Simultaneous estimation of chlorophyll a and lipid contents in microalgae by three-color analysis.

A method of rapid determination of chlorophyll a and lipid contents of microalgae based on colorimetric analysis of the digital images of the microalgae is proposed. The color variation of microalgae during cultivation is evaluated by the brightness of the three primary colors (red, green, and blue). The brightness values of the three primary colors are modeled as two linear correlation functions (RGB model) for microalgal chlorophyll a and lipid contents, respectively. The chlorophyll a and lipid contents predicted by the proposed model are compared with that determined by the standard methods. The good agreement of the model predictions with experimental results is demonstrated with a squared correlation coefficient (R(2)) of 0.99 for chlorophyll a and lipid. The reliability of the RGB model was verified in real cultivations of the microalgae in a photobioreactor. Growth dynamics, contents of chlorophyll a and lipid corresponded very well with previously reported studies.

A novel approach for medium formulation for growth of a microalga using motile intensity.

Motile intensity of the cells, defined as the specific mean kinetic energy, was measured by image analysis and used to formulate a suitable medium for the cultivation of a motile microalga. Nitrogen source at 60 mg/L was used as the target component. The cells grown in a medium containing urea showed the highest motile intensities during cultivation when compared to the cells grown with NH(4)Cl or (NH(4))(2)SO(4) as the nitrogen source. The urea culture gave the highest biomass yield and lipid content of 1.06+/-0.12 g/L and 22.34+/-2.56%, respectively after 150 h of cultivation. These results indicated a strong dependence of motile intensity on the nitrogen source used in the growth medium. This concept, which requires only a small droplet of the sample, can find potential application in the design and optimization of culture media.

Production of ascorbyl palmitate by surfactant-coated lipase in organic media.

The surface of a lipase from Burkholderia cepacia was coated with a nonionic surfactant, propylene glycol monostearate, and was used as a biocatalyst in the production of ascorbic acid in tert-butyl alcohol. The influence of various factors such as the type of surfactant, the pH of the buffer used for coating, the amount of surfactant in the coating, the organic solvent, and the temperature and molar ratio of the substrates used in the reaction on the conversion of ascorbyl palmitate were studied. After 24 h of reaction at 50 degrees C, a conversion of 47% was obtained using an ascorbic acid to palmitic acid molar ratio of 1:6. The native lipase showed only 6% conversion.

Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups.

A method for immobilization of Candida rugosa lipase to two types of chitosan beads by activating the hydroxyl groups of chitosan using carbodiimide coupling agent has been successfully developed. The ability of carbodiimide to activate the hydroxyl groups of chitosan was confirmed using the electron spectroscopy for chemical analysis (ESCA) technique. The properties of lipase immobilized using dry and wet chitosan beads were also investigated and compared. Immobilization enhanced the enzyme stability against changes of pH and temperature. High storage stability of 30 days and an increased enzyme activity of 2,110% were observed in wet immobilized lipase. Immobilized lipase using dry and wet chitosan beads retained 78% and 85% of its initial activity after 10 batch hydrolytic cycles. The kinetic parameters Km and Vmax were determined for the free and immobilized lipase. The activation energy (Ea) was found to decrease for immobilization of lipase on chitosan beads.

Regeneration of immobilized Candida antarctica lipase for transesterification.

Immobilized lipase from Candida antarctica was employed to convert triglycerides to biodiesel using alcohol. Immobilized lipase is frequently deactivated by lower alcohols with deactivation being caused by the immiscibility between triglycerides and methanol or ethanol. When the lower alcohol is adsorbed to the immobilized enzyme, the entry of triglycerides is blocked, which causes the reaction to stop. An alcohol with three or more carbon atoms, preferably 2-butanol or tert-butanol, can regenerate the deactivated immobilized enzyme. The present work established that the activity of immobilized lipase could be significantly increased when such alcohols were used for an immersion pretreatment of the enzyme. The activity of the commercially available immobilized enzyme, Novozyme 435, increased about tenfold in comparison to the enzyme not subjected to any pretreatment. Following complete deactivation of the enzyme by methanol, washing with 2-butanol and tert-butanol successfully regenerated the enzyme and restored it to about 56% and 75% of its original activity level, respectively.

A nanopatterned cell-seeded cardiac patch prevents electro-uncoupling and improves the therapeutic efficacy of cardiac repair.

