Nitrite (not free nitrous acid) is the main inhibitor of the anammox process at common pH conditions.

Nitrite is a substrate but also an inhibitor of anaerobic ammonium oxidation (anammox).There is currently no consensus on whether ionized nitrite (INi) or free nitrous acid (FNA) is the actual inhibitor of the process. The inhibition by INi and FNA on the anammox process has been analysed using a wide range of INi and FNA concentrations and by altering the pH and total nitrite conditions. The inhibitory impacts of both species were quantified through a rational inhibition equation, considering INi and FNA as substrate inhibitor and non-competitive inhibitor, respectively. Inhibitory constants were calculated with strong statistical support as 561 mg INi-N l(-1) and 0.117 mg FNA-N l(-1). Based on the model, INi is the main inhibiting species of the anammox process at pH > 7.1, which are the most common conditions occurring in field applications of anammox.

High pH (and not free ammonia) is responsible for Anammox inhibition in mildly alkaline solutions with excess of ammonium.

Ammonium is a substrate of the anaerobic ammonium oxidation (Anammox) process but it has been suggested as a substrate-inhibitor because of the action of its unionized form, free ammonia. High pH of the medium is also an important limiting factor of the Anammox bacteria. Both effects are difficult to discriminate. In this work the inhibitory effects of high pH, total ammonia (TA) and NH3 on the Anammox process were investigated simultaneously. Results confirmed that TA caused no inhibition and high pH is a much more important inhibiting factor than NH3 in mildly alkaline conditions, based on a multi-factorial analysis. Values of pH higher than 7.6 caused Anammox inhibition >10 % and should be avoided during the application of the Anammox process in practice.

Kinetics and thermodynamics of anaerobic ammonium oxidation process using Brocadia spp. dominated mixed cultures.

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial process commonly applied to treat ammonium pollution in effluents with low organic carbon content. Modeling anammox processes is important for simulating and controlling full-scale plants. In this study, the anammox process was simulated using three models, and substrate and growth parameters obtained by different research groups. Two Brocadia spp.-dominated mixed cultures, one granular and the other flocculent, were used for this purpose. A very good correlation between experimental data using both sludges and model predictions was achieved by one of the models, obtaining correlation coefficients higher than 0.997. Other models and stoichiometric equations tested were unable to predict the anammox kinetics and stoichiometry. Furthermore, the thermodynamic behavior of the two mixed cultures was compared through the determination of the energy of activation of the anammox conversion at temperatures ranging from 9 to 40 °C. Optimum temperature for anammox activity was established at 30-35 °C in both cases. The energy of activation values calculated for granular sludge and flocculent sludge were 64 and 124 kJ mol(-1), respectively.

Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis.

Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein A (OspA) of Borrelia burgdorferi, the agent of Lyme disease, and the major histocompatibility complex class II allele DRB1*0401. The immunodominant epitope of OspA for T helper cells was identified. A homology search revealed a peptide from human leukocyte function-associated antigen-1 (hLFA-1) as a candidate autoantigen. Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, hLFA-1, and their highly related peptide epitopes. Identification of the initiating bacterial antigen and a cross-reactive autoantigen may provide a model for development of autoimmune disease.

Compared microbiology of granular sludge under autotrophic, mixotrophic and heterotrophic denitrification conditions.

Water contamination by nitrate is a wideworld extended phenomena. Biological autotrophic denitrification has a real potential to face this problem and presents less drawbacks than the most extended heterotrophic denitrification. Three bench-scale UASB reactors were operated under autotrophic (R1, H2S as electron donor), mixotrophic (R2, H2S plus p-cresol as electron donors) and heterotrophic (R3, p-cresol as electron donor) conditions using nitrate as terminal electron acceptor. 16S rDNA genetic libraries were built up to compare their microbial biodiversity. Six different bacteria phyla and three archaeal classes were observed. Proteobacteria was the main phyla in all reactors standing out the presence of denitrifiers. Microorganisms similar to Thiobacillus denitrificans and Acidovorax sp. performed the autotrophic denitification. These OTUs were displaced by chemoheterotrophic denitrifiers, especially by Limnobacter-like and Ottowia-like OTUs. Other phyla were Bacteroidetes, Chloroflexi, Firmicutes and Actinobacteria that--as well as Archaea members--were implicated in the degradation of organic matter, as substrate added as coming from endogenous sludge decay under autotrophic conditions. Archaea diversity remained low in all the reactors being Methanosaeta concilii the most abundant one.

Direct residue analysis of systemic insecticides and some of their relevant metabolites in wines by liquid chromatography - mass spectrometry.

