A sulfur autotrophic fluidized bed reactor was established to overcome mass transfer problems and shorten the co-reduction time for perchlorate and nitrate from aqueous solution. Response surface methodology (RSM) was used to predict the subsequent extent of sulfur (S) disproportionation based on three variable parameters, namely the hydraulic retention time (HRT), co-existing nitrate concentration (C_〖inf 〗〖〖NO〗_3^–N〗 ) and recirculation ratio (R). The extent of sulfur (S) disproportionation was determined by sulfate generation and alkalinity consumption. RSM results show that a long HRT and high R promoted whereas co-existing nitrate inhibited sulfate production from S disproportionation. For complete perchlorate and nitrate reduction (>98.45%) with relatively low sulfate generation (236.07 mg/L), the optimal HRT, C_〖inf 〗〖〖NO〗_3^–N〗 and R were 0.50 h, 10 mg/L and 14, respectively. High-throughput sequencing revealed that Chlorobaculum was associated with S disproportionation while the abundance of Sulfurovum, an effective denitrification/perchlorate reducing bacteria, decreased over the height of the reactor.
Response surface methodology (RSM) was used to optimize the oxidation of the omeprazole sulfide to (S)-omeprazole catalyzed by environmentally friendly catalyst soybean pod peroxidase (SPP) in cetyltrimethylammonium bromide (CTAB)/isooctane/n-butyl alcohol/water water-in-oil microemulsions. With the initial concentration of SPP of 3200 U ml-1, the conversion of the omeprazole sulfide, the (S)-omeprazole yield and ee were 93.75%, 91.56% and 96.08%, respectively, under the optimal conditions: Wo of 15.85, the concentration of H2O2 of 22.44 mM and reaction temperature of 49.68 ℃, respectively. The proposed mechanism of asymmetric sulfoxidations catalyzed by SPP involves three concomitant mechanisms as follows: (1) a two-electron reduction of SPP-I, (2) a single-electron transfer to SPP-I and (3) nonenzymatic reactions, including five enzymatic and two nonenzymatic reactions, which is reasonable and can express the oxidations. With 5.44% of the average relative error, a kinetic model based on the mechanisms fitting observed data very well was established, and the SPP-catalyzed reactions including both the two-electron reduction and the single-electron transfer mechanisms obey ping-pong mechanism with substrate and product inhibition, while nonenzymatic reactions follow a power law. This study has also demonstrated the feasibility of SPP as a substitute with low cost, excellent enantioselectivity and better thermal stability.
This study investigated the mutagenic tolerance of eight Bambara groundnut genotypes to Sodium azide (NaN3) using SDS-PAGE. The seeds of six genotypes; TVSu-86, TVSu-91, TVSu-186, TVSu-235, TVSu-242, TVSu-350 were collected from IITA and two landraces from Abia and Enugu local markets. The seeds were treated with five concentrations: 0.00%(control), 0.01%, 0.03%, 0.05% and 0.07% of NaN3 after pre-soaking for 6hrs in distilled water and sown in pots arranged in a Complete Randomized Design with three replicates. There was reduction in germination percentage and growth characters as concentrations of NaN3 increases. Early flowering was recorded at 37 days mutated with 0.07% of NaN3 compared to control which flowered late at 42 days. NaN3(0.07%) caused lethal effect on Abia and Enugu landraces. There was no significant (P>0.05) difference in yield traits among mutants and control. Mutant seeds significantly (P<0.05) increased protein content (19.12%) at 0.05% of NaN3 compared to control(18.5%). The number of seeds(0.99), seed yield(0.89) and pod yield(0.96) strongly correlated with seeds per pod (0.85). The SDS-PAGE revealed the presence of polypeptide bands in mutants compared to control. TVSu-235 and TVSu-350 genotypes had higher tolerance and yield traits to 0.01% concentration of NaN3, thus could be further improved in subsequent breeding.
Virus filtration is a critical process in the production of biotherapeutics and drug products derived by plasma fractionation. The filterability of process solutions on virus removal filters is largely dependent on preceding downstream process steps, and column chromatography can have a particularly large impact on the throughput and flux of virus filtration. Filterability (throughput and flux) on Planova BioEX was greatly reduced for mAb and plasma IgG spiked with aggregate, and filterability improvement achieved by processing with chromatography resins (modified CEX, mixed-mode AEX and normal AEX column) was specific to protein solution. The various protein solutions spiked with aggregate showed distinct solution characteristics and by using the resulting filtration volume and flow rate, experimental and calculated filtration parameters were compared and evaluated with four clogging models: cake filtration, intermediate blocking, standard blocking and complete blocking. Effective purification of feed solutions by column chromatography has the capability to improve virus filtration processes. Further, the application of filtration parameters to the appropriate clogging model makes it possible to extrapolate filtration behavior to larger processing volumes.