The heart is an extremely sophisticated organ with nanoscale anisotropic structure, contractility and electro-conductivity; however, few studies have addressed the influence of cardiac anisotropy on cell transplantation for myocardial repair. Here, we hypothesized that a graft's anisotropy of myofiber orientation determines the mechano-electrical characteristics and the therapeutic efficacy. We developed aligned- and random-orientated nanofibrous electrospun patches (aEP and rEP, respectively) with or without seeding of cardiomyocytes (CMs) and endothelial cells (ECs) to test this hypothesis. Atomic force microscopy showed a better beating frequency and amplitude of CMs when cultured on aEP than that from cells cultured on rEP. For the in vivo test, a total of 66 rats were divided into six groups: sham, myocardial infarction (MI), MI + aEP, MI + rEP, MI + CM-EC/aEP and MI + CM-EC/rEP (n ≥ 10 for each group). Implantation of aEP or rEP provided mechanical support and thus retarded functional aggravation at 56 days after MI. Importantly, CM-EC/aEP implantation further improved therapeutic outcomes, while cardiac deterioration occurred on the CM-EC/rEP group. Similar results were shown by hemodynamic and infarct size examination. Another independent in vivo study was performed and electrocardiography and optical mapping demonstrated that there were more ectopic activities and defective electro-coupling after CM-EC/rEP implantation, which worsened cardiac functions. Together these results provide comprehensive functional characterizations and demonstrate the therapeutic efficacy of a nanopatterned anisotropic cardiac patch. Importantly, the study confirms the significance of cardiac anisotropy recapitulation in myocardial tissue engineering, which is valuable for the future development of translational nanomedicine.

Controlled delivery of recombinant adeno-associated virus serotype 2 using pH-sensitive poly(ethylene glycol)-poly-L-histidine hydrogels.

Loading of viral vectors in synthetic polymers is a promising strategy for overcoming hurdles associated with viral gene delivery. For enhanced gene expression at a specific site, gene transfer by using hydrogels represents a versatile approach. In this study, adeno-associated virus serotype 2 containing the green fluorescent protein gene (rAAV2-GFP) were loaded into poly(ethylene glycol) (PEG) hydrogels, with and without incorporation of poly-L-hisditine (polyHis). Inclusion of polyHis created pH responsive hydrogels in a physiological range of tissues, containing the damaged vasculature and activated phagocytosis. The fraction of polyHis used controlled the degree of swelling, water uptake and subsequent degradation of the hydrogels and release rate of rAAV2-GFP. The swelling ratio of the PEG-polyHis hydrogels increased inversely with environment pH. As pH declined from 7.4 to 6.0, PEG-polyHis hydrogel swelling ratio and degradation rate increased 875% and 135%, respectively. As a result, release and transduction efficiency of the rAAV2-GFP from PEG-polyHis hydrogel in human HT-1080 fibrosarcoma cells increased significantly compared to a PEG hydrogel. Transduction rate can be controlled by the hydrogels' polyHis concentration and is sensitive to localized decreases in pH consistent with inflammation. This is relevant to optimizing parameters for wound care and regenerative medicine applications.

Immobilization of cellulase onto electrospun polyacrylonitrile (PAN) nanofibrous membranes and its application to the reducing sugar production from microalgae.

This study demonstrates a method to prepare an immobilized cellulase by using an electrospun polyacrylonitrile (PAN) nanofibrous membrane as the support. To obtain an immobilized cellulase with high hydrolytic activity, the immobilization conditions including activation time, enzyme concentration, immobilization time, and temperature were optimized. Under those conditions, the immobilized cellulase possessed a protein loading of 30 mg/g-support and a specific activity of 3.2U/mg-protein. After immobilization, the enzymatic stability of cellulase against pH and thermal stresses was improved. Fourier transform infrared spectroscopy (FTIR) measurements also revealed that the cellulase was covalently bonded to the supports. The immobilized cellulase was then used to hydrolyze cell wall of microalgae for the production of reducing sugars. Analyses using response surface methodology (RSM) show that the hydrolysis yield was affected by the reaction temperature, pH, and substrate/cellulase mass ratio, and a hydrolysis yield of 60.86% could be obtained at 47.85°C, pH 5.82, and a substrate/cellulase mass ratio of 40 g-substrate/g-cellulase. This result suggests that the proposed scheme for the cellulase immobilization has great potential for the application to the reducing sugar production.

Hydrolysis of microalgae cell walls for production of reducing sugar and lipid extraction.

In this study, cell walls of microalgae were hydrolyzed for the production of reducing sugar by cellulase, which was immobilized onto an electrospun polyacrylonitrile (PAN) nanofibrous membrane. Since the nitrile groups of the PAN membrane were activated by the amidination reaction and covalent binding to the amino groups of the cellulase, electrospun PAN nanofibrous membranes with a high specific surface area were applied as supports for the immobilization. Under the optimal hydrolysis conditions, the immobilized cellulase performed its hydrolyzing conversion at 62%, and the hydrolysis yield remained at 40% after five times of reuse. Additionally, microalgal lipid extraction efficiency increased to around 56% from 32% dramatically after cell wall hydrolysis. These results demonstrate the efficacy and feasibility of the proposed applications in hydrolysis process followed with lipid extraction.

Estimation of fungal biomass and lipid production by morphological characteristics of Mucor rouxii.

This study presents methodology for estimating biomass and lipid production by Mucor rouxii via image analysis. Morphological variations in M. rouxii in relation to its biomass and lipid production were investigated using image analysis. Two parameters were used to characterize fungal morphology: fungal area per unit volume of the culture broth (A(f); cm(2)/ml) and fungal pellet circularity skewing (S(K)). These two parameters were correlated with both fungal biomass and lipid production and were used to develop an empirical model. This empirical model was further applied to estimate biomass yield and lipid production by M. rouxii under diverse culture conditions. Model predictions for fungal biomass and lipid production were in agreement with experimental results, indicating the reliability of image-based methodology. Thus, image-based methodology can be used to estimate fungal biomass and lipid production.

