Sequential lignin depolymerization by combination of biocatalytic and formic acid/formate treatment steps.

Lignin, a complex three-dimensional amorphous polymer, is considered to be a potential natural renewable resource for the production of low-molecular-weight aromatic compounds. In the present study, a novel sequential lignin treatment method consisting of a biocatalytic oxidation step followed by a formic acid-induced lignin depolymerization step was developed and optimized using response surface methodology. The biocatalytic step employed a laccase mediator system using the redox mediator 1-hydroxybenzotriazole. Laccases were immobilized on superparamagnetic nanoparticles using a sorption-assisted surface conjugation method allowing easy separation and reuse of the biocatalysts after treatment. Under optimized conditions, as much as 45 wt% of lignin could be solubilized either in aqueous solution after the first treatment or in ethyl acetate after the second (chemical) treatment. The solubilized products were found to be mainly low-molecular-weight aromatic monomers and oligomers. The process might be used for the production of low-molecular-weight soluble aromatic products that can be purified and/or upgraded applying further downstream processes.

Arsenic Mobilization from Historically Contaminated Mining Soils in a Continuously Operated Bioreactor: Implications for Risk Assessment.

Concentrations of soil arsenic (As) in the vicinity of the former Zloty Stok gold mine (Lower Silesia, southwest Poland) exceed 1000 µg g(-1) in the area, posing an inherent threat to neighboring bodies of water. This study investigated continuous As mobilization under reducing conditions for more than 3 months. In particular, the capacity of autochthonic microflora that live on natural organic matter as the sole carbon/electron source for mobilizing As was assessed. A biphasic mobilization of As was observed. In the first two months, As mobilization was mainly conferred by Mn dissolution despite the prevalence of Fe (0.1 wt % vs 5.4 for Mn and Fe, respectively) as indicated by multiple regression analysis. Thereafter, the sudden increase in aqueous As[III] (up to 2400 µg L(-1)) was attributed to an almost quintupling of the autochthonic dissimilatory As-reducing community (quantitative polymerase chain reaction). The aqueous speciation influenced by microbial activity led to a reduction of solid phase As species (X-ray absorption fine structure spectroscopy) and a change in the elemental composition of As hotspots (micro X-ray fluorescence mapping). The depletion of most natural dissolved organic matter and the fact that an extensive mobilization of As[III] occurred after two months raises concerns about the long-term stability of historically As-contaminated sites.

Production of superparamagnetic nanobiocatalysts for green chemistry applications.

Immobilization of enzymes on solid supports is a convenient method for increasing enzymatic stability and enabling enzyme reuse. In the present work, a sorption-assisted surface conjugation method was developed and optimized to immobilize enzymes on the surface of superparamagnetic nanoparticles. An oxidative enzyme, i.e., laccase from Trametes versicolor was used as model enzyme. The immobilization method consists of the production of superparamagnetic nanoparticles by co-precipitation of FeCl2 and FeCl3. Subsequently, the particle surface is modified with an organosilane containing an amino group. Next, the enzymes are adsorbed on the particle surface before a cross-linking agent, i.e., glutaraldehyde is added which links the amino groups on the particle surface with the amino groups of the enzymes and leads to internal cross-linking of the enzymes as well. The method was optimized using response surface methodology regarding optimal enzyme and glutaraldehyde amounts, pH, and reaction times. Results allowed formulation of biocatalysts having high specific enzymatic activity and improved stability. The biocatalysts showed considerably higher stability compared with the dissolved enzymes over a pH range from 3 to 9 and in the presence of several chemical denaturants. To demonstrate the reusability of the immobilized enzymes, they were applied as catalysts for the production of a phenoxazinone dye. Virtually, 100 % of the precursor was transformed to the dye in each of the ten conducted reaction cycles while on average 84.5 % of the enzymatic activity present at the beginning of a reaction cycle was retained after each cycle highlighting the considerable potential of superparamagnetic biocatalysts for application in industrial processes.

Enzyme Shielding in an Enzyme-thin and Soft Organosilica Layer.

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.

Laccases to take on the challenge of emerging organic contaminants in wastewater.

The removal of emerging organic contaminants from municipal wastewater poses a major challenge unsatisfactorily addressed by present wastewater treatment processes. Enzyme-catalyzed transformation of emerging organic contaminants (EOC) has been proposed as a possible solution to this major environmental issue more than a decade ago. Especially, laccases gained interest in this context in recent years due to their broad substrate range and since they only need molecular oxygen as a cosubstrate. In order to ensure the stability of the enzymes and allow their retention and reuse, either immobilization or insolubilization of the biocatalysts seems to be the prerequisite for continuous wastewater treatment applications. The present review summarizes the research conducted on EOC transformation with laccases and presents an overview of the possible immobilization techniques. The goal is to assess the state of the art and identify the next necessary steps that have to be undertaken in order to implement laccases as a tertiary wastewater treatment process in sewage treatment plants.

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.