Methanotrophic denitrification in wastewater treatment: microbial aspects and engineering strategies.

Anaerobic technologies are consolidated for sewage treatment and are the core processes for mining marketable products from waste streams. However, anaerobic effluents are supersaturated with methane, which represents a liability regarding greenhouse gas emissions. Meanwhile, anaerobic technologies are not capable of nitrogen removal, which is required to ensure environmental protection. Methane oxidation and denitrification processes can be combined to address both issues concurrently. Aerobic methane oxidizers can release intermediate organic compounds that can be used by conventional denitrifiers as electron donors. Alternatively, anoxic methanotrophic species combine methane oxidation with either nitrate or nitrite reduction in the same metabolism. Engineered systems need to overcome the long doubling times and low NOx consumption rates of anoxic methanotrophic microorganisms. Another commonly reported bottleneck of methanotrophic denitrification relates to gas-liquid mass transfer limitations. Although anaerobic effluents are supersaturated with methane, experimental setups usually rely on methane supply in a gaseous mode. Hence, possibilities for the application of methane-oxidation coupled to denitrification in full scale might be overlooked. Moreover, syntrophic relationships among methane oxidizers, denitrifiers, nitrifiers, and other microorganisms (such as anammox) are not well understood. Integrating mixed populations with various metabolic abilities could allow for more robust methane-driven wastewater denitrification systems. This review presents an overview of the metabolic capabilities of methane oxidation and denitrification and discusses technological aspects that allow for the application of methanotrophic denitrification at larger scales.

A new device to select carriers for biomass immobilization and application in an aerobic/anaerobic fixed-bed sequencing batch biofilm reactor for nitrogen removal.

This study proposes a new approach to selecting a biofilm carrier for immobilization using dissolved oxygen (DO) microsensors to measure the thickness of aerobic and anaerobic layers in biofilm. The biofilm carriers tested were polyurethane foam, mineral coal (MC), basaltic gravel, and low-density polyethylene. Development of layers in the biofilm carrier surface was evaluated using a flow cell device, and DO profiles were conducted to determine the size of the layers (aerobic and anaerobic). MC was the biofilm carrier selected due to allowing the development of larger aerobic and anaerobic layers in the biofilm (896 and 1,058 µm, respectively). This ability is supposed to improve simultaneous nitrogen removal by nitrification and denitrification biological processes. Thus, as a biofilm carrier, MC was used in a fixed-bed sequencing batch biofilm reactor (FB-SBBR) for treatment of wastewater with a high ammonia concentration (100-400 mgNH4+-N L-1). The FB-SBBR (15.0 L) was filled with matrices of the carrier and operated under alternating aeration and non-aeration periods of 6 h each. At a mean nitrogen loading rate of 0.55 ± 0.10 kgNH4+-N m-3 d-1, the reactor attained a mean nitrification efficiency of 95 ± 9% with nitrite as the main product (aerobic period). Mean denitrification efficiency during the anoxic period was 72 ± 13%.

Poultry slaughterhouse wastewater treatment plant for high quality effluent.

This paper assesses a wastewater treatment plant (WWTP) regarding the technology used, as well as organic matter and nutrient removal efficiencies aiming to optimize the treatment processes involved and wastewater reclamation. The WWTP consists of a dissolved air flotation (DAF) system, an upflow anaerobic sludge blanket (UASB) reactor, an aerated-facultative pond (AFP) and a chemical-DAF system. The removal efficiencies of chemical oxygen demand (COD) (97.9 ± 1.0%), biochemical oxygen demand (BOD) (98.6 ± 1.0%) and oil and grease (O&G) (91.1 ± 5.2%) at the WWTP, the nitrogen concentration of 17 ± 11 mg N-NH3 and phosphorus concentration of 1.34 ± 0.93 mg PO4(-3)/L in the final effluent indicate that the processes used are suitable to comply with discharge standards in water bodies. Nitrification and denitrification tests conducted using biomass collected at three AFP points indicated that nitrification and denitrification could take place in the pond.

Sulfidogenesis interference on methane production from carbohydrate-rich wastewater.

