Effect of carbon source and metal toxicity for potential acid mine drainage (AMD) treatment with an anaerobic sludge using sulfate-reduction.

This study compares sulfate-reduction performance in an anaerobic sludge with different carbon sources (ethanol, acetate, and glucose). Also, the toxic effect of copper was evaluated to assess its feasibility for possible acid mine drainage (AMD) treatment. Serological bottles with 1.5 g VSS/L and 150 mL of basal medium (0.67 g COD/g SO42- at a 7-8 pH) were used to determine the percentage of electron equivalents, maximum specific methanogenic (SMA), and sulfide generation activities (SGA). The copper effect was evaluated in a previously activated sludge in batch bioassays containing different concentrations of copper (0-50 mg/L), 3 gVSS/L, and 150 mL of basal medium (0.67 g COD/g SO42-). Carbon source bioassays with glucose obtained the best results in terms of the SGA (1.73 ± 0.34 mg S2-/g VSS•d) and SMA (10.41 mg COD-CH4/g VSS•d). The electron flow in the presence of glucose also indicated that 21.29 ± 5.2% of the metabolic activity of the sludge was directed towards sulfidogenesis. Copper toxicity bioassays indicated that a considerable decline in metabolic activity occurs above 10 mg/L. The 20%IC, 50%IC, and 80%IC were 4.5, 14.94, and 35.31 mg Cu/L. Compared to the other carbon sources tested, glucose proved to be a suitable electron donor since it favors sulfidogenesis. Finally, copper concentrations above 15 mg/L inhibited metabolic activity in the toxicity bioassays.

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.

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.

Improved aerobic biodegradation of abietic acid in ECF bleached kraft mill effluent due to biomass adaptation.

Kraft pulp mill effluents contain elevated concentrations of resin acids, chiefly abietic and dehydroabietic acid, and other lipophilic wood constituents. Resin acids, if not efficiently removed during wastewater treatment, can be responsible for chronic toxicity in aquatic systems. The objective of this study was to investigate the biological removal of abietic acid (AbA) during the treatment of elemental chlorine free (ECF) kraft mill effluents in aerobic lagoons and to assess its improvement with time as a result of biomass adaptation. Under these conditions, the average removal efficiencies of AbA and BOD(5) attained in the aerobic lagoon were high and exceeded 80% and 95%, respectively. Microbial inhibition of non-acclimated and acclimated biomass by AbA was not detected in batch bioassays. Kinetic studies showed that the K(s) and V(m) values equalled 76.7 mg AbA/l and 0.011 l/h, respectively, for the non-acclimated biomass, and 1678 mg AbA/l and 0.13 l/h, respectively, for the acclimated biomass.

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.

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.

Role of Organic Acids in the Manganese-Independent Biobleaching System of Bjerkandera sp. Strain BOS55

Bjerkandera sp. strain BOS55 is a white rot fungus that can bleach EDTA-extracted eucalyptus oxygen-delignified kraft pulp (OKP) without any requirement for manganese. Under manganese-free conditions, additions of simple physiological organic acids (e.g., glycolate, glyoxylate, oxalate, and others) at 1 to 5 mM stimulated brightness gains and pulp delignification two- to threefold compared to results for control cultures not receiving acids. The role of the organic acids in improving the manganese-independent biobleaching was shown not to be due to pH-buffering effects. Instead, the stimulation was attributed to enhanced production of manganese peroxidase (MnP) and lignin peroxidase (LiP) as well as increased physiological concentrations of veratryl alcohol and oxalate. These factors contributed to greatly improved production of superoxide anion radicals, which may have accounted for the more extensive biobleaching. Optimum biobleaching corresponded most to the production of MnP. These results suggest that MnP from Bjerkandera is purposefully produced in the absence of manganese and can possibly function independently of manganese in OKP delignification. LiP probably also contributed to OKP delignification when it was present.

Elimination and detoxification of softwood extractives by white-rot fungi.

The ability of several white-rot fungal strains to remove and detoxify acetone extractives (pitch or resin) in Scots pine sapwood was investigated in stationary laboratory batch assays. Fungal pretreatment provided up to 62% total pitch reduction and significant decreases in pitch toxicity. The best strains were Bjerkandera sp. strain Stereum hirsutum and Trametes versicolor that eliminated over 93% of the problematic triglyceride fraction and 58-87% of other lipophilic extractive classes in only 2 weeks. Fungal removal of the wood extractives was accompanied by a 7.4-16.9-fold decrease in their inhibitory effect, as determined in the Microtox bioassay. Wood pretreatment by Bjerkandera sp. and T. versicolor caused limited losses of woody mass (less than 4% in 4 weeks); whereas S. hirsutum led to somewhat higher mass losses (7% in 4 weeks). These results indicate the potential of white rot fungi to control pitch deposition problems in pulping and to reduce the aquatic toxicity caused by naturally-occurring lipophilic extractives in forest industry effluents.

Oxidation of lignin in eucalyptus kraft pulp by manganese peroxidase from Bjerkandera sp. strain BOS55.