L-cysteine is a ubiquitous and unique sulfur-containing amino acid with important physiological functions. The efficient L-cysteine production via microbial fermentation is interesting and has been paid great attention. In this study, different Escherichia coli K-12 strains (JM109, BW25113, MG1655, W3110) were investigated on their suitability to cysteine-producing plasmid pLH03. The enhancement of precursor synthetic pathway and thiosulfate assimilation pathway resulted in the good performance of BW25113. The expressions of synthetic pathway genes were optimized by two constitutive promoters to assess their effects on L-cysteine production. Main degradation pathway genes were also deleted coordinately for more efficient production of cysteine. The L-cysteine production was further increased through the manipulation of sulfur transcription regulator cysB and sulfur supplement. After the process optimization in a 1.5 L bioreactor, the final engineered strain LH2A1M0B△YTS-pLH03 [BW25113Ptrc2-serA-Ptrc1-cysM- Ptrc-cysB△yhaM△tnaA△sdaA-(pLH03)] accumulated 8.34 g/L of cysteine, laying a certain foundation for cysteine fermentation industry.
Cyanobacterial carbonate precipitation induced by cells and extracellular polymeric substances (EPS) enhances the mortar durability. The percentage of cell/EPS attachment regulates the effectiveness of the mortar restoration. This study investigates the cell coverage on mortar and microbially induced carbonate precipitation. Statistical analysis of results from scanning electron and fluorescence microscopy show that the cell coverage was higher in the presence of UV-killed cells than living cells. Cells preferably attached to cement paste than sand grains, with a difference of one order of magnitude. The energy dispersive X-ray spectroscopy analyses and Raman mapping suggest cyanobacteria used atmospheric CO2 to precipitate carbonates.
Nanotechnology plays a promising role in biomedical applications, particularly tissue engineering. Recently, the application of magnetic scaffolds and pulsed electromagnetic field (PEMF) exposure has been considered in bone tissue regeneration. In this study, 3rd generation dendrimer-modified superparamagnetic iron oxide nanoparticles (G3-SPIONs) are synthesized comprehensively characterized. Magnetic polycaprolactone (PCL) nanofibers are prepared by incorporating G3-SPIONs within the electrospinning process ,and physicochemical characteristics ,as well as cytocompatibility and cell attachment are assessed. Eventually, the osteogenic differentiation ability of adipocyte-derived mesenchymal stem cells (ADMSCs) cultured on the magnetic scaffold with and without PEMF exposure was investigated by measurement of alkaline phosphatase (ALP) activity and calcium content. The expression of specific bone markers was studied using the Real-time PCR method. According to the results, G3-SPIONs with mean size and zeta potential of 17.95 ± 3.57 nm and 22.7 mV, respectively, show a high saturation magnetization (57.75 emu/g). Adding G3-SPIONs to PCL significantly decrease nanofibers size to 495±144 nm and improves cell attachment and growth. The ADMSCs cultured on the G3-SPION-PCL scaffold in the presence of osteogenic media (OM) and exposure to PEMF expressed the highest Osteocalcin and Runx2 and showed higher calcium content as well as ALP activity. It can be concluded that the synthesized G3-SPION incorporated PCL nanofibers serve as a promising magnetic scaffold for bone regeneration. Also, utilizing the magnetic scaffold in the presence of OM and PEMF provides a synergistic effect toward osteogenic differentiation of ADMSCs. Key Words: Superparamagnetic iron oxide nanoparticles, Dendrimer, Polycaprolactone, Pulsed electromagnetic field, Bone tissue engineering
Several hundred U mL-1 of Nattokinase (NK), a fibrinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking flask. For use as a nutraceutical, large-scale preparation and a simple purification process is required. The present study utilized a fed-batch process with a pH-stat and low-glycerol-level-maintain feeding strategy to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). Finally, a NK activity of 7778 ±17.28 U mL-1 was obtained, which represented a 26-fold increase of NK activity by high cell density cultivation compared to the flask culture. Furthermore, fermentation supernatant was successively purified by ammonium sulfate precipitation and nickel column affinity chromatography with a total NK recovery rate of 65.2%.