Cultivation of microalgae for oil production with a cultivation strategy of urea limitation.

The microalgae, Chlorella sp., were cultivated in various culture modes to assess biomass and lipid productivity in this study. In the batch mode, the biomass concentrations and lipid content of Chlorella sp. cultivated in a medium containing 0.025-0.200 g L(-1) urea were 0.464-2.027 g L(-1) and 0.661-0.326 g g(-1), respectively. The maximum lipid productivity of 0.124 g d(-1)L(-1) occurred in a medium containing 0.100 g L(-1) urea. In the fed-batch cultivation, the highest lipid content was obtained by feeding 0.025 g L(-1) of urea during the stationary phase, but the lipid productivity was not significantly increased. However, a semi-continuous process was carried out by harvesting the culture and renewing urea at 0.025 g L(-1) each time when the cultivation achieved the early stationary phase. The maximum lipid productivity of 0.139 g d(-1)L(-1) in the semi-continuous culture was highest in comparison with those in the batch and fed-batch cultivations.

Effects of biomass weight and light intensity on the performance of photosynthetic microbial fuel cells with Spirulina platensis.

Microalgae Spirulina platensis were attached to the anode of a membrane-free and mediator-free microbial fuel cell (MFC) to produce electricity through the consumption of biochemical compounds inside the microalgae. An increase in open circuit voltage (OCV) was observed with decreasing light intensity and optimal biomass area density. The highest OCV observation for the MFC was 0.39 V in the dark with a biomass area density on the anode surface of 1.2 g cm(-2). Additionally, it was observed that the MFC with 0.75 g cm(-2) of biomass area density produced 1.64 mW m(-2) of electrical power in the dark, which is superior to the 0.132 mW m(-2) produced in the light. Which also means the MFC can be applied to generate electrical power under both day and night conditions.

High cell density cultivation of Escherichia coli with surface anchored transglucosidase for use as whole-cell biocatalyst for alpha-arbutin synthesis.

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for alpha-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of alpha-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.

Immobilization of lipase to chitosan beads using a natural cross-linker.

Genipin, a reagent of plant origin was used for the immobilization of lipase by cross-linking to chitosan beads. The catalytic properties and operational and storage stabilities of the immobilized lipase were compared with the soluble lipase. Under optimum conditions, 198 microg protein was bound per g chitosan with a protein-coupling yield of 35%. The hydrolytic activity was 10.8 U/g chitosan and the relative specific activity was 108%. The immobilized lipase showed better thermal and pH stabilities compared to the soluble form. The immobilized enzyme exhibited mass transfer limitations as reflected by a higher apparent K(m) value and a lower energy of activation. The immobilized enzyme retained about 74% of its initial activity after five hydrolytic cycles.

Production of mouse embryoid bodies with hepatic differentiation potential by stirred tank bioreactor.

Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.

Production of ascorbic acid glucoside by alginate-entrapped mycelia of Aspergillus niger.

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l(-1) maltose, 66 g l(-1) yeast extract, and 5 g l(-1) K(2)HPO(4) at 30 degrees C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l(-1) ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l(-1) h(-1) and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l(-1), and the productivity was 1.5 g l(-1) h(-1). The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l(-1) of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l(-1) h(-1) was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.

Surface display of transglucosidase on Escherichia coli by using the ice nucleation protein of Xanthomonas campestris and its application in glucosylation of hydroquinone.

A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12,846 for display of transglucosidase has been developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12,608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expressed transglucosidase fusion protein to the cell surface of E. coli with apparent high enzymatic activity. The localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40 degrees C for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83%. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16% only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.

Synthesis of mixed esters of ascorbic acid using methyl esters of palm and soybean oils.

Mixed esters of ascorbic acid were synthesized using methyl esters of palm and soybean oils as acyl donors, in acetone at 50 degrees C, and catalyzed by Novozym 435. A conversion of 62% was obtained with palm oil methyl ester at an ascorbic acid to acyl donor molar ratio of 1:4; the mixed ester contained 45.89% ascorbyl palmitate, 42.59% ascorbyl oleate and 10.1% ascorbyl linoleate. Acylation with soybean oil methyl ester resulted in 17% conversion, yielding a mixed ester containing 10.08% ascorbyl palmitate, 20.68% ascorbyl oleate, and 64.96% of ascorbyl linoleate. The mixed esters of ascorbic acid can find direct use in food and cosmetics.

Fusion protein of the hyaluronan binding domain from human TSG-6 with luciferase for assay of hyaluronan.

The gene expression plasmid, pET-Lmluc, for the fusion protein of the hyaluronan binding domain from human TSG-6 [product of tumor necrosis factor (TNF)-stimulated gene-6] and luciferase from Renilla reniformis was constructed. The fused gene was expressed in Escherichia coli and the resulted insoluble Lm-luc fusion protein was purified and refolded to recover both the hyaluronan binding capability and the luciferase activity. Hyaluronan as low as 1 ng ml(-1) was detected by using the indirect enzymatic immunological assay with the refolded Lm-luc fusion protein.