Two anaerobic fixed-structured bed reactors were fed with synthetic wastewater simulating the soluble fraction of sugarcane vinasse to evaluate the interference of sulfidogenesis on methanogenesis. The reactors running in parallel were subjected to the same operating conditions. The influent organic matter concentration (in term of chemical oxygen demand (COD)) remained close to 4,000 mgCOD L(-1) and the hydraulic retention time was 24 hours. One reactor, the methanogenic (control reactor), received sulfate only to provide the sulfur required as a nutrient to the methanogenic biomass. The other one, the sulfidogenic/methanogenic reactor (SMR), received sulfate concentration corresponding to COD/sulfate ratios of 4, 5 and 3. In the last phase, the COD removal efficiencies were higher than 96% in both reactors and the SMR achieved 97% of sulfate removal efficiency (COD/sulfate ratio of 3 and influent sulfate concentration close to 1,300 mgSO4(2-) L(-1)). Both reactors also had similar methane yields in this phase, close to 350 mLCH4 gCODremoved(-1) at standard temperature and pressure. These results indicated no significant inhibition of methanogenic activity under the sulfidogenic conditions assessed.

Continuous anaerobic bioreactor with a fixed-structure bed (ABFSB) for wastewater treatment with low solids and low applied organic loading content.

This paper describes a new type of anaerobic bioreactor with a fixed-structure bed (ABFSB) in which the support for the biomass consists of polyurethane foam strips placed along the length of the bioreactor. This configuration prevents the accumulation of biomass or solids in the bed as well as clogging and channeling effects. In this study, complex synthetic wastewater with a chemical oxygen demand of 404.4 mg O(2) L(-1) is treated by the reactor. The ABFSB, which has a working volume of 4.77 L, was inoculated with anaerobic sludge obtained from an upflow anaerobic sludge blanket bioreactor. A removal efficiency of 78 % for organic matter and an effluent pH of 6.97 were achieved. An analysis of the organic volatile acids produced by the ABFSB indicated that it operated under stable conditions during an experimental run of 36 days. The stable and efficient operation of the bioreactor was compared with the configurations of other anaerobic bioreactors used for complex wastewater treatment. The results of the study indicate that the ABFSB is a technological alternative to packed-bed bioreactors.

Microbial communities and metabolic pathways involved in reductive decolorization of an azo dye in a two-stage AD system.

Multiple stage anaerobic system was found to be an effective strategy for reductive decolorization of azo dyes in the presence of sulfate. Bulk color removal (56-90%) was achieved concomitant with acidogenic activity in the 1st-stage reactor (R1), while organic matter removal (≤100%) and sulfate reduction (≤100%) occurred predominantly in the 2nd-stage reactor (R2). However, azo dye reduction mechanism and metabolic routes involved remain unclear. The involved microbial communities and conditions affecting the azo dye removal in a two-stage anaerobic digestion (AD) system were elucidated using amplicon sequencing (16S rRNA, fhs, dsrB and mcrA) and correlation analysis. Reductive decolorization was found to be co-metabolic and mainly associated with hydrogen-producing pathways. We also found evidence of the involvement of an azoreductase from Lactococcus lactis. Bacterial community in R1 was sensitive and shifted in the presence of the azo dye, while microorganisms in R2 were more protected. Higher diversity of syntrophic-acetate oxidizers, sulfate reducers and methanogens in R2 highlights the role of the 2nd-stage in organic matter and sulfate removals, and these communities might be involved in further transformations of the azo dye reduction products. The results improve our understanding on the role of different microbial communities in anaerobic treatment of azo dyes and can help in the design of better solutions for the treatment of textile effluents.

Broken into pieces: The challenges of determining sulfonated azo dyes in biological reactor effluents using LC-ESI-MS/MS analysis.