A direct large volume injection (DI-LVI) high performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the quantitative determination of 16 systemic insecticides and their main plant metabolites. The assays were conducted on commercial red and white wines made from grapes grown in major wine-producing regions nationally and internationally. Using a 1:20 dilution and an injection volume of 800µL, a limit of quantitation (LOQ) of 1µgL-1 for all analytes was achieved. Matrix-matched standards (MM) were used for accurate quantitation. Imidacloprid (IMI) and methoxyfenozide (MET) were the most frequently detected parent insecticides in the wines reaching concentrations of 1-132µgL-1. Two important plant metabolites imidacloprid-olefin (IMI-OLE) and spirotetramat-enol (SPT-EN) were found at higher concentrations. In five samples SPT-EN was detected in the mgL-1 range with a maximum concentration of 16.3mgL-1 measured in a conventional white wine sample. Most "organic" wines contained no detectable or low insecticide residues, except for one sample, which showed the highest IMI (14.7µgL-1) and IMI-OLE (331µgL-1) concentrations. Considering the maximum residue limit (MRL) definition for the different insecticides, three "conventional" wine samples were non-compliant for SPT. This study highlights the importance to determine both parent and metabolite forms of systemic insecticides in the finished product.

Biological treatment of heavy metals in acid mine drainage using sulfate reducing bioreactors.

The uncontrolled release of acid mine drainage (AMD) from abandoned mines and tailing piles threatens water resources in many sites worldwide. AMD introduces elevated concentrations of sulfate ions and dissolved heavy metals as well as high acidity levels to groundwater and receiving surface water. Anaerobic biological processes relying on the activity of sulfate reducing bacteria are being considered for the treatment of AMD and other heavy metal containing effluents. Biogenic sulfides form insoluble complexes with heavy metals resulting in their precipitation. The objective of this study was to investigate the remediation of AMD in sulfate reducing bioreactors inoculated with anaerobic granular sludge and fed with an influent containing ethanol. Biological treatment of an acidic (pH 4.0) synthetic AMD containing high concentrations of heavy metals (100 mg Cu(2+)l(-1); 10 mg Ni(2+)l(-1), 10 mg Zn(2+)l(-1)) increased the effluent pH level to 7.0-7.2 and resulted in metal removal efficiencies exceeding 99.2%. The highest metal precipitation rates attained for Cu, Ni and Zn averaged 92.5, 14.6 and 15.8 mg metal l(-1) of reactor d(-1). The results of this work demonstrate that an ethanol-fed sulfidogenic reactor was highly effective to remove heavy metal contamination and neutralized the acidity of the synthetic wastewater.

Microbiological and structural aspects of granular sludge from autotrophic denitrifying reactors.

Denitrification is applied in the tertiary treatment of wastewater to reduce N-pollutants. Fluorescence in situ hybridisation (FISH), CARD (catalyzed reporter deposition)-FISH, cloning, and scanning electron microscopy (SEM) were applied to follow the evolution of the microbial composition and structure of granular sludge in autotrophic denitrifying bioreactors fed with nitrate and thiosulfate. With this goal, FISH oligonucleotide probes for the autotrophic denitrifiers, Thiobacillus denitrificans and Thiomicrospira denitrificans, were designed and their utility tested. CARD-FISH and cloning data showed that bacterial diversity changed with bioreactor operation time. After 110 days of operation, the abundance of Thiobacillus denitrificans cells increased considerably: from 1 to 35% of total DAPI-stained cells and from no isolated clones to 30% of the total positives clones. This fact strongly suggests that this microorganism played a dominant role in the autotrophic denitrification. The Archaeal diversity remained almost unchanged and it was mainly represented by Methanosaeta soehngenii. Scanning electron microscopy results indicated a considerable loss in the integrity of the sludge granules during the operation, with risk of sludge buoyancy.

The role of sulphate reduction on the reductive S decolorization of the azo dye reactive orange 14.

The aim of this study was to investigate the impact of a broad range of sulphate concentrations (0-10g SO4(-2) L(-1)) on the reduction of an azo dye (reactive orange 14 (RO14)) by an anaerobic sludge. An increase in the sulphate concentration generally stimulated the reduction of RO14 by sludge incubations supplemented with glucose, acetate or propionate as electron donor. Sulphate and azo dye reductions took place simultaneously in all incubations. However, there was a decrease on the rate of decolorization when sulphate was supplied at 10g SO4(-2) L(-1). Abiotic incubations at different sulphide concentrations (0-2.5 g sulphide L(-1)) promoted very poor reduction of RO14. However, addition of riboflavin (20 microM), as a redox mediator, accelerated the reduction of RO14 up to 44-fold compared to a control lacking the catalyst. Our results indicate that sulphate-reduction may significantly contribute to the reduction of azo dyes both by biological mechanisms and by abiotic reductions implicating sulphide as an electron donor. The contribution of abiotic decolorization by sulphide, however, was only significant when a proper redox mediator was included. Our results also revealed that sulphate-reduction can out-compete with azo reduction at high sulphate concentrations leading to a poor decolorising performance when no sufficient reducing capacity is available.

