Immobilization of defined laccase combinations for enhanced oxidation of phenolic contaminants.

Immobilization is an important method to increase enzyme stability and allow enzyme reuse. One interesting application in the field of environmental biotechnology is the immobilization of laccase to eliminate phenolic contaminants via oxidation. Fumed silica nanoparticles have interesting potential as support material for laccase immobilization via sorption-assisted immobilization in the perspective of applications such as the elimination of micropollutants in aqueous phases. Based on these facts, the present work aimed to formulate laccase-nanoparticle conjugates with defined laccase combinations in order to obtain nanobiocatalysts, which are active over a broad range of pH values and possess a large substrate spectrum to suitably address pollution by multiple contaminants. A multi-enzymatic approach was investigated by immobilizing five different types of laccases originating from a Thielavia genus, Coriolopsis polyzona, Cerrena unicolor, Pleurotus ostreatus, and Trametes versicolor onto fumed silica nanoparticles, separately and in combinations. The laccases differed concerning their pH optima and substrate affinity. Exploiting their differences allowed the formulation of tailor-made nanobiocatalysts. In particular, the production of a nanobiocatalyst could be achieved that retained a higher percentage of its relative activity over the tested pH range (3-7) compared to the dissolved or separately immobilized enzymes. Furthermore, a nanobiocatalyst could be formulated able to oxidize a broader substrate range than the dissolved or separately immobilized enzymes. Thereby, the potential of the nanobiocatalyst for application in biochemical oxidation applications such as the elimination of multiple target pollutants in biologically treated wastewater has been illustrated.

Advanced enzymatic elimination of phenolic contaminants in wastewater: a nano approach at field scale.

The removal of recalcitrant chemicals in wastewater treatment systems is an increasingly relevant issue in industrialized countries. The elimination of persistent xenobiotics such as endocrine-disrupting chemicals (EDCs) emitted by municipal and industrial sewage treatment plants remains an unsolved challenge. The existing efficacious physico-chemical methods, such as advanced oxidation processes, are resource-intensive technologies. In this work, we investigated the possibility to remove phenolic EDCs [i.e., bisphenol A (BPA)] by means of a less energy and chemical consuming technology. To that end, cheap and resistant oxidative enzymes, i.e., laccases, were immobilized onto silica nanoparticles. The resulting nanobiocatalyst produced at kilogram scale was demonstrated to possess a broad substrate spectrum regarding the degradation of recalcitrant pollutants. This nanobiocatalyst was applied in a membrane reactor at technical scale for tertiary wastewater treatment. The system efficiently removed BPA and the results of long-term field tests illustrated the potential of fumed silica nanoparticles/laccase composites for advanced biological wastewater treatment.

Determination of oxidoreductase activity using a high-throughput microplate respiratory measurement.

High-throughput multiparallel activity profiling for oxygen consuming cell layers has been recently developed for extracellular flux analysis. This technology has great potential for determining the enzymatic activity of oxidoreductases (i.e., laccase) both in vivo and in vitro, which is usually measured using photometrical tests monitoring the colored oxidation products. Improvements in terms of sample throughput, comparability, and gain of information (i.e., stoichiometry, electron transfer rate) can be achieved by means of a multiwell plate-based fluorimetric oxygen sensor. In the present study, various laccases have been applied to develop protocols that allow the multiparallel measurement of O(2)-consumption by enzymatic reactions. The developed and validated method enables the comparative quantitation of laccase characteristics (i.e., profiles of activity at various pH values) and minimizes the time it usually takes to collect respiratory data of oxygen-consuming enzymes. Furthermore, the possibility to assess differences between single and multisubstrate kinetics of laccases has been demonstrated.

Multi-catalysis reactions: new prospects and challenges of biotechnology to valorize lignin.

