Biochemical Engineering Journal (v.78, #C)

Editorial (iii).

BEJ Keywords (IV).

Microalgae-based carbohydrates for biofuel production by Chun-Yen Chen; Xin-Qing Zhao; Hong-Wei Yen; Shih-Hsin Ho; Chieh-Lun Cheng; Duu-Jong Lee; Feng-Wu Bai; Jo-Shu Chang (1-10).
Microalgae are considered as the most promising renewable feedstock for biofuel production and biorefineries, due to their advantages of fast growth, efficient carbon dioxide fixation, not competing for arable lands and potable water, and potentially accumulating high amounts of lipids and carbohydrates. Since carbohydrates in microalgae biomass are mainly cellulose in the cell wall and starch in the plastids without lignin and low hemicelluloses contents, they can be readily converted into fermentable sugars. However, to date there are very few studies focusing on the use of microalgae-based carbohydrates for biofuel production, which requires more understanding and knowledge to support the technical feasibility of this next-generation feedstock. This review article elucidates comprehensive information on the characteristics and metabolism of main fermentable microalgal carbohydrates (e.g., starch and cellulose), as well as the key factors and challenges that should be addressed during production and saccharification of microalgal carbohydrates. Furthermore, developments on the utilization of microalgae-based feedstock in producing liquid and gaseous biofuels are summarized. The objective of this article is to provide useful knowledge and information with regard to biochemistry, bioprocess engineering, and commercial applications to assist in the viable technology development of for biofuels generation from microalgae-based carbohydrates.
Keywords: Microalgae; Biogas; Growth kinetics; Integrated Processing; Biofuels; Biorefinery;

Marine microbes have the potential for accumulating large quantities of lipids and are therefore suitable candidate as feedstock in unsaturated fatty acid production. The efficient utilisation of glycerol as an alternative carbon source to glucose was demonstrated in the fermentation of newly isolated thraustochytrid strains from the Queenscliff, Victoria, Australia. The isolates exhibited the presence of omega-3 and omega-6 polyunsaturated fatty acids, with the major fatty acids for all isolates being (as percent total fatty acid), palmitic acid (25.1–40.78%), stearic acid (4.24–13.2%), eicosapentaenoic acid EPA (2.31–8.5%) and docosapentaenoic acid (7.24–10.9%). Glycerol as a carbon source gave promising biomass growth with significant lipid and DHA productivity. An approximate three-fold increase in carotenoid content in all isolates was achieved when glycerol was used as a carbon source in the production medium.
Keywords: β-Carotene; Marine microalgae; Astaxanthin; Canthaxanthin; Biofuel;

► The influence of various CO2 absorbents was compared on microalgal CO2 fixation. ► Microalgal growth rate were best enhanced by triethanolamine (TEA) among tested CO2 absorbents. ► The repeated addition of TEA enhanced CO2 fixation rate by 39.3% from no-addition case.The influence of alkanolamine CO2 absorbents on the CO2 fixation in photoautotrophic culture of green alga Scenedesmus sp. was investigated using monoethanolamine (MEA), 2-amino-2-methyl-1-propanol (AMP), diethanolamine (DEA) and triethanolamine (TEA). The dissolved inorganic carbon (DIC) contents increased when alkanolamine compounds existed in the medium. Utilizing the increased DIC for algal uptake, TEA exhibited a best enhancement in CO2 fixation performance compared to other absorbents, while primary ethanolamines displayed inhibition on cell growth due to the formation of relatively stable carbamate intermediate. By adding 5 mM TEA, the cell growth and CO2 fixation rate increased by 30.5% compared to the case with no TEA. TEA was supplemented when cell density was doubled, tripled and quadrupled to maintain the TEA to cells ratio constant pacing the increase of cell density. The repeated addition of TEA exhibited the enhancement of CO2 fixation rate by 39.3% and 18.5% from no-addition and one-time addition cases, respectively.
Keywords: Microalgae; CO2 fixation; CO2 absorption; Alkanolamines; Triethanolamine;

Characterization, extraction and purification of lutein produced by an indigenous microalga Scenedesmus obliquus CNW-N by Ming-Chang Chan; Shih-Hsin Ho; Duu-Jong Lee; Chun-Yen Chen; Chieh-Chen Huang; Jo-Shu Chang (24-31).
► Microalga Scenedesmus obliquus CNW-N is a suitable candidate for lutein production. ► The developed method shows higher selectivity for extracting lutein from microalgae. ► Optimizing saponfication conditions reduced the operation time from 3 to 6 h. ► Low temperature and antioxidant addition led to higher storage stability for lutein.This study aimed to improve the commercial viability of microalgae-based lutein production using an isolated microalga Scenedesmus obliquus CNW-N possessing a high lutein content of over 0.25%. Effective lutein extraction protocols, appropriate storage methods, and purification procedures were developed. Disruption of microalgae cells was most efficient with a bead-beater. The conventional saponification step was modified to reduce the overall extraction time by 24 h. Diethyl ether exhibited the best lutein extraction efficiency. Storage of the lutein extract at low temperature (4 or −20 °C) with antioxidant addition (around 0.01% BHT) can maintain 90% lutein stability after 80 days. Addition of a suitable amount of the antioxidant could promote the stability of lutein extracts under the exposure of light. The protocol developed in this work allows efficient lutein extraction from S. obliquus CNW-N at a lower cost. Further purification was employed to elevate the purity of lutein and its commercial value.
Keywords: Microalgae; Scenedesmus obliquus; Lutein; Extraction kinetics; Bioseparations; Purification;

