soil extracted humic and fulvic acids.

Humic and fulvic acids extracted from soils of different genesis were investigated for their ability to photoinduce the transformation of fenuron (2 x 10(-4) mol(-1)) at 365 nm. The ratio of the initial rate of fenuron consumption over the rate of light absorption by humic substances was found to be higher for fulvic acids (range 2.0 x 10(-3) to 9.0 x 10(-5)) than for humic acids (range 1.7 x 10(-4) to - 3.6 x 10(-5)). Within the FAs population, this ratio decreased as the specific absorption coefficient at 365 nm increased. It seems therefore that most of 365-nm absorbing components have no photoinductive activity and even reduce that of photoinductive chromophores.

Electron paramagnetic resonance (EPR) studies on stable and transient radicals in humic acids from compost, soil, peat and brown coal.

Quantitative EPR method was applied to characterise four types of humic acids (HA) derived from composts, soil, peat and soft brown coal. For each sample of HA the level of native (indigenous) radicals was estimated. Interactions of the HA with various gaseous agents and organic solvents were investigated. Strong effects of gaseous ammonia and aliphatic amines on spin concentration enhancement were observed; additionally, higher values of g-value were found to be associated with the formed 'transient' radicals. Correlation of copper(II) ions uptake by different HA with effect diminishing primary spin concentration was established. It was recognised that the radical centres, which are enhanced by 'ammonia effect' are quenched in the formed HA-Cu(II) complexes. The both opposite effects are competitive from each other, where 'copper(II) quenching effect' prevails. Reaction of nitrogen dioxide with the humic acids was also examined. The presence of diketones and/or other compounds with active methylene group results in formation of the iminoxy radicals; these radicals are immobilized in the solid (macromolecular) matrix of the humic acids.

Potentiometric-spectroscopic evaluation of metal-ion complexes by humic fractions extracted from corn tissue.

Humic fraction (HF) functional group-type and content are expected to depend on molecular size, which in turn, is expected to influence formation of heavy-metal complexes. In this study, corn (Zea mays L.) stalks and leaves were decomposed for an 8-month period to produce water-soluble humic substances. These substances were separated into three water-soluble fractions, HF1, HF2 and HF3, from highest to lowest relative molecular size. Functional group determination showed that carboxylic, and phenolic OH acidity increased as relative molecular size of humic fractions decreased. We also observed decreasing C/O ratios from larger to smaller corn tissue-derived humic fractions, whereas N/C and H/C ratios remained relatively unaffected. Furthermore, using potentiometric titration and FTIR spectroscopy we studied formation of Ca2+-, Cd2+-, and Cu2+-humic fraction complexes and how they were affected by pH and molecular size. We determined that metal-humic complexes exhibited at least two types of functional group-sites with respect to Ca2+, Cd2+, and Cu2+ complexation. Strength of metal-ion humic complexes followed the order Cu2+ > Cd2+ > Ca2+ and was affected by pH, especially for low affinity sites. Carboxylic groups were most likely the dominant group-sites involved in complex formation. Magnitude of the metal-humic formation constants in the logarithmic form at the lowest equilibrium metal-ion concentration, under the various pH values tested, varied from 5.39 to 5.90 for Ca2+, 5.36 to 6.01 for Cd2+, and 6.93 to 7.71 for Cu2+. Furthermore, the formation constants appeared to be positively influenced by decreasing molecular size of water-soluble humic fraction, and increasing pH. However, our molecular spectra showed that the pKa of corn humic fractions increased with decreasing relative molecular size and that Cu2+ was more covalently bonded by humic fractions than were Ca2+ and Cd2+, and the nature of the covalent bond character was independent of pH.

Effect of solution parameters on the removal efficiency of humic substances by a reuse material.

This paper provides an overview of the ability of polypropylene (PPL), a synthetic polymeric adsorbent made from reused plastic material. The removal of humic substances (HS) in a solution pretreated with dedoxyltrimethylammoniumbromide (DDTMAB), a cationic quaternary ammonium compound (QAC), was studied under various conditions. The removal of HS was achieved in its solute complexed form but also as suspended solids. The conditions of the HS solution, which fluctuation was suspected to have an impact on the performance of the adsorbent, were tested. The parameters investigated included the pH, the initial concentration of the surfactant, and the presence of suspended solids in solution. The results of the laboratory scale experiments showed that PPL removal efficiency of HS is strongly pH dependant with higher removal achieved at pH greater than 7. However, the shift for pH above 7 induced an increase in the optimum doses of DDTMAB as more and more deprotonated HS become available to react with DDTMAB. Ionic strength did have the opposite effect: the increase of ionic strength by addition of NaCl decreased PPL removal of HS.

Photocatalytic oxidation technology for humic acid removal using a nano-structured TiO2/Fe2O3 catalyst.

A novel TiO2 coated haematite photocatalyst was prepared and used for removal of colored humic acids from wastewater in an UV bubble photocatalytic reactor. XRD analysis confirmed that nano-size anatase crystals of TiO2 were formed after calcination at 480 degrees C. SEM results revealed that nano-size particles of TiO2 were uniformly coated on the surface of Fe2O3 to form a bulk of nano-structured photocatalyst Fe2O3/TiO2. The porous catalyst had a BET surface area of 168 m2/g. Both the color and total organic carbon (TOC) conversion versus the residence time were measured at various conditions. The effects of pH value, catalyst loaded, initial humic acid concentration and reaction temperature on conversion were monitored. The experimental results proved that the photocatalytic oxidation process was not temperature sensitive and the optimum catalyst loading was found to be 0.4 g/l. Degradation and decolorization of humic acids have higher efficiency in acidic medium and at low initial humic acid concentration. The new catalyst was effective in removing TOC at 61.58% and color400 at 93.25% at 180 minutes illumination time and for 20 mg/l neutral humic acid aqueous solution. The kinetic analysis showed thatthe rate of photocatalytic degradation of humic acids obeyed the first order reaction kinetics.

[Isolation and characterization of humin-like substances produced by wood-degrading fungi causing white rot]. / Poluchenie i kharakteristika guminopodobnykh veshchestv, sinteziruemykh derevorazrushaiushchimi gribami, vozbuditeliami "beloi gnili".

Three samples of high-molecular-weight humin-like substances were obtained by solid-phase cultivation of Coriolus hirsutus and/or Cerrena maxima on oat straw. The yield of humin-like substances amounted to 1.38-2.26% of the weight of the plant substrate consumed. These substances, produced both by individual and mixed cultures of the basidiomycetes, were shown to be similar in their structure and physicochemical properties. According to the data of IR and 13C-NMR spectroscopy, the substances contained aromatic fragments and were close to soil humic acids. Studies of the dynamics of laccase production suggested that the humin-like substances were produced bia direct degradation of lignin macromolecules with direct involvement of extracellular laccase.