Implementation of cytochrome c proteins and carbon nanotubes hybrids in bioelectrodes towards bioelectrochemical systems applications.

Multiheme cytochrome c (Cyt c) can function as a redox protein on electrode to accomplish bioelectrocatalysis. However, the direct electron transfer (DET) between the redox site of Cyt c and electrode is low due to the large coupling distance. A close proximity or a connection pathway from the deeply buried active site to the protein surface can be established by modifying the electrode with carbon nanotubes (CNTs) to improve the DET. Therefore, the isolated Cyt c has been assembled or casted with CNTs by various processes to form Cyt c-CNTs bioelectrodes that can be further applied to biosensing and bioanalysis. These strategies can be transplanted to the fabrication of biofilm-CNTs based electrodes by complexing the out membrane (OM) Cyt c of natural electricigen with CNTs to realize the application of the electrochemical properties of "in vivo" Cyt c to bioelectrochemical systems (BESs). This review intends to highlight the preparation strategies of bioelectrodes that have been well studied in electrochemical biosensors and improving approaches of the DET from the CNTs surface to Cyt c in their hybrids. The efficient fabrication processes of the biofilm-CNTs based electrodes that can be considered as "in vivo" Cyt c-CNTs based electrodes for BES designs are also summarized, aiming to provide an inspiration source and a reference to the related studies of BES downstream.

Research advances of SERS analysis method based on silent region molecules for food safety detection.

Food safety is a critical issue that is closely related to people's health and safety. As a simple, rapid, and sensitive detection technique, surface-enhanced Raman scattering (SERS) technology has significant potential for food safety detection. Recently, researchers have shown a growing interest in utilizing silent region molecules for SERS analysis. These molecules exhibit significant Raman scattering peaks in the cellular Raman silent region between 1800 and 2800 cm-1 avoiding overlapping with the SERS spectrum of biological matrices in the range 600-1800 cm-1, which could effectively circumvent matrix effects and improve the SERS accuracy. In this review, the application of silent region molecules-based SERS analytical technique for food safety detection is introduced, detection strategies including label-free detection and labeled detection are discussed, and recent applications of SERS analysis technology based on molecules containing alkyne and nitrile groups, as well as Prussian blue (PB) in the detection of pesticides, mycotoxins, metal ions, and foodborne pathogens are highlighted. This review aims to draw the attention to the silent region molecules-based SERS analytical technique and to provide theoretical support for its further applications in food safety detection.

Lysozyme regulates the extracellular polymer of activated sludge and promotes the formation of electroactive biofilm.

The formation of electroactive biofilm from activated sludge on electrode surface is a key step to construct a bio-electrochemical system, yet it is greatly limited by the poor affinity between the bacteria and the electrode interface. Herein, we report a new method to promote the formation of electroactive biofilm by regulating the extracellular polymeric substance (EPS) content in activated sludge with lysozyme. The investigation of the effect of lysozyme treatment on the content of extracellular polymers and the biofilm formation of electroactive bacteria suggests that lysozyme can improve the permeability of the positive bacterial cell membrane and thus increase the EPS content in the activated sludge. The characterizations of electrochemical activity, surface morphology and community structure of the anode biofilm indicate that increasing EPS content promotes the adhesion of the mixed bacteria in the activated sludge on the electrode and results in denser biofilms with better conductivities. The microbial fuel cell (MFC) inoculated with the sludge of high EPS content exhibits the power density up to 2.195 W/m2, much higher than that inoculated with the untreated sludge (1.545 W/m2). The strategy of adjusting EPS content in activated sludge with a biological enzyme can effectively enhance the ability of the bacterial community to form biofilms and exhibits great application potentials in the construction of high efficiency bio-electrochemical systems.

A sensitive and simple competitive nanozyme-linked apta-sorbent assay for the dual-mode detection of ochratoxin A.

The enzyme-linked apta-sorbent assay (ELASA) is widely used for the detection of small-molecule compounds as a result of low cost and reagent stability of aptamers. However, enzyme labels used in ELASA still suffer from some drawbacks, such as high production cost and limited stability. To overcome the drawbacks, we reported a nanozyme-linked apta-sorbent assay (NLASA) coupled with surface-enhanced Raman scattering (SERS)-colorimetric dual-mode detection. For nanozyme labels, Pd-Pt bimetallic nanocrystals (Pd-Pt NRs) could catalyze 3,3',5,5'-tetramethylbenzidine (TMB) to blue TMB2+, whose color variation could not only be distinguished by naked eyes but also had a strong SERS signal. The NLASA method was employed to detect ochratoxin A (OTA) with a limit of detection values of 0.097 nM (0.039 ppb) and 0.042 nM (0.017 ppb) via the colorimetric and SERS methods, respectively. This method was applied for the determination of OTA in wine and grape samples, and the detection results were in a satisfied agreement with those determined by the high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method. The proposed NLASA method provided a rapid and sensitive detection for OTA and could also be broadened for other small-molecules.