Cell growth and lipid production of a marine microalga Nannochloropsis oceanica DUT01 were investigated, and fresh medium replacement with different ratios to promote long term cell growth and lipid accumulation was also tested. The highest lipid content reached 64% in nitrogen deplete f/2 medium containing 37.5 mg/L NaNO3 combined with 1/5 fresh medium replacement, however, the highest lipid titer (0.6 g/L) and lipid productivity (31 mg/L/d) were achieved using BG11 medium containing 1.5 g/L NaNO3, taking advantage of 1/5 fresh medium replacement as well, which corresponded to the maximum biomass production of 1.4 g/L, highlighting the importance of high biomass accumulation for efficient lipid production. When biomass compositions were monitored throughout the culture, decreased protein content was found to be coupled with increased lipid production, whereas relatively stable carbohydrate content was observed. The fatty acids in the lipid of N. oceanica DUT01 comprise over 65% saturated fatty acids and monounsaturated acids (i.e. palmitic acid (C16:0) and oleic acid (C18:1)), suggesting that N. oceanica DUT01 is a promising candidate for biodiesel production. Interestingly, very high content of hexadecadienoic acid (C16:2, about 26–33%) was produced by DUT01, which distinguished this microalga with other microalgae strains reported so far.
Keywords: Bioconversion; Bioprocess monitoring; Lipid production; Nannochloropsis oceanica; Microalgae; Optimization;

► The monochromatic blue and red LED lights at various light intensities were employed for PMFC study. ► Increasing light intensity improved PMFC performance. ► Cell under red LED Illumination presented higher power density. ► The chlorophyll pigments under PFMC operations were assayed.Photosynthetic microbial fuel cells (PMFCs) are devices that convert chemical energy into the form of electricity through the catalytic activity of photosynthetic microorganisms. Power densities produced by the photosynthetic microalgae are greatly dependant on light sources and light intensities because these two factors can affect the chlorophyll formation, photosynthesis processes and stomata opening in the microalgae cells. In the present study, Chlamydomonas reinhardtii transformation F5 was used as biocatalyst in photo microbial fuel cells (PMFCs) and were illuminated with monochromatic blue and red LED lights at various light intensities (100, 300, 600 and 900 lx), respectively. The kinetic analysis was successfully employed to describe the intracellular and extracellular electron transfer mechanism of the cells. The results demonstrate that the performance of PMFCs increased in terms of maximum power density and exchange current density (i o) with the tendency of decreasing in internal resistance (R int) and over potential (η) values as increasing monochromatic blue and red LED light intensities. However the PMFCs performed better under red LED light as compared to operating under blue LED light. The maximum power density can reach 12.947 mW m−2, which could be a potential micro-power supply.
Keywords: Photosynthetic microbial fuel cell (PMFC); Blue light intensities; Red light intensities; Chlorophyll content; Microalgae; Kinetic parameters;

Subcritical water and dilute acid pretreatments for bioethanol production from Melaleuca leucadendron shedding bark by Ibrahim Nasser Ahmed; Sylviana Sutanto; Lien Huong Huynh; Suryadi Ismadji; Yi-Hsu Ju (44-52).
The feasibility of bioethanol production using the lignocellulose of the shedding bark of Melaleuca leucadendron (Paper bark tree) was investigated. The effects of pretreatment parameters (temperature, time and acid concentration) on the yields of sugars and inhibitors, and optimal pretreatment conditions were determined. At very low severity conditions (combined severity factor, CSF ≤ 0.335), 28% of xylan was recovered and this recovery increased with increasing CSF till it peaked to 64.4% (11.2 g xylose L−1) at a CSF of 1.475. However, at CSF > 2.0, xylose yield declined due to degradation. Mild and progressive glucose yield was detected in prehydrolysate at CSF ≥ 1.514, and subsequent enzymatic hydrolysis allowed complete glucan solubilization. Implementing environmentally friendly subcritical water pretreatment at CSF ≤ 0.335 on the shedding bark, about 85% of glucan solubilization was achieved after enzymatic hydrolysis. An industrial Saccharomyces cerevisiae strain readily fermented crude hydrolysate within 12 h, yielding 24.7 g L−1 ethanol at an inoculum size of 2% (v/v), representing a glucose to ethanol conversion rate of 0.475 g g−1 (91% ethanol yield). Based on our findings, the shedding bark is a potential feedstock for bio-ethanol production.
Keywords: Glucose; Cellulase; Fermentation; Ethanol; Subcritical water; Melaleuca leucadendron;

