Earticle

Klebsiella oxytoca stains are excellent 2,3-butanediol producers. To use this strain in industry area, 2,3-butanediol must be produced more than now. In order to achieve a high production, the optimization of fermentation condition is necessary. Among the various factors for 2,3-BDO fermentation, pH is the most important factor affecting 2,3-BDO production. 2,3-BDO production was measured in time, performing the fermentation with pH control and non-controlled pH at different initial pH values(5, 6, 7). 2,3-BDO was more produced without pH control than with pH control. Increasing pH values, 2,3-BDO was producued less but organic acid was produced more. Especially, 2,3-BDO was not yielded in the medium with pH maintained at 7. Metabolic flux analysis was perfored and the different results were shown according to the variable pH conditon. In conclution, pH is the important variable affecting 2,3-BDO metabolic pathway.

The autotrophic system in Haematococcus pluvialis has two major merits compared with the other systems, prevention of contamination and highly enhanced total astaxanthin yield. That system, however, needs for a lot of time to enter the induction stage, particularly under the outdoor, in which the light source is not fixed and continuous. And two stress-inducible factors, salt stress and high temperature, were proposed to enhance the efficiency of it, but costly and high energy-consuming, respectively. Here we present the highly inducible Haematococcus mutant. This mutant has the lower photosystem II(PSII) efficiency compared with wild-type, improved the photosensitivity and seems to be suffered from the more increased photodamage like ROS(reactive oxygen stress) by photoinhibition than wild-type from the same light treatment. Light supply is the most energy-consuming factor in the microalgal culturing systems. This mutant should decrease the requirement of light energy source for the light-dependent induction, and particularly be useful to such an outdoor culturing system of Haematococcus cells.

Porcine epidemic diarrhea virus (PEDV) causes a devastating enteric disease with acute diarrhea, dehydration and significant mortality in swine. Because of the high mortality which is up to 100% in suckling piglets, PED is an important porcine disease in Korea. PEDV, a member of the family Coronaviridae, is an enveloped virus possessing a singlestranded positive-sense approximately 28 kb RNA genome with a 5’cap and a 3’polyadenylated tail. Reading in the 5’to 3’direction, the PEDV genome contains genes for pol1 (P1) protein, spike (S) protein, an open reading frame (ORF3), envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. Especially, ORF3 protein has been suggested as an important determinant for PEDV biological properties. In this study, ORF3 gene of attenuated DR13 strain was amplified by RT-PCR. Then, the ORF3 gene was cloned into pET-21a vector. The pET recombinants harboring ORF3 gene were transformed into E. coli BL21(DE3) host cells by electroporation. Protein expression was induced by isopropyl-beta-Dthiogalactoside and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE showed a specific band of about 23.5 kDa in the whole cell lysate. The purified recombinant protein was evaluated for its antigenicity and reactivity through western blotting. The results indicated the recombinant ORF3 protein should be candidate as a feasible recombinant diagnostic reagent.

Porcine epidemic diarrhea virus (PEDV) is a single stranded RNA virus that belongs to the Coronaviridae family. The envelope contains three main viral proteins, spike (S, 180–20 kDa), membrane (M, 27–2 kDa and small membrane(sM, 7 kDa). The M protein is the most abundant of three; it is a triple-spanning membrane protein with a short amino-terminal domain on the outside of the virus and a long carboxyterminal domain on the inside. In order to obtain and assess the recombinant M protein used for a diagnostic reagent of porcine epidemic diarrhea (PED). M protein gene of KPEDV-9 strain was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). Then, the M protein gene was cloned into prokaryotic expression vector pET-21a. The recombinant plasmid pET-21a-M was constructed and transformed into E. coli BL21(DE3) for expression. The M protein gene of KPEDV-9 strain consisted of 692 nucleotides containing a single open reading frame (ORF) of 681 nucleotides, which encoded a 226aa-long peptide. SDS-PAGE analysis showed recombinant M protein was highly expressed by pET-21a-M and the product fusion protein his-tag-M reached 40% in the total bacteria proteins. The preliminary purified recombinant protein was evaluated for its antigenicity and reactivity through western blotting. The results indicated the recombinant M protein should be candidate as a feasible recombinant diagnostic reagent for preventing and control of PED.