Most studies on the biodegradation of textile azo dyes use color as parameter for measuring the efficiency of degradation. Although widely employed, spectrophotometric methods are susceptible to the interference of metabolites or degradation products from the biological treatment. We propose a method for determination of a model sulfonated azo dye (Direct Black 22, DB22) in wastewater using solid-phase extraction (SPE) and liquid chromatography - electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). MS analysis in negative electrospray ionization mode showed DB22 as the most abundant precursor ion, corresponding to [M-3Na + H]2-, which yields two radical anions of m/z 370.1 and m/z 645 after MS/MS fragmentation by collision-induced dissociation (CID). Calibration curve presented adequate linearity and precision in the range of 120-1500 ng mL-1, and recovery and detection limit were appropriate to the typically employed working concentrations. Nevertheless, we observed that standard heating of DB22 under alkaline conditions to simulate the production of wastewater during dye-baths resulted in loss of MS/MS signal, without affecting color. Further analysis showed that DB22 undergoes hydrolysis and does not remain unaltered in solution. Alternative methods of hydrolysis evaluated resulted in no MS/MS signal as well. SPE-LC-ESI-MS/MS analysis evidenced the structural change of DB22 in aqueous solution while the dyeing-capacity was preserved. This technique has also the potential of being tailored to consider the detection of the hydrolyzed fragments of azo dyes in wastewater for appropriate quantification, but it was not the scope of the current step of this research. Color remains as a more reliable parameter for monitoring azo compounds which are unstable in aqueous solution, while a more robust and holistic method needs to be developed for the speciation of the DB22 products of thermal hydrolysis.

Toxic effects of cadmium (Cd²âº) on anaerobic biomass: kinetic and metabolic implications.

Cadmium ion (Cd(2+)) toxicity on anaerobic systems, used for organic matter removal, was assessed by studying its effect on kinetic parameters and metabolic changes. This fundamental study was performed in a continuous fixed bed anaerobic bioreactor that treated synthetic wastewater simulating domestic sewage. The biomass was immobilized on a fixed bed made of polyurethane foam. Under influent cadmium concentrations of 0.0, 0.4, 4.4 and 6.2 mg Cd(2+) L(-1) the organic matter removal efficiencies were 84%, 82%, 72% and 52%, respectively. At influent concentration of 6.2 mg Cd(2+) L(-1) the reactor had reached its limit for cadmium toxicity. In the removal of dissolved organic matter, the first-order apparent kinetic coefficients (k(1)(app)) were 0.84, 0.67 and 0.10 h(-1) for the operations with 0.0, 0.4 and 4.4 mg Cd(2+) L(-1), respectively. The apparent inhibition coefficient for cadmium (k(i)(app)) was 1.69 mg L(-1). Despite the toxic effects of cadmium on anaerobic organic matter removal at large Cd(2+) concentrations, the results demonstrated that the anaerobic process was suitable for cadmium concentrations below 29.8 mg Cd(2+) L(-1), considering the bioavailable fraction of adsorbed cadmium in the support when the cadmium influent concentration was 6.2 mg Cd(2+) L(-1).

Characterization and kinetics of sulfide-oxidizing autotrophic denitrification in batch reactors containing suspended and immobilized cells.

Sulfide-oxidizing autotrophic denitrification is an advantageous alternative over heterotrophic denitrification, and may have potential for nitrogen removal of low-strength wastewaters, such as anaerobically pre-treated domestic sewage. This study evaluated the fundamentals and kinetics of this process in batch reactors containing suspended and immobilized cells. Batch tests were performed for different NOx-/S2- ratios and using nitrate and nitrite as electron acceptors. Autotrophic denitrification was observed for both electron acceptors, and NOx-/S2- ratios defined whether sulfide oxidation was complete or not. Kinetic parameter values obtained for nitrate were higher than for nitrite as electron acceptor. Zero-order models were better adjusted to profiles obtained for suspended cell reactors, whereas first-order models were more adequate for immobilized cell reactors. However, in the latter, mass transfer physical phenomena had a significant effect on kinetics based on biochemical reactions. Results showed that sulfide-oxidizing autotrophic denitrification can be successfully established for low-strength wastewaters and have potential for nitrogen removal from anaerobically pre-treated domestic sewage.

The thermal transformation of Man Made Vitreous Fibers (MMVF) and safe recycling as secondary raw materials (SRM).