Cloning and expression of Cel8A from Klebsiella pneumoniae in Escherichia coli and comparison to cel gene of Cellulomonas uda by I-Son Ng; Xiaoqin Chi; Xiaomin Wu; Ziwei Bao; Yinghua Lu; Jo-Shu Chang; Xueping Ling (53-58).
► Cel8A from Klebsiella pneumoniae XM-4 has firstly been cloned and expressed in Escherichia coli. ► The highest recombinant protein was obtained by used pET-32a(+) without signal peptide to cel8A. ► 95% similarity of Cel8A to Cellulomonas uda showed the biological evolution between both strains. Klebsiella pneumoniae XM-4 is first isolated from a bacterial consortium. According to the 16S rRNA sequence, it has only 75% sequence similarity to Cellulomonas sp. However, a 999-bp open reading frame of K. pneumoniae, which encodes a putative endo-glucanase of 333 amino acids belonging to GH8 family and designated as Cel8A, is found closely to Cellulomonas uda with 95% similarity in amino acid sequence. This implies that the biological evolution between both strains has occurred. The cel8A is constructed in vectors of pET-22b(+) and pET-32a(+) with or without containing a signal peptide, and then cloned and expressed in Escherichia coli. The best endo-glucanase production of 25.4 mg/l is then obtainable by applying pET-32a(+) without the signal peptide. The recombinant CelA8 has an optimal specific activity of 62.4 U/mg against CMC or 23.3 U/mg against β-d-glucan at 55 °C and pH 5.0, indicating good potential for the industrial application.
Keywords: Klebsiella pneumoniae; Cellulomonas uda; Endo-glucanase; Signal peptide; Recombinant expression; Biological evolution;

Antrodia cinnamomea has recently become popular as a drug remedy in Taiwan because of the mushroom's physiologically beneficial properties. However, because of its host specificity, slow growth rate and rarity in nature, the fruiting bodies of A. cinnamomea have become the most expensive mushrooms in Taiwan in recent years. Artificial cultivation of A. cinnamomea basidiomes to satisfy market demand is considered to be the most effective solution.In this study, solid-state fermentation was carried out in petri dishes for mycelia growth and basidiomatal formation of A. cinnamomea. Different kinds of citrus peel were added to the medium to investigate the feasibility of enhancing the production of bioactive compounds. The crude triterpenoid content of mycelia in the control test reached the highest level of 9.66 mg/g DW on day 30. Among various kinds of citrus peel, grapefruit peel was the most effective in enhancing the content of crude triterpenoid. Moreover, at the addition ratio of 4 g per petri dish, the amount of crude triterpenoid reached 47.10 mg/g DW on day 30, with more than a fourfold increase. Furthermore, compared with the mycelia of the control culture, the profiles of HPLC analysis show that the mycelia cultured with the grapefruit-peel addition contained more kinds of triterpenoid and was similar to natural basidiomes. This study demonstrates that additions of some kinds of citrus peel could effectively accelerate the formation of basidiomes and enhance the production of bioactive metabolites.
Keywords: Antrodia cinnamomea; Triterpenoid; Solid-state fermentation; Citrus peel;

Pentose-rich hydrolysate from acid pretreated rice straw as a carbon source for the production of poly-3-hydroxybutyrate by Raveendran Sindhu; Nagapoosam Silviya; Parameswaran Binod; Ashok Pandey (67-72).
► First report on biopolymer production from mild acid pretreated rice straw. ► B. firmus can grow in hydrolysate media without detoxification. ► Maximum PHB production was 1.697 g/l from 1.9 g/l biomass. ► Highest value (89% of biomass) reported from Bacillus species.The aim of this work was to evaluate three different bacterial strains for their ability to accumulate poly-3-hydroxybutyrate (PHB) using pentose sugar rich hydrolysate generated from acid pretreated rice straw as the sole carbon source. Out of these, Bacillus firmus NII 0830 showed maximum PHB production. Acid pretreated black liquor contained sugars and sugar degradation products such as formic acid, acetic acid, furfural and hydroxymethyl furfural. The bacterium grew in the hydrolysate medium without any detoxification and it could accumulate 1.9 g/l biomass with 1.697 g/l PHB and the PHB content in the cell was 89%. This was the highest value ever reported from Bacillus species. The optimum conditions for the fermentation media were an inoculum concentration of 6.5%, 90 h of incubation and 0.75% of xylose concentration. The characterization of extracted polymer was carried out by FTIR, 1H and 13C NMR which showed characteristics similar to that of the standard PHB from Sigma.
Keywords: Fermentation; Microbial growth; Biosynthesis; Submerged culture; Poly-3-hydroxybutyrate; Bacillus firmus;

► PHBV are biopolymers produced by microorganisms. ► Increasing the 3HV monomer can reduce the crystallinity, resulting in more flexibility. ► 3HV fraction in PHBV increased when prpE was expressed.Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) are biopolymers produced by microorganisms and have commercial potential as bioplastics. The compositions of polymers influence their physical and mechanical properties. PHBV polymers with high composition of 3-hydroxybutyrate (3HB) are brittle. Increasing the fraction of 3-hydroxyvalerate (3HV) monomer in the PHBV can reduce the crystallinity, resulting in more flexibility. To increase the fraction of 3HV in PHBV and reduce the antibiotic supplementation to maintain the recombinant plasmid, we cloned the prpE gene encoding propionyl-CoA synthase, the vgb gene encoding bacterial hemoglobin (VHb), and the polyhydroxyalkanoates (PHAs) synthesis operon (phaCAB) in one recombinant plasmid transformed into Escherichia coli XL1-blue, termed CT-2E. The prpP gene encoding propionate permease and the bktB gene encoding beta-ketothiolase were also cloned to evaluate their effects on PHBV accumulated in E. coli containing phaCAB-vgb, termed CT-2-BP. The results showed that the 3HV fraction in PHBV and the molecular weight of PHBV increased when prpE was expressed in CT-2E, compared with E. coli containing phaCAB-vgb (termed CT-2). The polydispersity index (PDI) of the PHBV derived from CT-2-BP was 1.33, indicating that polymer uniformity was greater than that from CT-2, which had a PDI of 1.68. Compared with CT-2, the enthalpies and melting temperature of PHBV from CT-2E and CT-2-BP decreased, indicating the advantages of processing.
Keywords: PHA; PHBV; PrpE; PhaCAB; Genetic engineering; Escherichia coli;