Although the propanediol utilization: CoA dependent propionaldehyde dehydrogenase (PduP) has been suggested to be involved in the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol- fermenting process, it is still poorly understood. Thus in here, we disrupted the pduP gene on the chromosomal DNA of K. pneumoniae for investigating the enzyme effect on 3-HP production. The mutant strain still produce 3-HP but accumulates 3-hydroxypropionaldehyde (3-HPA) after consumed glycerol completely that resulted in cell death as well as decreased final titer of 3-HP production. To analyze the enzyme properties, pduP was cloned and expressed in E. coli. Recombinant K. pneumoniae PduP exhibited broad substrate specificity including 3-HPA and utilized both NAD+ and NADP+ as cofactors. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde (PA), at pH 8.0 and 37°C. The Km and Vmax values for PA in the presence of NAD+ were 0.22 mM and 0.09 U mg-1, respectively. When pduP was overexpressed in K. pneumoniae, 3-HP production remarkably increased from 1.0g L-1 to 1.8 g L-1. The presentation demonstrated firstly in the role of PduP in cellular biosynthesis of 3-HP from glycerol.

gTME (global Transcriptional Machinery Engineering) is a technique that introduces mutations into genes that affect global gene expression to obtain various phenotypes. Previous research has successfully developed an ethanol-tolerant strain by mutation of Spt15, a transcription factor.Histone deacetylase (HDAC) is an enzyme for nucleosome modification involved in global gene regulation HDAC consists of 3 classes among which HDAC class II is relevant to carbon metabolism and stress resistance. Hda1, one of the members of the HDAC class II, influences gene expression in HAST (Hda1 Affected SubTelomeric) region. In this region, there exist some genes related to stress tolerance including ethanol tolerance.3) We generated a mutant library expressing mutant Hda1 in addition to the endogenous wild type Hda1, and screened for ethanol-resistant strains. Gene expression patterns of the isolated ethanol-resistant strains will be further analyzed to elucidate the mechanisms for ethanol tolerance by the mutant Hda1

Many lactic acid bacteria produce bacteriocins which exhibit antimicrobial activity against closely related to the producer strains and against food-borne spoilage. Therefore bacteriocin may have great potential as natural biopreservatives. The purpose of this study was to identify and characterize a bacteriocin produced by lactic acid bacteria isolated from kimchi. Bacteriocin activity was measured by well diffusion assay. Gram-positive and Gram-negative strains were used as indicator strains. As result microbes with broad antimicrobial spectrum were isolated and they were identified as Leuconostoc citreum J1 and J17. Bacteriocins produced by isolates are active at pH values between 2.0 and 9.0 and retain activity after treatment at 121℃ for 15min. Proteolytic enzyme inactivated the bacteriocin after 4h of incubation. Moreover bacteriocins have antagonistic activity against a broad spectrum of microorganisms such as Listeria monocytogenes ATCC 19115, Streptococcus mutans KCTC 3056, Lactobacillus plantarum KCTC 3104 and so on. This preliminarily study shows the potentiality as a natural preservative in food industry, although further studies needs to be performed.

In human intestinal tract is one of the most varied and complex ecosystems, and dietary life an important role in the presence and prevalence of probiotics (lactobacilli and bifidobacterium). The composition and dynamics of fecal microbial communities during the study were analyzed by the PCR-denaturing gradient gel electrophoresis technique using group and genus-specific primers. DGGE of fecal 16S rDNA amplicons from 6 volunteer individuals showed host-specific populations of lactobacilli and bifidobacterium that were stable over a period of 5 weeks. Group specific Lactobacilli such as Lactobacillus sp., Leuconostoc sp., and Weisella sp. and genus specific bifidobacterium such as B. longum, B. coryneforme, B. adolescentis, B. breve, and Bacteroides fragilis were detected in human feces. These results indicate that the community of Lactobacilli and Bifidobacterium in human feces is unique because of the presence of specific species for human feces and a minority of the Lactobacilli and Bifidobacterium group may be caused by dietary life. In the future, intestinal tract microbial research can be regarded as a lactic acid bacteria-rich food providing various probiotics.