Improving the anode performance of microbial fuel cell with carbon nanotubes supported cobalt phosphate catalyst.

Microbial fuel cell (MFC) is a sustainable technology that can convert waste to energy by harnessing the power of exoelectrogenic bacteria. However, the poor biocompatibility and low electrocatalytic activities of surface usually cause weak bacterial adhesion and low electron transfer efficiency, which seriously hampers the development of MFCs. Herein, a novel carbon nanotube supported cobalt phosphate (CNT/Co-Pi) electrode is fabricated by assembling CNTs on carbon cloth, followed by the electrodeposition of Co-Pi catalyst. The deposited amorphous Co-Pi thin film contains phosphate and the cobalt ions of multiple oxidation states. The hydrophilic phosphate can promote the adhesion of microorganisms on electrode. The strong conversion ability of multiple states of cobalt offers excellent electrocatalytic activity for the electron transfer across biotic/abiotic interface. Therefore, the highly conductive CNTs substrate, along with the Co-Pi catalyst, provide an effective electron transfer between the electrogenic bacteria and the electrode, which endows MFC high power densities up to 1200 mW m-2. Our work has demonstrated for the first time that CNT/Co-Pi catalyst can promote the interfacial electron transfer between electrogenic bacteria and electrode, and highlighted the application potentials of Co-Pi as an anode catalyst for the fabrication of high performance MFC anodes.

Expression of an alkaline feruloyl esterases from thermophilic Chaetomium thermophilum and its boosting effect on delignification of pulp.

Exploration of feruloyl esterase (FAE) with the resistance to heat and alkali conditions in biobleaching process to improve the separation efficiency of lignocellulose is the key to achieving green papermaking. Herein, we expressed FAEB of C. thermophilum and obtained a thermostable alkaline FAE that can effectively promote the removal of lignin from pulp. The faeB gene was successfully obtained through genomic Blast strategy and high-efficiency expressed under the control of strong alcohol oxidase promoter in Pichia pastoris. The recombinant CtFAEB has an optimal temperature of 65 °C and pH of 7.0. After treated at 65 °C for 1 h, CtFAEB can still retain 63.21 % of its maximum activity, showing a good thermal stability. In addition, the recombinant CtFAEB has broad pH stability and can retain about 56 % of the maximum activity even at pH 11.0. Compared with the effect of mesophilic FAE, pretreatment with thermostable CtFAEB can promote the delignification by laccase and alkaline hydrogen peroxide from the pulp at 70 °C and pH 9.0. Alignment of the protein sequences of CtFAEB and mesophilic FAE suggested that the percentage of amino acids that easily form alpha helix in CtFAEB increases, which enhances its structural rigidity and thereby improves its thermal stability and alkali tolerance. Our study provides an effective method to obtain thermostable and alkaline FAEs, which will promote its application in biobleaching and other biorefining industries.

Boosting the anode performance of microbial fuel cells with a bacteria-derived biological iron oxide/carbon nanocomposite catalyst.

Modifying the electrodes of microbial fuel cells (MFCs) with iron oxides can improve the bacterial attachment performances and electrocatalytic activities for energy conversion, which is of significance in the fabrication of MFCs. However, the conventional modification methods usually result in the aggregation of iron sites, producing the electrodes of poor qualities. Herein, we report a novel method for the modification of electrochemical electrodes to boost the anode performance of MFC. The Shewanella precursor adhered on carbon felt electrode was directly carbonized to form a bacteria-derived biological iron oxide/carbon (Bio-FeOx/C) nanocomposite catalyst. The large spatial separation between the bacteria, as well as those between the iron containing proteins in the bacteria, deliver a highly dispersed Bio-FeOx/C nanocomposite with good electrocatalytic activities. The excellent microbial attachment performance and electron transfer rate of the Bio-FeOx/C modified electrode significantly promote the transfer of produced electrons between bacteria and electrode. Accordingly, the MFC with the Bio-FeOx/C electrode exhibits the maximum power density of 797.0 mW m-2, much higher than that obtained with the conventional carbon felt anode (226.1 mW m-2). Our works have paved a new avenue to the conversion of the natural bacterial precursors into active iron oxide nanoparticles as the anode catalyst of MFCs. The high catalytic activity of the prepared Bio-FeOx endows it great application potentials in the construction of high-performance electrodes.