With consideration of sustainable development, this study explored the fermentation strategy of cost-effective production of biodegradable polymer- polyhydroxyalanoates (PHAs) for feasibility of eco-friendly materials recycling during wastewater treatment. As prior studies showed that Aeromonas hydrophila NIU01 was a promising PHA-producing bacterium, this follow-up study tended to seek for optimal nutrient-supplementation strategy to stimulate maximal PHA accumulation of A. hydrophila NIU01 for cellular production. As maximal PHA production took place at growth-limiting conditions, two-stage fermentation was much more appropriate for practical applications compared to batch mode of operation. Moreover, this optimal two-stage operation strategy maximized cellular PHA production under nitrogen-limiting conditions at C/N molar ratio of 60/1. For materials recycling, this operation strategy could be applicable to simultaneous PHB production and wastewater decolorization using A. hydrophila.
Keywords: Bioremediation; Biostimulation; Fermentation; Polyhydroxyalkanoates (PHAs); Aeromonas hydrophila; Poly-3-hydroxybutyrate (PHB);

Studies on biosurfactants from Pseudozyma sp. NII 08165 and their potential application as laundry detergent additives by Kuttuvan Valappil Sajna; Rajeev K. Sukumaran; Himani Jayamurthy; Kunduru Konda Reddy; Sanjit Kanjilal; Rachapudi B.N. Prasad; Ashok Pandey (85-92).
► Mannosylerythritol lipids production by a novel Pseudozyma strain. ► Mannosylerythritol lipids production was 34 g/l from media containing 8% soybean oil. ► MEL-C with unique hydrophobic structure. ► Application of Pseudozyma crude biosurfactants as laundry detergents additives.The novel isolate Pseudozyma sp. NII 08165 produced glycolipid biosurfactants, which was a combination of all the three mannosylerythritol lipids (MELs) isomers along with some unknown glycolipids. The strain produced 34 g/l MELs in medium containing 8% (w/v) soybean oil as carbon source after nine days of fermentation. The structural characterization of purified MEL revealed the hydrophobic structure of MEL-C consisting of short chain fatty acid (C2 or C4) at the C-2′ position and a long chain fatty acid (C14, C16 or C18) at the C-3′ position of the mannose moiety. The MEL-C showed good surface activity with critical micelle concentration (CMC) of 4.5 × 10−6  M and surface tension of 33 mN/m at CMC. The crude biosurfactants were stable at high temperature and over the alkaline pH range which favour their scope of application as laundry detergent additives. Fabric wash analysis revealed that crude biosurfactants from Pseudozyma sp. NII 08165 removed stains efficiently and can be used in laundry detergent formulations.
Keywords: Pseudozyma; Mannosylerythritol lipid; Yeast; Fermentation; Purification; Submerged culture;

Enhancing production of prodigiosin from Serratia marcescens C3 by statistical experimental design and porous carrier addition strategy by Wei-Chuan Chen; Wan-Ju Yu; Chia-Che Chang; Jo-Shu Chang; Shih-Horng Huang; Chih-Hung Chang; Shan-Yu Chen; Chih-Ching Chien; Chao-Ling Yao; Wen-Ming Chen; Yu-Hong Wei (93-100).
► Maximal prodigiosin production of 7.07 g/L by statistical experimental design methodology. ► First report on prodigiosin production (15.6 g/L) by addition of porous carrier. ► The fermentation strategy in this study effectively increases prodigiosin production above that achieved elsewhere. Serratia marcescens C3 produces a natural red-pigment, prodigiosin, which exhibits immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. This work seeks to improve the production of prodigiosin by S. marcescens C3 using various strategies. Starch and peptone were identified as the optimized carbon and nitrogen sources for the production of prodigiosin, yielding a prodigiosin concentration of 2.3 g/L. This value was significantly increased to 6.7 g/L using a carbon/nitrogen ratio of 6/4 (starch/peptone = 16 g/L/10.67 g/L). To enhance prodigiosin production even further, a statistical experimental design methodology was utilized to optimize the composition of the culture medium that is utilized in the production of prodigiosin. Prodigiosin production of 7.07 g/L was achieved when the concentrations of two trace compounds, FeSO4·4H2O and MnSO4·4H2O, were optimized using the statistical experimental design methodology. Their optimal concentrations were 0.56 mM and 3.25 mM, respectively. Ultimately, the production of prodigiosin was increased from 2.3 g/L to 15.6 g/L, or by a factor of nearly seven by immobilizing microorganisms in 3% calcium alginate beads.
Keywords: Prodigiosin; Antibiotic; Fermentation; Immobilization; Microbial growth;