The white rot fungus Bjerkandera sp. strain BOS55 was shown in previous studies to cause high levels of kraft pulp bleaching and delignification under culture conditions in which manganese peroxidase (MnP) occurs as the dominant oxidative enzyme. In this study, the MnP of Bjerkadera was isolated and tested in vitro with eucalyptus oxygen-delignified kraft pulp (ODKP) based on measuring the reduction in kappa number as an indicator of lignin oxidation. The MnP preparation applied at 60 U/g pulp for 6 h caused a significant decrease of 11-13% in the kappa number in the ODKP under optimal conditions compared to parallel-incubated controls lacking enzyme. The effects of MnP dosage, Mn2+ concentration, organic acid buffer selection, pH and H2O2 addition were evaluated. The optimal Mn2+ concentration range for lignin oxidation in ODKP was 100-500 microM. In the presence of low oxalate concentrations (0.3-2 mM) the Bjerkandera MnP also significantly reduced the kappa number of ODKP by 6% without any Mn. This observation is in agreement with the fact that purified Bjerkandera MnP has Mn-independent activities. Under incubation conditions with added Mn2+, buffers composed of metal-complexing organic acids provided two-fold better kappa number reductions compared to the inert acetic acid. The optimal H2O2 dosage was found to be 0.017 micromol/min ml when added as semi-continuous pulses (every 30 min) or 0.2 micromol/min ml when generated continuously by glucose oxidase. Excess H2O2 caused severe inactivation of MnP during the incubations. Factors that improved the turnover of the enzyme, such as Mn2+ and metal-chelating acids, stabilized MnP against rapid inactivation.

Kinetic characterization of Brocadia spp.-dominated anammox cultures.

In this study, kinetic analyses were conducted for two Brocadia-dominated enrichment cultures, granular and flocculent, retrieved from lab-scale anaerobic ammonium oxidation (anammox) reactors. Substrate KS ranged from 0.35 to 0.69 mMN and VSmax ranged from 0.67 to 0.88 mmol Ng(-1)VSSh(-1). The model respected the experimentally measured stoichiometry of N-compounds, serving as an independent validation. Growth kinetics of the flocculent sludge was also studied, which indicates a µmax of 0.0984 d(-1) and a YX/S of 0.105 mol C-biomass mol(-1)NH4(+). The flocculent enrichment culture was used to determine the stoichiometric equation. The kinetic parameters of the Brocadia spp. cultures measured here can be used for optimizing applications of the anammox process.

Ab initio study of the structural, electronic, and thermodynamic properties of linear perfluorooctane sulfonate (PFOS) and its branched isomers.

Structural, electronic, and thermodynamic properties of linear perfluorooctane sulfonate (PFOS) and its trifluoromethyl-branched isomers (i.e. 1-CF(3)- to 6-CF(3)-PFOS) were theoretically studied by means of ab initio density functional theory (DFT) calculations with the B3LYP functional and a 6-31++G(d,p) basis set. The anionic form of linear PFOS and its trifluromethyl-branched isomers were considered for the initial construction of the computational models; subsequently, H(+), Li(+), and Na(+) were added as counter-ions to study their effect on the properties under investigation. Insignificant changes with respect to the anions were observed in the structure of both the protonated and salt forms due to the presence of these counter-ions. However, important differences in the electrostatic potential maps as well as HOMO and LUMO molecular orbitals were observed for the various forms of PFOS. The linear and branched PFOS ions were identified as the most suitable compounds for interacting with charged species. Furthermore, in the linear anion, it was observed that the LUMO orbital is diffused along the whole fluoro-carbon chain, whereas it is localized to the region close to the ternary carbon in the 4-CF(3)-PFOS, 5-CF(3)-PFOS, and 6-CF(3)-PFOS isomers. The higher accessibility of the LUMO orbital in these branched anions suggests that they have a higher probability of reacting with free radicals when compared with the linear counterpart. This behavior is reflected in the experimental observation that only the branched PFOS isomers were susceptible to reductive defluorination by reduced vitamin B(12) as we previously reported. The relative stability of the linear and branched PFOS in their different forms computed by comparing their calculated Gibbs free energy showed that 1-CF(3)-, 6-CF(3)-, and linear PFOS are the most favorable structures from a thermodynamic point of view.

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Continuous anaerobic treatment of autoxidized bark extracts in laboratory-scale columns.

Debarking wastewaters of the forest industry contain high concentrations of tannins that are inhibitory to methane bacteria. The tannins can be polymerized to nontoxic colored compounds by the applications of an autoxidation pretreatment, enabling the anaerobic treatment of easily biodegradable components in the wastewater. The continuous anaerobic treatment of untreated and autoxidized pine bark extract was studied in laboratory-scale columns packed with a granular sludge bed. The autoxidation doubled the conversion efficiency of bark extract COD to methane (from 19 to 40%). After 5 months of operation, anaerobic treatment of the autoxidized extracts was feasible at high influent concentrations (14 g COD/L) and loading rates (26 g biodegradable COD/L . d) with 98% elimination of the biodegradable fraction. The detoxification pretreatment polymerized the toxic tannins to poorly biodegradable high molecular weight tannins and humic compounds which were not eliminated during anaerobic treatment. Although the original tannins of the untreated extract were eliminated by 60%, they were not biodegraded to volatile fatty acids and methane but instead were transformed to phenolic degradation intermediates (phenol, p-cresol, 3-phenyl-propionate, and carboxycyclohexane). Therefore, the autoxidation pretreatment did not decrease the content of readily biodegradable substrates which accounted for 53% of the extract COD. The recalcitrant COD expected in the effluents of reactors treating autoxidized debarking waste-water can be effectively separated by calcium precipitation prior to anaerobic treatment.