Catalytic effects of different redox mediators on the reductive decolorization of azo dyes.

The catalytic effects of redox mediators, with distinct standard redox potentials (E'0), were evaluated on the first-order rate constant of decolorization (Kd) of recalcitrant azo dyes by an anaerobic granular sludge. The dyes studied included mono-azo (Reactive Orange 14, RO14), di-azo (Direct Blue 53, DB53), and tri-azo (Direct Blue 71, DB71) compounds. Toxicity and auto-catalytic aspects seemed to play a role in determining the rate of decolorization. Addition of riboflavin, anthraquinone-2,6-disulphonate (AQDS) or lawsone as a redox mediator, increased the Kd value for all dyes studied, although their impact varied in every case. Kd values were increased from 1.1-fold up to 3.8-fold depending on the redox mediator applied. Moreover, catalysts with moderately similar E'0 value caused distinct stimulation on the rate of decolorization. These results should be considered for selecting the proper redox mediator to be applied during the anaerobic treatment of textile wastewaters and effluents containing electron-withdrawing pollutants, such as nitro-aromatic and polychlorinated compounds.

Biotransformation and biodegradation of N-substituted aromatics in methanogenic granular sludge.

N-Substituted aromatic compounds are environmental contaminants associated with the production and use of dyes, explosives, pesticides and pharmaceuticals. In this article, we examine the potential of anaerobic granular sludge from anaerobic treatment systems towards the detoxification, transformation, and mineralization of nitroaromatic and azo compounds. Nitroaromatics and azo dyes with strong electron withdrawing are highly inhibitory to acetoclastic methanogenic bacteria. However, nitro and azo substituted aromatics are readily reductively detoxified in methanogenic consortia to their respective aromatic amines, which are several orders of magnitude less toxic. This reductive detoxification has allowed the successful operation of anaerobic reactors for the treatment of highly toxic aromatic compounds. In the course of the experiments it was discovered that some aromatic amines were mineralized. These results indicate that some N-substituted aromatic compounds can be completely mineralized and serve as a carbon and energy source for anaerobic bacteria.

Comparison of chemo-, hetero- and mixotrophic denitrification in laboratory-scale UASBs.

This study investigated removal of sulfide and p-cresol linked to denitrification in laboratory-scale upflow anaerobic granular sludge bed (UASB) bioreactors. Three parallel denitrification bioreactors were run for nine months, which were operated under chemolithoautotrophic conditions (i.e., using sulfide as electron donor -e-donor- and bicarbonate as C source); heterotrophic conditions (with p-cresol as e-donor and C source), and mixotrophic conditions (utilizing both sulfide and p-cresol as electron donors), respectively. The average hydraulic retention time and nitrate load applied to the bioreactors was 13.4 h and 1,240 mg N-NO3/l/day, respectively. The nitrate removal efficiency was 89, 95 and 99%, respectively, for the chemo-, hetero- and mixotrophic reactors. The mixotrophic UASB removed both sulfide and p-cresol almost completely, indicating that simultaneous removal of the inorganic and organic e-donors occurred. Nitrite was seldom observed as an intermediate. N2O gas and methane concentrations in the biogas were also negligible. These results indicate that mixotrophic denitrification with phenols and sulfide is feasible in high rate UASB reactors.

2-Chloro-1,4-dimethoxybenzene as a mediator of lignin peroxidase catalyzed oxidations.

Poly R478, 4-methoxymandelic acid and oxalic acid were oxidized by lignin peroxidase (LiP) in the presence of the fungal metabolite 2-chloro-1,4-dimethoxybenzene (2Cl-14DMB), whereas no oxidation occurred in the absence of 2Cl-14DMB. These substrates clearly inhibited the consumption of 2Cl-14DMB and the formation of 2-chloro-1,4-benzoquinone from 2Cl-14DMB by LiP. The results suggest that 2Cl-14DMB can replace the function of veratryl alcohol as a redox mediator in lignin peroxidase catalyzed oxidations.

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.

Purification and characterization of two lignin peroxidase isozymes produced by Bjerkandera sp. strain BOS55.