Polylactic acid (PLA) is considered to be one of the most promising biosource-derived polymer replacing current petrochemical ones. Optically pure lactic acid is favorable as a feedstock of polymer since even the presence of small amounts of the other isomer in PLA polymerization highly affects the properties of polymer. The fermentative production of lactic acid leads to optically pure isomer of lactic acid compared to the racemic mixtures of L and D isomers from chemical synthesis. The lactic acid fermentation is well documented in the literature, but reports on the optical purity of lactic acid during the fermentation are limited. In this study, the fermentation characteristics of L. paracasei KM2, ldhD knock-out cell, were compared with those of Lactobacillus paracasei KCTC 3510. In batch cultures and fed-batch cultures, the optical purity of produced lactic acid by L. paracasei KM2 was improved up to 99.8% compared to 97% by KCTC 3510. Fed-batch culture gave higher productivity while keeping the optical purity of lactic acid.

Three recombinant Escherichia coli strains expressing styrene monooxygenase (styAB) under T7 promoter were developed for the use in the conversion of styrene to (S)-styrene oxide, an important chiral building block in organic synthesis. By coexpression of chaperone proteins (GroEL-ES and DnaK-DnaJ-GrpE in plasmid pG-KJE8), disruption of NADH dehydrogenases (nuo-) involved in electron transport chain and over expression of styAB, individually or in combinations, the whole-cell StyAB activity was improved greatly. The highest activity (270 U/g CDW) was obtained with E. coli DnuoG haboring the plasmids for styAB and chaperon genes, which was around 2.5 folds higher than the one with the E. coli strain containing styAB in the pET plasmid. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 392 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 5.4 mmol/g dry cells/h and 65 mM/h, respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance.

Joseph Bigger discovered more than 60 years ago, at the very beginning of the antibiotic era, that populations of homogenous antibiotic-sensitive bacteria contained a very small fraction of antibiotic-tolerant cells. Accumulating evidence suggests that these disparate phenomena result from the ability of bacteria to enter into a dormant state. These non-dividing cells, persisters, are different from antibiotic-resistant mutants in that their antibiotic tolerance is non-heritable and reversible. Clinically, persisting bacterial pathogens is known to cause secondary infection. We screened for novel substances that kill the persister cell which survived despite the excessive antibiotic. For the inhibition of bacterial persistence, we screened 6,800 compounds in chemical library, 9 hits were selected. After batch test, ultimately 4 chemical compounds were nominated for inhibitor of bacterial persistence. And one chemical is absolutely kill the persister with norfloxacin. The dramatic evolution of resistance after the introduction of antibiotics now threatens our society with a post-antibiotic era. We suggest it is very important that partnering a traditional antibiotic with a drug that prevents bacteria from evolving resistance or from persisting during treatment might dramatically improve the therapy and protect the efficacy of these precious drugs.

The water that is passed through the magnetic field is changed into magnetized water. The magnetized water is known to have a unique pattern of hydrogen bond between water molecules, thereby producing different physicochemical properties from the ordinary water. Although it has been known that the magnetized water shows different physicochemical properties, the exact nature of the magnetized water is not clearly elucidated yet. We investigated the effect of the magnetized water in antimicrobial activity. The magnetized water was tested for its antimicrobial activity against gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Escherichia coli. Bacteria were cultured on LB broth at 37°C. The bacterial growth was determined by measuring the optical density (OD) at 600 nm and counting the number of colony forming units (CFU) at 1 hour intervals. The magnetized water showed stronger antimicrobial activities than not-magnetized water and more sensitive antimicrobial activity in Escherichia coli than Staphylococcus aureus. The nature of how the magnetized water can reduce bacteria is still unclear and needs further investigation.