This work describes the high temperature reaction sequence of commercial Man Made Vitreous Fibers (MMVF) Cerafiber, Superwool, Rock wool and Glass wool which may be used as substitute for asbestos in some industrial applications. Knowledge of the reaction path and transformation sequence is very important to assess whether carcinogenic crystalline phases are formed during devitrification, which may occur when used as insulators. In addition, knowledge about the nature of the phases formed at high temperature is mandatory to assess if thermally transformed MMVF can be safely recycled as secondary raw material (SRM). In this scenario, this study provides useful information for the optimization of the industrial annealing process aimed to attain a safe, recyclable product. The results of this work show that one of the high-temperature products of Cerafiber and Superwool is cristobalite which is classified as a carcinogenic. It was possible to define the temperature interval at which Cerafiber and Superwool fibers can be safely used as thermal insulators (e.g. insulators in tunnel and/or roller kilns, etc.). As cristobalite is formed in both synthetic fiber products at temperatures higher than 1200 degrees C, their use should be limited to devices operating at lower temperatures. Rock and Glass wool melt upon thermal treatment. As far as the industrial process of inertization is concerned, a maximum firing temperature of 1100 and 600 degrees C is required to melt Rock wool and Glass wool, respectively, with the high-temperature products that can be safely recycled as SRM. Recycling of these products in stoneware tile mixtures were subsequently attempted. The addition of 1-2 wt.% of the melts of Rock and Glass wool gave promising results in terms of viscous sintering reactions and resistance to staining with the only weak characteristic being the color properties of the fired bodies which tend to worsen.

Mitochondrial changes induced by natural and synthetic asbestos fibers: studies on isolated mitochondria.

Asbestos fibers, such as chrysotile and crocidolite, are known to have cytotoxic effects on different cell types. In vivo exposure to asbestos fibers can induce both fibrotic and malignant lung diseases , however, the mechanisms linking exposure to the subsequent development of the diseases are unknown. Numerous investigations suggest the involvement of reactive oxygen species (ROS). ROS are known to damage biological macromolecules including proteins, cell membrane lipids and nucleic acids; alterations of these essential cellular components can alter cell function and can drive the cell to neoplastic transformation or to cell death. Because the mitochondrial respiratory chain is an important source of ROS and RNS (reactive nitogen species) in the cells, we have investigated the effects of aqueous extracts of asbestos (natural and synthetic) fibers on some mitochondrial activities. Our data show that crocidolite fibers release substances in solution that may interfere directly with the mitochondrial cytochrome oxidase complex. Moreover, the calcium ions released from these fibers induce opening of the permeability transition pore of the inner membrane leading to a possible cytotoxic effect due to the release of apoptotic factors normally localized in the mitochondrial intermembrane space. In addition, crocidolite extracts enhance the mitochondrial production of ROS. No significant biochemical effects are exerted by chrysotile, either natural or synthetic, on isolated mitochondria. Nevertheless, all asbestos fibers tested induce morphological alterations visualized by transmission electron microscopy and morphometric analysis.

Effects of solid-phase mass transfer on the performance of a stirred anaerobic sequencing batch reactor containing immobilized biomass.

This work reports on experiments for an anaerobic sequencing batch reactor containing immobilized biomass which aimed at verifying the effects of solid-phase mass transfer on the reactor's overall performance. Four experiments were carried out at 30 degrees C with cubic polyurethane foam particles previously inoculated with anaerobic biomass. Different solid-phase mass transfer conditions were reached in each experiment by varying the size of the bioparticle from 0.5 to 3.0 cm. The reactor was fed with a low-strength synthetic wastewater containing protein, carbohydrates and lipid and the effects of mass transfer were evaluated through dynamic substrate concentration profiles during 8-hour batch cycles. A modified first-order kinetic model provided a good representation of the behavior of the dynamic concentration profiles. The solid-phase mass transfer was found to slightly affect the concentration of effluent organic matter expressed as chemical oxygen demand (COD). The concentration of residual effluent substrate increased as the size of the bioparticle was increased. The cycle time was not affected as the size of the bioparticle was increased from 0.5 to 2.0 cm. However, it was found that the cycle time in a reactor with 3.0-cm cubic particles should be higher than that required in systems with smaller particles. The apparent first-order kinetic parameter was estimated as 0.59+/-0.01 h(-1) for experiments with bioparticle sizes ranging from 0.5 to 2.0 cm, while a value of 0.48 h(-1) was obtained in the experiment with 3.0-cm bioparticles.