Alpinia oxyphylla Miq. bioactive extracts from supercritical fluid carbon dioxide extraction by Chih-Chen Lee; Chien-Chih Chiu; Wei-Ting Liao; Pei-Fang Wu; Yen-Ting Chen; Kuo-Chuan Huang; Yi-Ting Chou; Zhi-Hong Wen; Hui-Min Wang (101-107).
In this study, supercritical fluid carbon dioxide extraction technology was developed to gain the active components from a native plant, Alpinia oxyphylla Miq. We studied the biological effects of A. oxyphylla extracts via multiple assays and demonstrated bio-functions at various concentration ranges. Investigations of A. oxyphylla extracts indicated that anti-oxidative properties in dose-dependant manners on radical scavenging activities, reducing power and metal chelating power. The cultured human normal peripheral blood mononuclear cells (PBMCs) were used to test the properties of the extracts in inflammatory cytokine release, and the data did not induce inflammatory molecule releases from un-stimulated PBMCs. A. oxyphylla extracts were able to induce Th1 cytokine IFN-γ released, but not Th2 cytokine IL-13, showing an enhanced anti-bacterial/viral immune function without possible allergic response mediated by IL-13. The extracts also had in vitro mushroom tyrosinase inhibition and cellular tyrosinase melanin decreasing activities in B16F10 cells. In addition, the cell proliferation assay illustrated anti-growth and anti-migration effects in dose-dependent manners of the extracts on human skin melanoma cells, A375 and A375.S2, indicating that the extracts exerted the anti-cancer properties. To our knowledge, this was the first report presenting these bioactivities on A. oxyphylla extracts including antioxidant, anti-inflammation, de-pigmentation and anti-melanoma
Keywords: Alpinia oxyphylla Miq.; Biomedical; Enzyme activity; Enzyme technology; Immunoassay; Supercritical fluid carbon dioxide extraction;

Growing exclusively on stout camphor trees in Taiwan, Antrodia cinnamomea is known for its extraordinary antioxidant and antitumor activities. As an alternative to the limited supply of natural source, cultured A. cinnamomea from solid state or submerged liquid fermentation still offers many of its medicinal effects. To further enhance the production of functional compounds and corresponding activities, oat, wheat, buckwheat and pearl barley were used as substrates for solid state fermentation of A. cinnamomea in this study. Among these cereal-based culturing, the methanol extract of A. cinnamomea mycelia grown on oats showed stronger overall antioxidant properties. EC50 for the antioxidant activity (conjugated diene method), the DPPH radicals scavenging ability and reducing power were estimated to be around 0.57 mg/mL, 1.07 mg/mL and 0.31 mg/mL, respectively. Incubating cultured cells with 150 ppm of the oat-cultured mycelial extract for 24 h greatly reduced the viability of MCF-7 breast cancer cells and HepG2 hepatocellular carcinoma cells to 29% and 76%, while 3T3 normal fibroblasts were virtually unaffected. In general, cereal-based solid state fermentation of A. cinnamomea produced more of the secondary metabolites and their methanolic extracts showed stronger antioxidant and anti-tumor activities than extracts obtained from liquid fermentation at the same concentration.
Keywords: Solid-state fermentation; Filamentous fungi; Cereals; Animal cell culture; Antioxidant activity; Antitumor activity;

► We established cyanophycin production by Lactococcus lactis. ► An addition of one glycine codon onto the cphA gene can enhance the specific yield. ► A high specific yield of 20% dry cell weight can be obtained. ► The cyanophycin existed mostly in the insoluble form with a low content of lysine.Cyanophycin is a natural source of polypetide consisting of aspartic acid as a backbone and arginine as its side chain. After the removal of arginine, the remaining poly-aspartate can be served in numerous industrial and biomedical applications. The synthesis of cyanophycin is catalyzed by cyanophycin synthetase. In this study, we used lactic acid bacteria to produce cyanophycin by nisin-controlled gene expression system (NICE). The cyanophycin synthetase gene cphA of Synechocystis sp. strain PCC6803 was cloned to the vector pNZ8149 followed by transformation into Lactococcus lactis subsp. cremoris NZ3900. The effects of nisin concentrations and the amounts of supplemented aspartic acid and arginine were examined for the production of cyanophycin. Alterations of the terminus of cphA gene were also conducted in an attempt to increase the yield of cyanophycin. An optimal cyanophycin production was noted under a culture condition of log phase induced at 250 ng/mL nisin in M17L medium supplemented with 20 mM arginine and 10 mM aspartic acid. An insertion of glycine residue at the C terminus of cyanophycin synthetase resulted in a yield of 20% of dry cell weight, a 10-fold increase when compared with the wild type. The results showed that recombinant lactic acid bacteria, a GRAS system, could provide an alternative approach of producing cyanophycin suitable for agricultural and biomedical applications.
Keywords: Cyanophycin; Lactic acid bacteria; Recombinant DNA; Biopolymer; Culture condition;

To enhance protein production from osteoblasts by using micro-patterned surfaces by Swary Fransiska; Ming Hua Ho; Chung Hsing Li; Jia Lin Shih; Sheng Wen Hsiao; Doan Van Hong Thien (120-127).
In this study, microgrooved surfaces were used to culture osteoblast-like cells to promote the production of osteogenic proteins. First, modified photolithographic techniques were used on silicon wafers to create micro-grooved patterns. Groove width was continuously varied from 1 to 20 μm. Then, the cells were cultured for various periods to induce the contact guidance effect, and the cell behaviors, including the production of osteogenic proteins, were analyzed. The results indicated that the contact guidance effect was significant with suitable groove width. The guidance from topography promoted cell attachment and proliferation. Moreover, the secretion of osteogenic proteins, including alkine phosphate (ALP) and osteopontin (OPN), was greatly enhanced after cells were guided by grooved patterns. The grooved structure induced osteocalcin gene expression, revealed by the promotion of phosphorylation of Smad1. It was found that groove width less than 10 μm induced the alignment of osteoblasts, causing the increase in the production of osteogenic proteins.
Keywords: Grooved surface; Micro-patterned; Osteoblast; Topography; ALP; OPN; Protein production; Osteogenic differentiation;