Considerable effort has been dedicated to the chemical depolymerization of lignin, a biopolymer constituting a possible renewable source for aromatic value-added chemicals. However, these efforts yielded limited success up until now. Efficient lignin conversion might necessitate novel catalysts enabling new types of reactions. The use of multiple catalysts, including a combination of biocatalysts, might be necessary. New perspectives for the combination of bio- and inorganic catalysts in one-pot reactions are emerging, thanks to green chemistry-driven advances in enzyme engineering and immobilization and new chemical catalyst design. Such combinations could offer several advantages, especially by reducing time and yield losses associated with the isolation and purification of the reaction products, but also represent a big challenge since the optimal reaction conditions of bio- and chemical catalysis reactions are often different. This mini-review gives an overview of bio- and inorganic catalysts having the potential to be used in combination for lignin depolymerization. We also discuss key aspects to consider when combining these catalysts in one-pot reactions.

Sorption-assisted surface conjugation: a way to stabilize laccase enzyme.

Enyzme immobilization on solid surfaces is one of the most relevant methods to improve enzyme activity and stability under harsh conditions over extended periods. A typically interesting application is the immobilization of laccases, multicopper enzymes oxidizing aromatic compounds, to solid surfaces in order to develop valuable tools for the elimination of micropollutants in wastewater. Laccase of the white-rot fungus Coriolopsis polyzona has been successfully immobilized on fumed silica nanoparticles using a novel method. It consists in the sorption of the enzyme to amino-modified silica nanoparticles and the subsequent covalent cross-linking using glutaraldehyde as a homobifunctional linker. The so-produced nanoparticulate material has been characterized by means of scanning electron microscopy and Brunauer-Emmett-Teller surface area analysis revealing modifications of the surface structure and area during the coupling procedure. Laccase immobilization on spherical nanoparticles produced according to the method of Stöber has been shown to be much less efficient than on fumed silica nanoparticles. Long-term stability assays revealed that the novel developed method allows a drastic stabilization of the enzyme. In real wastewater, 77% of the laccase activity remained on the nanoparticles over 1 month, whereas the activity of free laccase dropped to 2.5%. The activity loss on the nanoparticles resulted from partial inactivation of the immobilized enzymes and additional release into the surrounding solution with subsequent fast inactivation of the free enzymes, since almost no activity was found in the supernatants.

Impact of bio-augmentation with Sphingomonas sp. strain TTNP3 in membrane bioreactors degrading nonylphenol.

This study evaluates the potential of bio-augmentation to improve the degradation of recalcitrant nonylphenol during the wastewater treatment in membrane bioreactors (MBR). One MBR containing activated sludge was bio-augmented using multistep inoculation with freeze dried Sphingomonas sp. strain TTNP3, whereas a second control reactor contained activated sludge solely. The (14)C-labeled-nonylphenol isomer (4-[1-ethyl-1,3-dimethylpentyl]phenol) was applied as a single pulse. Bio-augmentation resulted in an immediate increase of dissolved radioactivity in the effluent in comparison to the control reactor (13% and 2% of initially applied radioactivity after 1 day, respectively). After 5 days of operation, the retentate of the bio-augmented reactor contained only 7% of the initial radioactivity in contrast to 50% in the control reactor. The radioactivity associated to the mixed liquor suspended solids, i.e., the suspension of biomass and other solids on the retentate side of the membrane, was mainly found as non-extractable residues that were increasingly formed during prolonged reactor operation, especially for the control MBR. HPLC-LSC and GC-MS(n) analyses revealed that the bio-augmented reactor produced more polar hydroquinone as main degradation intermediate, whereas the control reactor effluent contained a complex mixture of apolar compounds with shortened oxidized alkyl chains. Thus, the apparent differences in the behavior of nonylphenol between the reactors were due to the catabolism of nonylphenol conferred by bio-augmentation with Sphingomonas sp. strain TTNP3.

Fate of a branched nonylphenol isomer in submerged paddy soils amended with nitrate.