The performance of an anaerobic sequencing batch biofilm reactor treating domestic sewage colonized by anoxygenic phototrophic bacteria.

There are few reports on morphological characterization of microbial population colonizing anaerobic bioreactors and the aim of this work was to access such variable in an anaerobic sequencing batch biofilm reactor treating the University of Sao Paulo (Sao Carlos city, Brazil) domestic sewage. This pilot-scale reactor (1.2m3) has been treating 0.65 m3 of liquid waste under cycles of 8h. The ASBBR has the distinct characteristics of being filled with support material for biomass attachment with the aim of skipping the sedimentation phase during the operational cycles, as it is commonly observed in anaerobic sequencing batch reactors (ASBR). Physical, chemical and physico-chemical variables were accessed in the influent and in the effluent for performance evaluation. Microbial characterization was made by means of direct microscopy and samples were taken over 150 d with a 25 d period interval. The ASBBR attained approximately 60% of COD removal efficiency. Microscopic analysis of biomass showed the presence of anoxygenic phototrophic bacteria probably influencing the ASBBR performance in the domestic sewage treatment. It is very likely that the exclusion of phototrophic sulfur bacteria by efficiently restraining the light would enhance the bioreactor efficiency.

Biofilm adaptation to sulfate reduction in anaerobic immobilized biomass reactors sujected to different COD/sulfate ratios.

Various aspects of biofilm adaptation to sulfate reduction in horizontal-flow anaerobic immobilized biomass (HAIB) reactors subjected to increasing sulfate concentrations and different COD/sulfate ratios are presented and discussed. Four bench-scale HAIB reactors filled with vegetal carbon (R1 and R2) and polyurethane foam matrices (R3 and R4) were utilized. Influent sulfate concentrations ranging from 500 to 3000 mg/L were applied at COD/sulfate ratios ranging from 5.0 to 1.7. Reactors R1 and R4 were operated with higher sulfate loads than those applied to R2 and R3. For the same COD/sulfate ratio, the highest sulfate reduction efficiency (approximately 80%) was displayed by the vegetal carbon reactor (R2) subjected to low sulfate loads. According to the results of our molecular biology analyses, the different support materials provided different biomass colonization conditions. The lowest diversity of sulfate-reducing bacteria was found in the HAIB filled with polyurethane foam matrices operating with high sulfate loads.

Influence of the addition of sulphate and ferric ions in a methanogenic anaerobic packed-bed reactor treating gasoline-contaminated water.

Benzene, toluene and xylene (BTX) are relatively soluble aromatic compounds of gasoline. Gasoline storage tank leakages generally lead to an extensive contamination of groundwater. In the natural environment for instance, these compounds might be biodegraded under a variety of reducing potentials. The objective of this work was to examine the influence of the addition of sulphate and Fe(OH)3 in a methanogenic horizontal-flow anaerobic immobilized-biomass reactor treating gasoline-contaminated water. Three different conditions were evaluated: methanogenic, sulphidogenic and sulphidogenic with the addition of ferric ions. Methanogenic condition showed the higher BTX degradation rates and the addition of sulphate negatively affected BTX removal rates with the production of H2S. However, the addition of ferric ions resulted in the precipitation of sulphur, improving BTX degradation by the consortium. Metanosphaera sp., Methanosarcina barkeri and Methanosaeta concilii were identified in the consortium by means of 16S and directly related to the addition of ferric ions.

Kinetics, mass transfer and hydrodynamics in a packed bed aerobic reactor fed with anaerobically treated domestic sewage.