The white-rot fungus Bjerkandera sp. strain BOS55 excretes at least seven lignin peroxidase (LiP) isozymes. Two of these, LiP-2 and LiP-5 (molecular weight 40-42 kDa), were purified to homogeneity. Both isozymes had the same N-terminal amino acid sequence which showed strong homology with LiP isozymes produced by other white-rot fungi. The kinetics of both isozymes were similar. LiP-5 oxidized veratryl alcohol optimally only in the presence of H2O2 near pH 3.0 (16.7 U/mg) and LiP-2 did this below pH 2.5 (33.8 U/mg). Also at normal physiological pHs for fungal growth (pH 5.0-6.5) both isozymes were still active. Further characterization of LiP-2 and LiP-5 revealed that the Km for H2O2 strongly decreased with increasing pH. As a result of this the catalytic efficiency (TN/Km) calculated on the basis of the Km for H2O2 in the oxidation of veratryl alcohol was constant over wide pH range.

Calculated ionisation potentials determine the oxidation of vanillin precursors by lignin peroxidase.

In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O-glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O-acetyl esters and glucosides of isoeugenol and coniferyl alcohol would be potential LiP substrates. Both O-acetyl esters were tested and were shown to be converted to O-acetyl vanillin in molar yields of 51.8 and 2.3%, respectively.

De-novo biosynthesis of chlorinated aromatics by the white-rot fungus Bjerkandera sp. BOS55. Formation of 3-chloro-anisaldehyde from glucose.

The white-rot fungus Bjerkandera sp. BOS55 produced de-novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi, other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde and a yet unknown compound containing two chlorine atoms. Additionally GC/MS analysis revealed the production of small amounts of anisyl alcohol and 3-chloro-anisyl alcohol. After 14 days, the extracellular fluid of Bjerkandera BOS55 contained 100 microM veratraldehyde and 50 microM 3-chloro-anisaldehyde. This is the first report of de-novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. Anisaldehyde and 3-chloro-anisaldehyde were also produced by Bjerkandera adusta but not by Phanerochaete chrysosporium.

Partially oxidized polycyclic aromatic hydrocarbons show an increased bioavailability and biodegradability.

Polycyclic aromatic hydrocarbons have a low water solubility and tend to adsorb on soil particles, which both result in slow bioremediation processes. Many microorganisms, known for their ability to degrade polycyclic aromatic hydrocarbons, only partially oxidize these compounds. White rot fungi, for instance, convert polycyclic aromatic hydrocarbons to more water soluble and bioavailable products. Polycyclic aromatic hydrocarbon metabolites were more readily mineralized by natural mixed bacterial cultures, like activated sludge and soil, than the parent polycyclic aromatic hydrocarbon compounds. These results suggest that sequential breakdown by white rot fungi followed by indigenous bacteria leads to an effective polycyclic aromatic hydrocarbon bioremediation process.

Coupling chemical derivatization reactions with supercritical fluid extraction.

Coupling chemical derivatization reactions with supercritical fluid extraction for the determination of trace levels of organic and organometallic compounds in liquid and solid matrices is reviewed. Derivatization is used to increase the solubility of analytes in supercritical carbon dioxide, to increase analyte volatility for gas chromatographic analysis and to integrate sample preparation steps in order to reduce analysis time and costs. Reactions that are covered in this review derivatize analytes possessing carboxyl, hydroxyl, sulfonic and amino groups to their alkyl, acyl and silyl derivatives. Derivatization is also used to derivatize active matrix sites to facilitate the release of analytes. Approaches used to couple derivatization with supercritical fluid extraction include reactions conducted prior to extraction, under in-site supercritical fluid conditions, or off-line under injection port conditions. Examples of applications are given for organic and organometallic compounds in environmental, pharmaceutical and agricultural product samples.

Determination of benzylsuccinic acid in gasoline-contaminated groundwater by solid-phase extraction coupled with gas chromatography-mass spectrometry.

Benzylsuccinic acid (BSA) and methylbenzylsuccinic acid (methyl-BSA) are unambiguous biotransformation products resulting from anaerobic toluene and xylene biodegradation, respectively. A solid-phase extraction method based on polystyrene-divinylbenzene sorbent was developed for the quantitative BSA determination in groundwater samples as an alternative to liquid-liquid extraction. Gas chromatography coupled with mass spectrometry was used for separation and detection. The recovery from spiked 11 groundwater samples was 88 to 100%. The precision of the method, indicated by the relative standard deviation, was +/- 4% and the method detection limit was 0.2 microg/l. The concentration of BSA and methyl-BSA in groundwater samples from anaerobic BTEX (benzene, toluene, ethylbenzene and xylenes)-contaminated sites ranged from below the detection limit (3 microg/l) to 155 microg/l.