The hupSLD-hypQ3Q4F gene cluster located in the pSOL1 megaplasmid of C. acetobutylicum ATCC 824 potentially encodes a [NiFe]-hydrogenase. The gene cluster is organized in an operon comprising six genes. The operon is expressed only in a solventogenic state, but neither in an alcohologenic nor acidogenic state.Generally, [NiFe]-hydrogenase are known as hydrogen consuming enzymes but little is known about the function of [NiFe]-hydrogenase in Clostridia. Thus, batch fermentation profiles were conducted with two different mutants with disruptions in the hupSLD-hypQ3Q4F operon and a control strain in MS medium without pH control.The product profiles of the hupL knockout mutant were found not to be significantly different from those of the control strain. In contrast, a deletion mutant of hypQ3Q4 produced more butanol, transiently accumulated less butyrate, and produced less hydrogen compared to the control strain. It seems that [NiFe]-hydrogenase in C. acetobutylicum is hydrogen producing enzyme, so accordingly the decrease of hydrogen from defective [NiFe]-hydrogenase during solventogenesis phase affected reducing equivalents of reduced ferredoxin and NAD(P)H.

Carrageenans are classified according to the number and the position of sulfate ester groups. They are made up of linear chains of galactose. The κ-carrageenase CgkA from Pseudoalteromonas carrageenovora is a hydrolase involved in the degradation of carrageenan. In this study, we expressed recombinant scaffolding protein mini-CbpA from Clostridium cellulovorans in Escherichia coli. To assemble the carrageenan-degrading complex via cohesin-dockerin interaction, a chimeric carrageenase cCgkA was constructed to containing the catalytic domain of CgkA fused with a dockerin domain of C. cellulovorans EngB. The resulting hydrolysis complex could degrade carrageenan efficiently with synergic effect of carrageenan-degrading enzyme activities. This is the first report on the formation of carrageenan-degrading complex using the cohesin-dockerin interaction system. The enhancement of carrageenandegrading complex activities will lead to the commercial production of useful products from agar biomass at low costs. [This Study was supported by Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea(No. 309016-5)]

A capnophilic rumen bacteria, Actinobacillus succinogenes can produce high level of succinic acid as a major fermentation end product. Succinic acid is a four-carbon dicarboxylic acid, produced as an intermediate of the tricarboxylic acid cycle and can be used to produce various industrially useful chemicals like 1,4-butandiol and tetrahydrofuran, as well as biodegradable polymers. During the anaerobic fermentation, the carbon dioxide availability is a key factor determining the succinic acid productivity. In this study, to increase the succinic acid productivity, optimization of the CO2 supply method and concentration of the magnesium carbonate (MgCO3) as CO2 source of the media was performed. In this case, batch-type fermentation is problematic because of high requirement and low solubility of the magnesium carbonate, especially in early phase of the fermentation. Therefor in fed-batch mode, the magnesium carbonate feeding time and the concentration were controlled according to the cell growth during the fermentation to maximize the succinic acid productivity. It was found that total magnesium carbonate requirement is lower in fed-batch fermentation than in batch culture, providing a way of cost reduction.

Flavin-containing monooxygenases (FMOs) have been identified from a variety of organisms. Our previous studies found that recombinant E. coli cells harboring fmo gene from Methylophaga aminisulfidivorans MPT produced indigo (920 mg/L) and indirubin (≤5 mg/L) in a 5 L fermentor containing tryptophan medium (2g/L tryptophan, 10g/L NaCl and 5g/L yeast extract). Interestingly, indirubin production greatly increased when cysteine was added to the tryptophan medium. Although growth of the recombinant E. coli harboring fmo gene was inhibited by cysteine, protein expression level and activity of FMO were not influenced. The optimum culture conditions for indirubin production in tryptophan medium were as follows: 2 g/L tryptophan, 5 g/L yeast extract, 10 g/L NaCl, 3 mM cysteine, pH 8.0 and at 30℃. Under these conditions, recombinant E. coli cells produced 223.6 mg/L of indirubin. In order to investigate the proteins involved in indirubin production in E. coli, proteomic analysis was performed using two-dimensional gel electrophoresis in conjunction with MALDI-TOF mass spectrometry. The analysis showed that the expression levels of 66 proteins were varied quantitatively with statistical significance under the optimum indirubin production condition. Through classification of physiological functions from Matrix Science Database analysis, nine proteins, which maybe involved in tryptophan synthesis and biofilm formation, were identified.