Manganese Is Not Required for Biobleaching of Oxygen-Delignified Kraft Pulp by the White Rot Fungus Bjerkandera sp. Strain BOS55.

The white rot fungus Bjerkandera sp. strain BOS55 extensively delignified and bleached oxygen-delignified eucalyptus kraft pulp handsheets. Biologically mediated brightness gains of up to 14 ISO (International Standards Organization units) were obtained, providing high final brightness values of up to 80% ISO. In nitrogen-limited cultures (2.2 mM N), manganese (Mn) greatly improved manganese-dependent peroxidase (MnP) production. However, the biobleaching was not affected by the Mn nutrient regimen, ranging from 1,000 (mu)M added Mn to below the detection limit of 0.26 (mu)M Mn in EDTA-extracted pulp medium. The lowest Mn concentration tested was at least several orders of magnitude lower than the K(infm) known for MnP. Consequently, it was concluded that Mn is not required for biobleaching in Bjerkandera sp. strain BOS55. Nonetheless, fast protein liquid chromatography profiles indicated that MnP was the predominant oxidative enzyme produced even under culture conditions in the near absence of manganese. High nitrogen (22 mM N) and exogenous veratryl alcohol (2 mM) repressed biobleaching in Mn-deficient but not in Mn-sufficient culture medium. No correlation was observed between the titers of extracellular peroxidases and the biobleaching. However, the decolorization rate of the polyaromatic dye Poly R-478 was moderately correlated to the biobleaching under a wide range of Mn and N nutrient regimens.

Infrared spectroscopy analysis of hemp (Cannabis sativa) after selective delignification by Bjerkandera sp. at different nitrogen levels.

Fourier-transform infrared (FT-IR) spectroscopy has been used to monitor changes in C/N-modified lignocellulosic substrates from Cannabis sativa L. in a 7-week solid-state fermentation with the white-rot fungus Bjerkandera sp. strain BOS55. The microbial transformation of hemp was considered as a pretreatment to pulping processes in paper industries. Special emphasis was paid on the N-content of the substrate, which was modified by: (i) external ammonium inputs, (ii) water extraction, and (iii) protease treatment.Selective delignification in the N-limited media was observed. The most diagnostic FT-IR spectral bands in relation to changes in the lignocellulosic substrate were those corresponding to alkyl structures (2920, 1460 cm(-1)), carboxyl groups (1720 cm(-1)), amides (1650, 1540 cm(-1)) and carbohydrate (mainly 1030 cm(-1)). Simple and multiple regression functions revealed the potential of FT-IR in accurately reflecting substrate composition features previously determined by wet chemical methods. Correspondence analysis suggests C/N-dependent degradation patterns, and discriminant analysis confirmed that the differences between N-limited, N-enriched and the original substrate were significant (P < 0.05) in terms of the intensities of five FT-IR diagnostic bands (1030, 1130, 1270, 1540 and 1650 cm(-1)).The results suggest that, in the system studied, the FT-IR spectroscopy is a reliable alternative to wet chemical analyses in the routine monitoring of the success of the biologic process since it reflects both qualitative and quantitative changes and it is very sensitive to lignin alteration and to carbohydrate and protein concentration.

Nitrogen-removal with protease as a method to improve the selective delignification of hemp stemwood by the white-rot fungus Bjerkandera sp. strain BOS55.

Certain white-rot fungi cause selective removal of lignin from woody substrates. Selective delignification can potentially be applied to biopulping and upgrading animal feeds. Nitrogen nutrient limitation is known to enhance the selectivity of lignin degradation. The relatively high N-content of annual fiber crops is an important drawback for utilizing white-rot fungi for their selective delignification. In this study, removal of N from hemp stemwood with protease was explored as a means of improving the selectivity of lignin degradation by the white-rot fungus Bjerkandera sp. strain BOS55. Various protease treatments followed by hot-water extraction were found to be suitable in lowering the N-content of hemp stemwood by up to 70%. The removal was significantly higher than with hot-water extraction alone, which caused a 39% N-removal. The selectivity of lignin degradation was compared in protease-treated, hot-water treated, untreated and ammonium-spiked hemp stemwood, providing N levels that were, respectively, 0.32-, 0.61-, 1.0- and 5.0-fold relative to the natural N-content in the substrate. Removal of N by hot-water extraction alone or in combination with protease greatly protected the holocellulose fraction from excessive decay during 10 weeks of solid state fermentation. However, the selectivity of lignin decay was only greatly enhanced (three-fold) by the protease treatment, due mostly to a highly improved lignin degradation at the lowest N-level.