This study presents an assessment of the kinetic, mass transfer and hydrodynamic parameters of a pilot-scale fixed bed reactor containing immobilized biomass in polyurethane matrices and fed with the effluent of a horizontal-flow fixed bed anaerobic reactor, which was used to treat domestic sewage. It was found that the liquid-solid and intra-particle mass transfer resistances significantly affected the overall oxygen consumption rate and that mechanical agitation could minimize such resistances. The volumetric oxygen transfer coefficient (kLa) values for superficial air velocities between 8.4 cm min(-1) and 57.0 cm min(-1) varied from 20.8 h(-1) to 58.8 h(-1) for tap water, and 16.8 h(-1) to 53.0 h(-1) for the anaerobic pre-treated effluent. The intrinsic oxygen uptake rate was estimated to be 19.9 mgO2 gVSS(-1) h(-1). A first-order kinetic model with residual concentration was considered to adequately represent the COD removal rate, whereas nitrogen conversion was considered to be well represented by a model of pseudo-first-order reaction in series. It was also found that the ammonium conversion to nitrite was the limiting step of the overall nitrogen conversion rate. The hydrodynamic behavior of the reactor was represented by three to four completely mixed reactors in series.

The influence of the buffering capacity on the production of organic acids and alcohols from wastewater in anaerobic reactor.

Some bacteria common in anaerobic digestion process can ferment a broad variety of organic compounds to organic acids, alcohols, and hydrogen, which can be used as biofuels. Researches are necessary to control the microbial interactions in favor of the alcohol production, as intermediary products of the anaerobic digestion of organic compounds. This paper reports on the effect of buffering capacity on the production of organic acids and alcohols from wastewater by a natural mixed bacterial culture. The hypothesis tested was that the increase of the buffering capacity by supplementation of sodium bicarbonate in the influent results in benefits for alcohol production by anaerobic fermentation of wastewater. When the influent was not supplemented with sodium bicarbonate, the chemical oxygen demand (COD)-ethanol and COD-methanol detected in the effluent corresponded to 22.5 and 12.7 % of the COD-sucrose consumed. Otherwise, when the reactor was fed with influent containing 0.5 g/L of sodium bicarbonate, the COD-ethanol and COD-methanol were effluents that corresponded to 39.2 and 29.6 % of the COD-sucrose consumed. Therefore, the alcohol production by supplementation of the influent with sodium bicarbonate was 33.6 % higher than the fermentation of the influent without sodium bicarbonate.

Structural and morphological characterization of synthetic chrysotile single crystals.

Stoichiometric chrysotile single crystals have been synthesized as a unique phase by hydrothermal reaction under controlled conditions; the synthesized monocrystals show a cylinder-in-cylinder morphology and can be used as a reference sample with definite chemical composition to investigate the factors responsible of the chrysotile cytotoxicities and carcinogenicities.

Thermal behavior of bone and synthetic hydroxyapatites submitted to magnesium interaction in aqueous medium.

The thermal behavior of the products obtained from magnesium interaction with powdered femoral bone and carbonate containing synthetic hydroxyapatite under conditions of pH fluctuation in aqueous medium has been investigated. The products, heat treated at different temperatures from 100 to 1300 degrees C, have been characterized by infrared spectroscopy and X-ray diffraction technique. The results show that the interaction with magnesium ion destabilizes the apatitic structure and favours its thermal conversion into beta-tricalcium phosphate (beta-TCP). The replacement of magnesium with calcium in the beta-TCP crystal lattice hinders its subsequent thermal conversion into the alpha form. The influence of magnesium on the thermal stability is much more evident for carbonate-containing synthetic hydroxyapatite than for bone apatite.

Structural and chemical characterization of inorganic deposits in calcified human mitral valve.

X-ray diffraction, i.r. absorption, and chemical analyses have been carried out on the mineral deposits of calcified human mitral valves and glutaraldehyde-preserved porcine aortic grafts. The mineral deposits isolated from highly calcified mitral valves and porcine aortic grafts are constituted of type B-carbonate apatite. Magnesium substituted beta-tricalcium phosphate is present, together with an apatitic phase similar to dahllite, in the ashes of poorly calcified mitral valves. The contraction of the unit cell of beta-tricalcium phosphate due to magnesium incorporation is compared with the variation of the lattice constants of synthetic beta-tricalcium phosphate at different degree of magnesium substitution for calcium. The results reveal the important role of magnesium on the calcification of human valves. In fact, the apatitic phase deposited at the beginning of the calcification process, when there is a high magnesium content, converts completely into beta-tricalcium phosphate by heat treatment at 1,000 degrees C. On the other hand, when the calcification becomes massive, magnesium content appears highly reduced, and the deposited apatitic phase is characterized by a high thermal stability.