Using a (14)C-labelled branched isomer of NP, the fate of NP was studied in two flooded typical paddy soils under anoxic conditions. Biodegradation of NP occurred under anoxic conditions and was accompanied by the production of polar metabolites and bound residues in alkaline soil extract and humin. The addition of nitrate (20mM) increased the degradation of NP in both soils, especially in soil derived from silt loam deposit, which contained high amounts of organic matter. Less than 1% NP mineralization was detected as free CO(2) in both soils, whereas up to 30, 21, 31% of NP residues comprised at least two polar metabolites, those extractable from humic substances and from the humin fraction, respectively. The denaturing gradient gel electrophoresis (DGGE) analysis of DNA extract of the soil microorganisms hinted differences of microbial community between soils with different degradation rates of NP. The present study provides for the first time information about the fate of a branched nonylphenol isomer in submerged soils amended with nitrate. The production of high amounts of polar metabolites under anoxic conditions suggests the necessity of also considering the fate and possible effects of the degradation products of NP in anoxic environment such as in waterlogged soil.

Selenate removal in methanogenic and sulfate-reducing upflow anaerobic sludge bed reactors.

This paper evaluates the use of upflow anaerobic sludge bed (UASB) bioreactors (30 degrees C, pH=7.0) to remove selenium oxyanions from contaminated waters (790 microg Se L(-1)) under methanogenic and sulfate-reducing conditions using lactate as electron donor. One UASB reactor received sulfate at different sulfate to selenate ratios, while another UASB was operated under methanogenic conditions for 132 days without sulfate in the influent. The selenate effluent concentrations in the sulfate-reducing and methanogenic reactor were 24 and 8 microg Se L(-1), corresponding to removal efficiencies of 97% and 99%, respectively. X-ray diffraction (XRD) analysis and sequential extractions showed that selenium was mainly retained as elemental selenium in the biomass. However, the total dissolved selenium effluent concentrations amounted to 73 and 80 microg Se L(-1), respectively, suggesting that selenate was partly converted to another selenium compound, most likely colloidally dispersed Se(0) nanoparticles. Possible intermediates of selenium reduction (selenite, dimethylselenide, dimethyldiselenide, H(2)Se) could not be detected. Sulfate reducers removed selenate at molar excess of sulfate to selenate (up to a factor of 2600) and elevated dissolved sulfide concentrations (up to 168 mg L(-1)), but selenium removal efficiencies were limited by the applied sulfate-loading rate. In the methanogenic bioreactor, selenate and dissolved selenium removal were independent of the sulfate load, but inhibited by sulfide (101 mg L(-1)). The selenium removal efficiency of the methanogenic UASB abruptly improved after 58 days of operation, suggesting that a specialized selenium-converting population developed in the reactor. This paper demonstrates that both sulfate-reducing and methanogenic UASB reactors can be applied to remove selenate from contaminated natural waters and anthropogenic waste streams, e.g. agricultural drainage waters, acid mine drainage and flue gas desulfurization bleeds.

Role of dissolved humic acids in the biodegradation of a single isomer of nonylphenol by Sphingomonas sp.

This study shows the important role of humic acids in the degradation of (14)C and (13)C labeled isomer of NP by Sphingomonas sp. strain TTNP3 and the detoxification of the resulting metabolites. Due to the association of NP with humic acids, its solubility in the medium was enhanced and the extent of mineralization of nonylphenol increased from 20% to above 35%. This was accompanied by the formation of significant amounts of NP residues bound to the humic acids, which also occurred via abiotic reactions of the major NP metabolite hydroquinone with the humic acids. Gel permeation chromatography showed a non-homogenous distribution of NP residues with humic acids molecules, with preference towards molecules with high-molecular-weight. Solid state (13)C nuclear magnetic resonance spectroscopy indicated that the nonextractable residues resulted exclusively from the metabolites. The chemical shifts of the labeled carbon indicated the possible covalent binding of hydroquinone to the humic acids via ester and possibly ether bonds, and the incorporation of degradation products of hydroquinone into the humic acids. This study provided evidences for the mediatory role of humic acids in the fate of NP as a sink for bacterial degradation intermediates of this compound.

Production of a robust nanobiocatalyst for municipal wastewater treatment.