► Denitrifying sulfide removal is a biorefinery process to produce elemental sulfur. ► Colloidal S0 from sulfide and nitrate-laden wastewaters was produced. ► Optimal hydraulic retention time for maximizing S0 conversion from wastewaters exists.The denitrifying sulfide removal (DSR) process is a biorefinery process that can produce colloidal S0 from sulfide and nitrate-laden wastewaters. At long reaction time the formed S0 produced is reduced back to sulfide by sulfate-reducing bacteria so the resulting in poor conversion rate of S0 is poor. The presence of optimal hydraulic retention time (HRT) for maximizing S0 conversion from DSR wastewaters was proposed by batch assays and then confirmed in continuous flow tests.
Keywords: Denitrifying sulfide removal; Elementary sulfur; Hydraulic retention time; Biorefinery;

Biofilm formation and heavy metal resistance by an environmental Pseudomonas sp. by Chih-Ching Chien; Bo-Chou Lin; Chun-Hsien Wu (132-137).
Pseudomonas sp. strain EJ01 can resist high concentration of heavy metals. ► Biofilm formation and exopolysaccharide are important for the heavy metal resistance. ► Biofilm mutant (strain m-3055) was generated by transposon mutagenesis. ► Strain m-3055 was less tolerance to heavy metals compared to strain EJ01. ► Metal removal from medium was significant in strain EJ01 but not in strain m-3055.We have isolated a heavy metal resistance Pseudomonas sp. (designated as strain EJ01) and demonstrated that this bacterium was able to tolerate cadmium ion (Cd2+) in the growth medium up to 7 mM. At stationary phase, cells of strain EJ01 aggregated and formed biofilm when concentrations of Cd was high (2 mM and above). This phenomenon might be related to the bacterium's ability to produce exopolysaccharides (EPS). Strain EJ01 also showed resistance to other toxic heavy metals examined. Concentrations of heavy metal was reduced significantly when Pseudomonas sp. EJ01 grown in the medium containing these toxic metal ions. Biofilm formation and EPS production were important for the bacterium's resistance against heavy metals. Exopolysaccharide was produced by strain EJ01 and 50% more EPS was produced when the cells grown in the medium containing 2 mM CdCl2 compared to those grown on the medium without heavy metals. A biofilm defective mutant (strain m-3055) was obtained by transposon mutagenesis. Although strain m-3055 produced comparable amount of EPS to that of strain EJ01, the mutant showed less tolerance to the toxic heavy metals. Heavy metal removal from the culture medium by strain m-3055 was also significantly reduced compared to strain EJ01.
Keywords: Bioremediation; Biofilms; Microbial growth; Environmental preservation; Heavy metals; Environmental Pseudomonas sp.;

Improvement of biofouling resistance on bacterial cellulose membranes by Hengky Kurniawan; Yun-Sheng Ye; Wei-Hsuan Kuo; Jinn-Tsyy Lai; Meng-Jiy Wang; Hwai-Shen Liu (138-145).
Bacterial cellulose possesses excellent biocompatibility and mechanical strength that show great potentials for biomaterial applications. In this study, the surface modifications of bacterial cellulose (BC) membranes were facilitated using either simple coating or chemical grafting methods. The surface coating method is to simply immobilize BC membranes with poly(ethylene glycol) (PEG) solutions of concentration from 1 to 10%, followed by post-treatment with argon (Ar) plasma. The chemical method involved grafting mPEG (monofunctional methyl ether PEG) on BCs. The outcomes of surface modifications were characterized by surface chemical compositions (electron spectroscopy for chemical analysis (ESCA), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetry analysis (TGA), and surface morphology (atomic force microscopy (AFM) and scanning electron microscopy (SEM)). The effects of resistance to biofouling were verified by quantifying the adsorption of proteins and mammalian cells. The results showed that the PEG coating on BCs improved the resistance to cell adhesion by more than 30%. On the other hand, the specific chemical grafting resulted in a particularly high resistance to biofouling that the density of adherent cells reduced by more than 70% when compared to that on pristine BC. We have demonstrated that the two proposed methods were effective for the preparation of bioinert BC membranes with great potentials for applications in biomaterials and tissue engineering.
Keywords: Acetobacter; Antifouling; Cellulose; Bioinert; Immobilization; Tissue cell culture;