The production of purified virus particles with high quality and quantity for vaccine preparation requires scalable purification procedure in downstream step. A purification scheme based on combined strong anion-exchange and size exclusion chromatography (2D -AEC×SEC) is developed for production non-structural protein (NSP) free foot and mouth diseases (FMD) vaccine and the whole procedure is accomplished with 78 % recovery, 85 % virus yield and more than 90 % of residual DNA (rDNA) is removed from the purified vaccine. Due to use AEC as the first column, the injection volume increases four times compare to previous report. Alternatively, a mathematical modeling and simulation approach based on plate model chromatography are developed and matched with the experimental chromatography data to obtain better perception in predicting retention behavior and saving time in downstream scale-up method development. The analysis of purified virus particles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) high performance size exclusion chromatography (HP-SEC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), transmission electron microscopy (TEM) and biological test provide to the best quality of purified FMD virus.
Electrospinning has been widely used for the development of fibrous scaffolds for cartilage tissue engineering (TE), however their small pores significantly limit cell infiltration throughout the scaffolds, particularly in three-dimensional (3D) designs. In this endeavor, a direct incorporation of chondrocytes into the fibers mesh during the electrospinning presents itself as a promising solution by use of bio-electrospraying. Yet, for this technology to be effectively employed for cartilage TE, it is necessary to assess if chondrocytes are in any way adversely affected. So, in this work, several electrospraying experiments were performed by adjusting various operational parameters to evaluate their influence on chondrocyte viability and function. A high percentage of post-electrosprayed chondrocytes remained viable upon the exposure of an external electric field generated by low needle to collector distances and low applied voltages. No obvious differences were found with non-electrosprayed chondrocytes in terms of viability, morphology and proliferation. The data reported here further suggest that bio-electrospraying under the optimal operational conditions might be a promising alternative to the existent cell seeding techniques, promoting not only cells safe delivery to the scaffold, but also the development of highly cellularized and uniform tissue constructs for cartilage repair.
Celery spices are an important raw material in the production of food and cosmetic, including carveol, carvone and dihydrocarvone. In this study, a new Klebsiella sp. O852 was isolated, which was capable of converting limonene to trans-dihydrocarvone. The yields of trans-dihydrocarvone reached up to 1058 mg/L when the strain O852 was incubated using LB-M medium for 4 h at 36ºC and 150 rpm and the process was monitored for 36 h after adding 1680 mg/L limonene dissolved in ethanol. Limonene was used as a 20% (v/v) solution in the ethanol. Besides, the genome of Klebsiella sp. O852 comprised 20 contigs and 19 scaffolds. The genome size was 5.49 Mb. A total of 5218 protein-encoding genes were predicted, these genes mainly distributed in some metabolism and biosynthesis categories. Finally, several genes involved in trans-dihydrocarvone biosynthesis were further analyzed, identified and verified by quantitative real-time polymerase chain reaction (RT-qPCR) and exogenous expression. These novel genes may find value in the bioconversion of inexpensive raw materials to natural flavors and fragrances.
L-ornithine is a basic amino acid, which shows significant value in food and medicine industries. There is a huge space for L-ornithine production with strains available for metabolic engineering, and it is urgent to develop a high-efficiency engineering strain for industrialization. Here, xylose isomerase and xylulose kinase were introduced into Corynebacterium glutamicum S9114 to establish xylose metabolism pathway, and then xylose became a substitute carbon source of glucose. In addition, the optimization and overexpression of phosphoenolpyruvate carboxylase and pentose transporter have been conducted to promote the synthesis of L-ornithine for the first time. Furthermore, though optimizing the concentration ratio of glucose and xylose (7:3), adding biotin and thiamine hydrochloride, we arrived at the highest L-ornithine yield 41.5g/L in shaking flask fermentation so far. Our results demonstrate that the combination of metabolic engineering and the optimization of fermentation process can make great potential for L-ornithine production by lignocellulose hydrolysate.