Improving the power generation performances of Gram-positive electricigens by regulating the peptidoglycan layer with lysozyme.

The power generation performance of a microbial fuel cell (MFC) greatly depends on the relative amount of electricigens in the anodic microbial community. Running the MFC multiple times can practically enrich the electricigens, and thus improve its power generation efficiency. However, Gram-positive electricigens cannot be enriched well because of their thick non-conductive peptidoglycan layer. Herein, we report a new Gram-positive electricigen enrichment method by regulating the peptidoglycan layer of the bacteria using lysozyme. Lysozyme can partially hydrolyze the peptidoglycans layer of Gram-positive Firmicutes to improve the permeability of cell wall, and thus enhance its electricity generation activity. The stimulation of Gram-positive electricigen endows MFCs a high power generation community structure, which results in the power density 42% higher than that of the control sample. Our work has provided a new and simple method for optimizing the anode community structure by regulating weak electricigens in the community with lysozyme.

Cloning, Characterization, and Functional Expression of a Thermostable Type B Feruloyl Esterase from Thermophilic Thielavia Terrestris.

Feruloyl esterases (FAEs) have great potential applications in paper and breeding industry. A new thermo-stable feruloyl esterase gene, TtfaeB was identified from the thermophilic fungus Thielavia terrestris h408. Deduced protein sequence shares the identity of 67% with FAEB from Neurospora crassa. The expression vector pPIC9K-TtfaeB was successfully constructed and electro-transformed into GS115 strain of Pichia pastoris. One transformant with high feruloyl esterase yield was obtained through plate screening and named TtFAEB1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TtFAEB1 showed a distinct protein band appearing at the position of about 35-kDa, indicating that TtfaeB gene has been successfully expressed in P. pastoris. The recombinant TtFAEB was purified by affinity chromatography and the specific activity of purified TtFAEB was 6.06 ± 0.72 U/mg. The optimal temperature and pH for purified recombinant TtFAEB was 60 °C and 7.0, respectively. TtFAEB was thermostable, retaining 96.89 and 84.16% of the maximum activity after being treated for 1 h at 50 °C and 60 °C, respectively. Additionally, the enzyme was stable in the pH range 4.5-8.0. The homology model of TtFAEB showed that it consists of a single domain adopting a typical α/ß-hydrolase fold and contains a catalytic triad formed by Ser117, Asp201, and His260. TtFAEB in association with xylanase from Trichoderma reesei could release 77.1% of FA from destarched wheat bran. The present results indicated that the recombinant TtFAEB with excellent enzymatic properties is a promising candidate for potential applications in biomass deconstruction and biorefinery.

Influence of Randomly Inserted Feruloyl Esterase A on ß-Glucosidase Activity in Trichoderma reesei.

As a well-known industrial fungus for cellulase production, the strain RUT-C30 of Trichoderma reesei was selected to produce the feruloyl esterase A (FAEA) by a random integration protocol. The strong promoter of cellobiohydrolase 1 (cbh1) gene was used to drive the expression of FAEA. Using double-joint PCR protocol, Pcbh1-faeA-TtrpC expression cassette was successfully constructed and co-transformed into RUT C30 strain of T. reesei. One transformant with high feruloyl esterase yield (3.44 ± 0.16 IU/mL) was obtained through plate screening and named TrfaeA1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TrfaeA1 showed a distinct protein band appearing at the position of about 34 kDa, indicating that faeA gene has been successfully expressed in T. reesei. Compared with that in original RUT C30 strain, ß-glucosidase production in transformant TrfaeA1 was significantly increased by about 86.4%, reaching 63.2 IU/mL due to the random insertion of faeA. Moreover, the total secretion protein and filter paper activities of the transformant TrfaeA1 were also improved by up to 5.5 and 4.3%, respectively. The present results indicated that the random insertion strategy could be an effective and feasible method to improve and optimize the cellulase system of filamentous fungi.

Enhanced phosphorus flux from overlying water to sediment in a bioelectrochemical system.

This report proposed a novel technique for the regulation of phosphorus flux based on a bioelectrochemical system. In the simulated water system, a simple in situ sediment microbial fuel cell (SMFC) was constructed. SMFC voltage was increased with time until it was 0.23V. The redox potential of the sediment was increased from -220mV to -178mV during the process. Phosphorus concentration in the water system was decreased from 0.1mg/L to 0.01mg/L, compared with 0.09mg/L in the control. The installation of a SMFC produced an external current and internal circuit, which promoted the transfer of phosphate in overlying water to the sediment, enhanced the microbial oxidation of Fe(2+), and increased the formation of stable phosphorus in sediment. In conclusion, phosphorus flux from the overlying water to sediment was enhanced by SMFC, which has the potential to be used for eutrophication control of water bodies.