The method for selective enzyme immobilization on arrayed microelectrodes has been investigated in order to fabricate a near-real time biosensor for potential ex vivo or in vivo applications (e.g. in fermentation processes, in neuroscience studies, etc.). In this study, chitosan is used as the enzyme immobilization material, permselective polymers (polypyrrole and Nafion®) are used as the exclusion layers for charged interferents, and glutamate oxidase is used as the model enzyme for immobilization. To fabricate a glutamate sensor in the array, glutamate oxidase is selectively immobilized on a microelectrode site which is closely arrayed to other microelectrodes (platinum microelectrodes with ∼100 μm separation) with the electrode dimension of ∼30 μm × 140 μm by adsorbing the enzyme on the electrodeposited chitosan matrix on the electrode surface. When testing the glutamate sensor with glutamate at physiological concentrations, it has a linear detection range up to 217 μM, a response time ∼1 s, a limit of detection 2.5 ± 1.2 μM, and a sensitivity 38.1 ± 5.4 nA/μM cm2 (n  = 8); on the other hand, the closely arrayed control sensor without the chitosan film showed no signal upon the addition of glutamate. Little interferent current was observed. The sensor can retain up to 70% of its initial sensitivity for at least 9 days and no observable decrease in sensor sensitivity was detected after 20 times of continuous operations. Successful selective glutamate oxidase immobilization on closely packed microelectrodes for monitoring glutamate is demonstrated.
Keywords: Biosensors; Immobilized enzymes; Glutamate; Microelectrode array; Chitosan; Biomedical; Bioprocess monitoring;

Because of ethical and scientific controversy, the utilization of fetal bovine serum (FBS) for cell culture medium must be minimized. This study develops porcine platelet-rich plasma (P-PRP) as a FBS substitute for human mesenchymal stem cell (hMSC) cultivation. Concentrating porcine blood by serial centrifugation to obtain P-PRP leads to activation by different agonist combinations to stimulate the secretion of growth factors. The concentration of growth factor in P-PRP is significantly increased by activation (p  < 0.05). The concentration of PDGF, KGF and TGF-β in activated P-PRP is significantly higher than that in FBS. Design-expert was used to decide whether Co−T+Ca−, Co+T−Ca−, and Co+T+Ca− are optimal agonist formulations. MSC cultivation shows that the attachment rate, proliferation rate and viability of P-PRP supplemented media are significantly higher than those for FBS-supplemented and commercial media (p  < 0.05). The results demonstrate that P-PRP is an optimal FBS substitute that supports in vitro h-MSCs expansion for subsequent biomedical applications.
Keywords: Platelet-rich plasma; Mesenchymal stem cell; Fetal bovine serum substitute; Porcine blood utilization; MSC viability; MSC proliferation;

Inhibitory effect of ectoine on melanogenesis in B16-F0 and A2058 melanoma cell lines by Chao-Ling Yao; Yu-Min Lin; Mahmoud Shaaban Mohamed; Jian-Haw Chen (163-169).
► Ectoine is a safe, effective and natural whitening agent for cosmetic application. ► Ectoine has no cytotoxicity on melanoma cell lines up to 500 μM. ► Ectoine inhibits melanin synthesis and tyrosinase activity in melanoma cell lines. ► Ectoine has various effects on expressions of melanogenesis-related genes. ► Ectoine has various effects on secretion of melanogenesis-related proteins.Skin injuries, congenital lesions, melasma, Addison's disease and many pigment abnormalities prompt us to search for an effective whitening agent. Ideal whitening agent is a natural compound that can inhibit melanogenesis and has no cytotoxic effects. In a previous study, we have developed an optimum method for the production and characterization of ectoine from a halophilic bacterium isolated from a salt environment in Taiwan was identified as Marinococcus sp. In the present study, we screened the whitening properties of the biosynthesized ectoine using mouse and human melanoma cell lines, B16-F0 and A2058. Here, we examined the cell viabilities of melanoma cells after ectoine treatment at various concentrations up to 500 μM. Also, we addressed the melanin synthesis of melanoma cells after treatment with ectoine. The inhibitory effects of ectoine on tyrosinase activity were assessed in both mushroom tyrosinase and cellular tyrosinase. Furthermore, we investigated the type of inhibition of mushroom tyrosinase using Lineweaver–Burk enzyme kinetic. The melanogenesis-related gene expression (tyrosinase, TRP1, TRP2 and MITF) and their protein secretion were determined by the assays of quantitative real-time PCR and western blots, respectively. Our results demonstrated that ectoine is a safe and effective whitening agent, inhibited melanin synthesis, reduced both mushroom tyrosinase and cellular tyrosinase, and had various inhibitory effects on the expressions of melanogenesis-related genes and secretion of proteins in mouse and human melanoma cell lines. Thus, we suggest that ectoine can serve as a useful and safe new agent in cosmetic and clinical applications.
Keywords: Ectoine; Melanogenesis; Animal cell culture; Enzyme activity; Viability; Product inhibition;

The incorporation of chemical and biological functions into synthetic polymers provides particular potential for advances in studying the complex interactions between biomolecules and materials. We developed a simple method to simultaneously modulate the biocompatibility and create micro features on surface of poly(methyl methacrylate) (PMMA). The microstructured PMMA was functionalized by blending with negatively charged sodium dodecyl sulfate (SDS), positively charged poly-lysine (PL), or zinc oxide nanoparticles (ZnO NPs) to form SDS/PMMA, PL/PMMA, and ZnO-NP/PMMA, respectively. The proliferation of L-929 fibroblasts increased significantly to ∼1.3 folds on the PL blended PMMA films when compared to that on the pristine sample. On the other hand, SDS and ZnO NPs significantly inhibited the growth of mammalian cells. The overall results showed that the integration of microenvironment and chemical functionalities into materials provide promising effects for modulating the growth of mammalian cells.
Keywords: Amphiphilic materials; Antibiotic; Biomimetics; Lysine; Microporous membranes;

