Fluorescently Labeled Cellulose Nanofibers for Environmental Health and Safety Studies.

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (µM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos.

Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition. RESULTS: Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L(-1) for PsLPMOA and >260 mg L(-1) for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stability and activity in the presence of sea salt. Both PsLPMOs were activated by a cellobiose dehydrogenase (CDH) from Neurospora crassa, with an apparent optimum of interaction at pH 5.5. Investigation into their regioselective mode of action revealed that PsLPMOA released C1- and C4-oxidized cello-oligosaccharide products, while PsLPMOB released only C4-oxidized products. PsLPMOA was found to cleave polymeric cellulose in the presence of up to 6 % sea salt, which emphasizes the use of sea water in the industrial saccharification process with improved ecological footprints. CONCLUSIONS: Two new LPMOs from the mangrove fungus Pestalotiopsis sp. NCi6 were found to be fully reactive against cellulose. The combined hydrolytic activities of these salt-responsive LPMOs could therefore facilitate the saccharification process using sea water as a reaction medium for large-scale biorefineries.

Fractionation of hemp hurds by organosolv pretreatment and its effect on production of lignin and sugars.

Fractionation of hemp hurds into its three main components, cellulose, hemicellulose, and lignin, was carried out using organosolv pretreatment. The effect of processing parameters, such as temperature, catalyst concentration, reaction time, and methanol (MeOH) concentration, on the dissolution and recovery of hemicellulose and lignin was determined. More than 75% of total hemicellulose and 75% of total lignin was removed in a single step with low amounts of degradation products under the following conditions: 165 °C, 3% H2 SO4 , 20 min reaction time, and 45% MeOH. Enzymatic hydrolysis of the residual pretreated biomass yielded up to 60% of cellulose-to-glucose conversion. The maximum recovery of the main components was obtained at a combined severity factor value of around one. Characterization of pretreated biomass and isolated lignin was carried out with FTIR and 2D (13) C-(1) H correlation HSQC NMR spectroscopy, the latter technique providing detailed structural information about the obtained methanol organosolv lignin (MOSL). Results suggested that xylopyranoside is the major carbohydrate associated with hemp lignin. The chemical properties of MOSL samples in terms of their phenolic group content and antioxidant capacity were also investigated. The results showed that MOSL samples have a high phenolic group content and antioxidant capacity relative to Klason lignin.

Wiring of pyranose dehydrogenase with osmium polymers of different redox potentials.

In this study, five different flexible osmium based redox polymers were investigated for their ability to efficiently "wire" the oxidoreductase pyranose dehydrogenase (PDH, EC 1.1.99.29) from Agaricus meleagris, on graphite electrodes for possible applications in biofuel cells. A series of newly synthesised osmium based redox polymers covering the potential range between -270 and +160 mV vs. Ag|AgCl (0.1M KCl) was used. The performance of the redox polymers for enzyme wiring was investigated using glucose as substrate. The optimal operational conditions such as pH and potential were investigated.

Increasing the coulombic efficiency of glucose biofuel cell anodes by combination of redox enzymes.

A highly efficient anode for glucose biofuel cells has been developed by a combination of pyranose dehydrogenase from Agaricus meleagris (AmPDH) and cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH). These two enzymes differ in how they oxidize glucose. AmPDH oxidizes glucose at the C(2) and C(3) carbon, whereas MtCDH at the C(1) carbon. Both enzymes oxidize efficiently a number of other mono- and disaccharides. They do not react directly with oxygen and produce no H(2)O(2). Electrodes were prepared by embedding (i) only AmPDH (in order to study this enzyme separately) and (ii) a mixture of AmPDH and MtCDH in an Os redox polymer hydrogel. Single-walled carbon nanotubes (SWCNTs) were added in order to enhance the current density. The electrodes were investigated with linear sweep and cyclic voltammetry in the presence of different substrates at physiological conditions. The electrochemical measurements revealed that the product of one enzyme can serve as a substrate for the other. In addition, a kinetic pathway analysis was performed by spectrophotometric measurements leading to the conclusion that up to six electrons can be gained from one glucose molecule through a combination of AmPDH and MtCDH. Hence, the combination of redox enzymes can lead to an enzymatic biofuel cell anode with an increased coulombic efficiency far beyond the usual yields of two electrons per substrate molecule.