Laccases are oxidoreductases with the outstanding ability to oxidize phenolic and non-phenolic substrates coupled to the reduction of O2 to H2O. Among them, bacterial enzymes are suitable biocatalysts for application in industrial processes under harsh conditions. However, to be active on high redox potential substrates, bacterial laccases requires of redox mediators: electron carriers between the laccase and other compounds not directly oxidizable by the enzyme. Here we demonstrate that β-(10-phenothiazyl)-propionic acid can be used as an efficient and low-cost redox mediator for decolorization of synthetic dyes by bacterial laccases. Using this laccase-mediator system, more than 80% of Indigo Carmine and Malachite Green decolorization was reached after 1 h or 2 h of incubation, respectively, both at pH 8 and in tap water (pH 6.8). Furthermore, more than 40% of Remazol Brilliant Blue R and 80% of Xylidine ponceau were decolorized after 5 h at pH 8 and 50°C. In addition, we showed this system supports at least 3 decoloration cycles without loss of activity, representing a promising biological process for cost-effective and environmentally friendly decolorization and degradation of synthetic dyes and for other industrial applications of laccases requiring neutral or alkaline pH.
The industrial production of active proteins from E. coli necessitates the refolding of high concentrations of protein over a short period of time. However, it is difficult to simultaneously achieve high concentration and short residence time. The dialysis method can refold high concentrations of proteins, but this process takes a long time. The dilution method can quickly refold proteins, but the resultant proteins are inevitably diluted. In the present study, by designing microchannels in dialysis membranes, which can enlarge the surface area for quickly removing protein denaturant, high concentrations of active carbonic anhydrase---eight times more concentrated than achieved using dilution method---were refolded in 20 minutes, which is orders of magnitude faster than the conventional dialysis method.
Human microbial alterations are associated with environmental stress, nutritional, genetic and triggering de-novo variations. Nevertheless, human gut microbiome at extreme altitude (>5800 m) remains unexplored. We aimed to demonstrate the microbial predominance in individuals with same ethnicity and dietary pattern at extreme altitude with unique challenges like cold, hypoxia, radiation etc. Different analysis pipelines were used for fecal whole genome sequencing at 210m, 3500m, 4420m and 5805m, and 16s rRNA V3-V4 regions amplification sequencing of 19 individuals belonging to the same ethnicity and dietary pattern, for presence of taxonomy & functional potential and confirming the prediction upto the strain level within the same cohort. Principal component analysis, revealed distinct microbiome changes at different altitudes, with varied and higher Bacteroides and Prevotella ratio. There was predominance of genus Prevotella at altitudes 4420m & 5805m than at 210m & 3500m. Appearance of species Prevotella copri strain 61740 was increasing significantly at extreme altitudes, whereas co-occurrence of other bacterial strains had different pattern than Prevotella. The extensive strain level analysis indicated alteration in the metabolic pathways. This study under stressful and hypoxic environment of extreme altitudes, associated microbial variation with altered metabolic pathways, reveals influence of extreme environment on human gut microbiota with predominance of Prevotella.
Production of specialty chemicals increasingly makes use of enzyme catalysts, and Novozym 435 (N435) is among most often applied. However, its polymeric skeleton is unstable in many solvents. In this context, we report results of a systematic study of the biocatalysts, fabricated using highly porous siliceous pellets/enzyme (MH), grafted with octyl (-O), amino (-A) and octyl and amino (-OA) groups, deployed in a rotating bed reactor and tested in hydrolysis and esterification reactions. N435 appeared the most active in both reactions, when activity was related to the catalyst’s mass, mainly owing to very large enzyme load. But its structure degraded in many typical solvents, whereas no such effect was detected in MH-O- and MH-OA-catalysts. MH-O showed the highest specific activity, however, a significant enzyme leaching was observed in a hydrolytic reaction, in contrast to MH-OA. In esterification reaction the MH-O-bound lipase was not only most active but also quite stable.
Flux experiments and fluorescence microscopy were combined and optimized to visualize the membrane surface during biofouling of two mixed cellulose ester membranes. Using flux measurements, the fouling by bovine serum albumin (BSA) was measured in the presence of 1 to 12% labeled BSA. By fitting the relative flux decays to an exponential decay for statistical analysis, the dye in this range of labeled protein was found to not affect the fouling nature of the protein. A 2.5% or 5% labeled protein sample was determined to be the best percent labeled protein for fluorescence imaging the membrane because the beginning of cake formation was observed within 25 min of experimental time. Finally, by fitting the flux data to four different biofouling mechanism equations, we conclude that both membranes, though at different rates, have BSA depositing inside the membrane pores restricting the flow eventually leading to cake formation. The combination of the two techniques allows for further insight into the biofouling mechanism of BSA, and this method can be applied to other biological molecules.