Denitrification of overlying water by microbial electrochemical snorkel.

A novel microbial electrochemical snorkel (MES) bioreactor was constructed by inserting an iron rod into the sediment of a simulated natural water body for the first time. Its nitrate removal performance and mechanism were investigated. The DNA high-throughput sequencing analysis indicates that denitrifying bacteria were grown on the iron rod in the overlying solution. The XRD analysis on the oxides formed on the surface of the iron rod indicates that they are goethite and green rust. In the MES system, the green rust on the iron rod can concentrate nitrate and denitrifying bacteria, forming an anaerobic biocathode. The denitrifying bacteria can reduce the nitrate into nitrogen with the electrons moved from the sediment. The nitrate removal efficiency reached 98% in 16days. This novel MES system showed excellent in-situ nitrate removal performance by moving and concentrating the electrons in sediment and the nitrate in overlying solution in an anaerobic microenvironment.

Preparation and antibacterial activity of lysozyme and layered double hydroxide nanocomposites.

It is necessary to develop "green" disinfection technology which does not produce disinfection by-products. Lysozyme-layered double hydroxide nanocomposites (LYZ-LDHs) were prepared by intercalating LYZ in LDH for the first time. Their antibacterial activity was evaluated using staphylococcus aureus as a target. The bacteria removal mechanism was also studied. Characterization of LYZ-LDHs by X-ray diffraction and Fourier transform infrared spectroscopy indicated that LYZ was successfully intercalated in LDH, compressed and deformed without secondary structural change. LYZ-LDHs showed excellent bactericidal effectiveness against staphylococcus aureus. The antibacterial performance of LYZ-LDHs was found to be affected by the LYZ/LDH ratio and the pH of the bacteria-containing water. The bacteria removal efficiency of LYZ-LDHs with LYZ/LDH mass ratio of 0.8 was consistently above 94% over the pH range of 3-9. LYZ-LDHs adsorbed bacteria to their surface by LDH and then killed them by the immobilized LYZ. This new material integrated the bactericidal ability of LYZ and adsorption ability of LDH. Moreover, the antibacterial ability of LYZ-LDHs was persistent and not limited by the adsorption capacity.

Molecular structure matters: PEG-b-PLA nanoparticles with hydrophilicity and deformability demonstrate their advantages for high-performance delivery of anti-cancer drugs.

Various carriers are being advanced for anti-cancer therapy, which can protect drugs and ferry them to the target site. However, little understanding exists regarding the effect of molecular structure on anti-cancer drug delivery efficiency. To fill this knowledge gap, we take poly(lactic acid) (PLA), poly(lactide-co-glycolide) (PLGA), and poly-ethylene glycol-co-poly-lactide (PEG-b-PLA) polymers as prototype materials and comparatively explore the inherent relationship between the molecular structure and the delivery ability. Compared with PLA and PLGA NPs, PEG-b-PLA ones possess the advantages of longer blood circulation time, more tumor accumulation, and better intratumoral delivery ability. Subsequent mechanism investigations reveal that the molecular structure will regulate the polymer arrangement and render NPs different hydrophilicity/deformability, which dictate the distinct delivery performances. Finally, the superior PEG-b-PLA NPs are further loaded with the anti-cancer drug paclitaxel (PTX) and functionalized with magnetic (M) Fe3O4 nanocrystals. As-designed PTX/M PEG-b-PLA NPs show much better tumor inhibition efficacy and fewer side effects than the commercialized Taxol® formulation, strongly supporting their use as high-performance carriers for anti-cancer therapy.

Mechanisms for the direct electron transfer of cytochrome c induced by multi-walled carbon nanotubes.

Multi-walled carbon nanotube (MWCNT)-modified electrodes can promote the direct electron transfer (DET) of cytochrome c (Cyt c). There are several possible mechanisms that explain the DET of Cyt c. In this study, several experimental methods, including Fourier transform infrared spectroscopy, circular dichroism, ultraviolet-visible absorption spectroscopy, and electron paramagnetic resonance spectroscopy were utilized to investigate the conformational changes of Cyt c induced by MWCNTs. The DET mechanism was demonstrated at various nano-levels: secondary structure, spatial orientation, and spin state. In the presence of MWCNTs, the secondary structure of Cyt c changes, which exposes the active site, then, the orientation of the heme is optimized, revolving the exposed active center to the optimum spatial orientation for DET; and finally, a transition of spin states is induced, providing relatively high energy and a more open microenvironment for electron transfer. These changes at different nano-levels are closely connected and form a complex process that promotes the electron transfer of Cyt c.