Synthesis of methotrexate-conjugated gold nanoparticles with enhanced cancer therapeutic effect by Ngoc Thi Thanh Tran; Tzu-Hao Wang; Chiao-Yun Lin; Yian Tai (175-180).
We have developed a simple approach to synthesize gold nanoparticle (AuNP) by utilizing the anti-cancer drug, methotrexate (MTX), as both the reducing agent and the capping molecule. MTX-conjugated AuNPs (MTX-AuNPs) of various sizes (∼3 and ∼20 nm) can be obtained in a one-pot synthesis. We utilized transmission electron microscopy, X-ray diffraction, and UV–vis spectroscopy to characterize these nanoparticles, and Fourier-transform infrared (FTIR) spectroscopy and Raman spectroscopy to investigate the chemical interactions between the MTX and AuNPs. To examine the cancer therapeutic effect of these nanoparticles, we applied the lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay tests. The results demonstrated that the MTX-AuNPs of small size (∼3 nm) exhibited considerably high cytotoxic effects in human choriocarcinoma cell lines compared to the effects of equal doses of free MTX; these results suggest that anti-cancer drug-conjugated AuNPs have an enhanced therapeutic effect.
Keywords: Gold nanoparticles; Methotrexate; In vitro; Cancer therapy;

Examining the inhibitory actions of copolypeptides against amyloid fibrillogenesis of bovine insulin by Chen-Yuan Kao; Jun-Kun Lai; Ta-Hsien Lin; Yu-Jiun Lin; Jeng-Shiung Jan; Steven S.-S. Wang (181-188).
Amyloid fibrillogenesis has been involved in at least 40 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been demonstrated to fibrillate in vitro. With bovine insulin as a model, the research presented here examines the influence of two simple, unstructured d,l-lysine-co-glycine (d,l-lys-co-gly) and d,l-lysine-co-L-phenylalanine (d,l-lys-co-phe) copolypeptides, on the in vitro fibril formation process of bovine insulin at pH 2.0 and 55 °C. Our results showed that amyloid fibrillogenesis of insulin may be suppressed by both copolypeptides in a concentration-dependent fashion. In addition, the copolypeptides with higher molar fractions of glycine or l-phenylalanine residue, which are considered to possess higher hydrophobic interacting capacities, demonstrated the superior inhibitory potency against insulin fibril formation. Our findings suggest that the association of insulin and copolypeptides, which is likely dominated by hydrophobic interactions and hydrogen bonding, may mitigate the extent of insulin fibrillogenesis. We believe the results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and amyloid-forming proteins.
Keywords: Aggregation; Biophysical chemistry; Protein; Polypeptides; Amyloid fibril; Inhibition; Amyloid diseases; Insulin;

Human γD-crystallin aggregation induced by ultraviolet C irradiation is suppressed by resveratrol by Josephine Wan-Ru Wu; Chen-Yuan Kao; Lilian Tsai-Wei Lin; Wen-Sing Wen; Jinn-Tsyy Lai; Steven Sheng-Shih Wang (189-197).
Human γD-crystallin, a 173-residue protein, is a major protein component of the human eye lens and associated with the development of juvenile and mature-onset cataracts. The study reported here is aimed at examining the effects of resveratrol on human γD-crystallin aggregation and/or precipitation triggered by ultraviolet-C (UV-C) light. Our turbidity, electron microscopy (TEM), and SDS–PAGE results showed that, under UV-C irradiation, resveratrol was able to suppress the formation of human γD-crystallin aggregates and the observed aggregation inhibition was dependent upon the concentration of resveratrol added. Through the use of Ellman reagent, the concentration of sulfhydryl group was observed to decrease upon prolonged UV-C irradiation. Also, there existed a positive correlation between the content of sulfhydryl group in human γD-crystallin and the concentration of resveratrol. Furthermore, we propose that resveratrol's free radical scavenging ability is likely to be correlated with its mechanism of action in inhibiting human γD-crystallin aggregation induced by UV-C irradiation. We believe this work may aid in the development of potential therapeutics for cataract.
Keywords: Resveratrol; Human γD-crystallin; Ultraviolet; Aggregation; Cataract; Free radical;

Two-dimensional amine-functionality gradient by plasma polymerization by Dave Mangindaan; Wei-Hsuan Kuo; Meng-Jiy Wang (198-204).
A two-dimensional (2D) gradient was created by employing two-step diffusion-controlled plasma polymerization of allylamine on polypropylene membrane, with varied deposition durations composed of two stages. The wettability gradient was examined by water contact angle measurements which clearly demonstrated that the gradient was well manipulated by varying the treatment duration, thus controlled the surface coverage and resulted in the average water contact angle ranged from 110° to 25°. On the created 2D gradients, the cell responses toward the distribution of nitrogen content were imaged by confocal laser microscopy. Moreover, a correlation based on experimental data revealed a linear relationship between the nitrogen content and water contact angle. The addition of serum proteins assisted also the adhesion and growth of L-929 cells. The methodology in fabricating the 2D gradient demonstrated the flexibility of the plasma technique which can be further used to create different types of well-defined gradients for applications in surface functionalization, biomedical devices, and material screening.
Keywords: Biokinetics; Biomedical; Diffusion-reaction; Two-dimensional gradient; Plasma polymerization; Viability;