Immobilization is a fundamental method to improve both enzyme activity and stability. In the present work, the process previously described for immobilizing laccase - an enzyme oxidizing phenolic compounds - onto fumed silica was optimized, in order to efficiently produce industrially relevant amounts of a nanobiocatalyst for biological micropollutant elimination, whilst saving 80% of surface modification agent (3-aminopropyl triethoxy silane) and 90% of cross-linker (glutaraldehyde). Minimized losses during preparation and favorable effects of immobilization yielded conjugates with drastically increased enzymatic activity (164% of invested activity). Long-term stability and activity regarding bisphenol A (2,2-bis(4-hydroxyphenyl)propane) removal of the synthesized biocatalyst were assessed under application-relevant conditions. With 81.1±0.4% residual activity after 7 days, stability of conjugates was drastically higher than of free laccase, which showed virtually no activity after 1.5 days. These results illustrate the huge potential of fumed silica nanoparticles/laccase-composites for innovative biological wastewater treatment.

Laccase-modified silica nanoparticles efficiently catalyze the transformation of phenolic compounds.

A new system based on laccase-modified silica nanoparticles has been developed and tested for its ability to degrade a major endocrine disrupting chemical, 4,4'-isopropylidenediphenol (bisphenol A). The nanoparticles have been produced using the Stöber method and characterized using scanning electron microscopy, dynamic light scattering and zeta-potential measurements. The introduction of primary amino groups at the surface of these particles has been achieved using an organo-silane (amino-propyl-triethoxy-silane). The use of glutaraldehyde as bi-functional coupling agent allowed the efficient conjugation of a laccase from Coriolopsis polyzona at the surface of the nanoparticles, as monitored by measuring the amount of proteins coupled and the zeta-potential of the produced nanoparticles. The oxidative activity of the so-produced bio-conjugate was tested using radioactive-((14)C) labeled bisphenol A. Analytical methods based on high performance liquid chromatography coupled to mass spectrometry and gas chromatography allowing a convenient and reliable study of the enzymatic activity of the produced bio-conjugates have been developed. It is demonstrated that even if a decrease of the specific catalytic activity of the immobilized enzyme is measured, the activity of the bio-conjugate remains compatible with the application of these systems to the transformation of phenolic pollutants. Additionally, the developed analytical methods allowed the identification of the transformation products formed during the enzymatic reaction.

Metabolism of the nonylphenol isomer [ring-U-14C]-4-(3',5'-dimethyl-3'-heptyl)-phenol by cell suspension cultures of Agrostemma githago and soybean.

The biotransformation of the nonylphenol isomer [ring-U-14C]-4-(3',5'-dimethyl-3'-heptyl)-phenol (4-353-NP, consisting of two diastereomers) was studied in soybean and Agrostemma githago cell suspension cultures. With the A. githago cells, a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v) was used, in order to produce higher concentrations and amounts of 4-353-NP metabolites for their identification; 4-353-NP was applied via the n-hexadecane phase. Initial concentrations of [14C]-4-353-NP were 1 mg L(-1) (soybean), and 5 and 10 mg L(-1) (A. githago). After 2 (soybean) and 7 days (A. githago) of incubation, the applied 4-353-NP was transformed almost completely by both plant species to four types of products: glycosides of parent 4-353-NP, glycosides of primary 4-353-NP metabolites, nonextractable residues and unknown, possibly polymeric materials detected in the media. The latter two products emerged especially in soybean cultures. Portions of primary metabolites amounted to 19-22% (soybean) and 21-42% of applied 14C (A. githago). After liberation from their glycosides, the primary 4-353-NP metabolites formed by A. githago were isolated by HPLC and examined by GC-EIMS as trimethylsilyl derivatives. In the chromatograms, eight peaks were detected which due to their mass spectra, could be traced back to 4-353-NP. Seven of the compounds were side-chain monohydroxylated 4-353-NP metabolites, while the remaining was a (side-chain) carboxylic acid derivative. Unequivocal identification of the sites of hydroxylation/oxidation of all transformation products was not possible. The main primary metabolites produced by A. githago were supposed to be four diastereomers of 6'-hydroxy-4-353-NP (about 80% of all products identified). It was concluded that plants contribute to the environmental degradation of the xenoestrogen nonylphenol; the toxicological properties of side-chain hydroxylated nonylphenols remain to be examined.