LTB consisted of five identical polypeptides and, as a pentameric form, has been demonstrated to bind to the GM1 ganglioside at cellular surface. The recombinant LTB has attracted much attention due to its non-toxicity and potential as a strong immunogenic antigen and immuno adjuvant for both system and mucosal immune responses. Actinobacillus pleuropneumoniae is the causal agent of swine pleuropneumoniae, a disease resulting in morbidity and mortality of pigs and accordingly economic losses within the swine industry. Toxins (APX) produced by A. pleuropneumoniae appear to be important virulence factors of swine pleuropneumoniae and are thought to be of particular importance in the induction of protective immunity. In order to use S.cerevisiae for effective delivery of APX to the gut-associated lymphoid tissue, a chimeric APX fused with LTB subunit (LTB::APX) was constructed and tested for the oligimerization, which enables the chimeric complex to bind the intestinal membrane GM1-ganglioside receptor. Westhern blot ananlysis and ELISA indicated that a chimeric fusion protein was produced in recombinant S. cerevisiae. However, assembly into the native pentameric structure did not occur probably due to the stearic hindrance caused by the increased size of LTB::APX fusion construct. Therefore, co-expression strategy using episomal and integrative vectors for LTB and LTB::APX, respectively, was adapted for the oligomerization of LTB and LTB::APX fusion construct. GM1-ELISA indicated that the LTB::APX fusion construct, along with the LTB, was oligomerized to make the functional pentameric form. APX-specific antibody was observed when the recombinant yeast expressing both LTB as well as LTB::APX was orally administered. Antibody titer of orally administered mouse was enhanced when using the recombinant yeast expressing the pentameric form instead of recombinant yeast expressing either LTB::APX fusion only or APX alone. Better protection against challenge of A. pleuropneumoniae was also observed in the case of recombinant yeast compared to others. This study clearly indicated that the coexpression strategy enable the LTB::APX fusion construct to participate the pentameric formation, which resulted in facilitating the uptake of the recombinant protein through the GM1-ganglioside. Through which, an improved induction of systemic immune responses was achieved, which resulted in better protective response against the A. pleuropneumoniae challenge.

Raw rice wine (RRW), called Makgeoli, is the most traditional alcoholic beverage in Korea. It has an alcoholic content of 6-7 percent. Traditional Korean RRW is entering the spotlight in domestic and international markets due to its good taste, nutritious quality, and reputation as being good for health. The market production of RRW brands in the region has increased in recent years. However, there has been no information regarding the inhibition of growth of RRW-related bacteria. Furthermore, rapid fermentation leads to deterioration in product quality, which is a serious problem for exporting of goods onto the world market. The object of this study was to improve the quality and prolong the shelf life of RRW. We isolated a lactic acid bacterium from RRW, which was identified as Lactobacillus fermentum (98%) based on the 16S rRNA sequence. Treatments of the RRW with various natural plant extracts changed its bacterial population. In particular, catechin products have proven to be very effective in the inhibition of bacterial population growth. The results suggest that natural plant extracts such as catechin products can be used as natural preservatives. The results can provide meaningful data for introducing RRW onto the competitive world market.

Conversion of cellulose to glucose is the most critical step for utilization of cellulose as biomass. Enzymatic hydrolysis of cellulose is achieved by cooperation of three types of cellulases including endoglucanase, exoglucanase, and β-glucosidase. Recently, yeast strains which are engineered to express cellulases have been developed to improve bioethanol production from lignocellulosic biomass. In this study, we focused on searching for endoglucanases that possess sufficient activity when expressed on yeast surface. We expressed various endoglucanases from fungi and bacteria on the surface of Sacchoromyces cerevisiae, and the activities of these recombinant enzymes were determined at various temperature conditions using Carboxymethylcellulose (CMC). As a result, Clostridium thermocellum CelD and Thermoascus aurantiacus endoglucanase I showed enzyme activities comparable with that of Trichoderma reesei endoglucanase II, which is widely used for bioethanol production studies. Therefore, these novel endoglucanases might be useful to generate yeast strains for consolidate bioprocessing of lignocellulosic biomass to ethanol.

Production of succinic acid was investigated in Escherichia coli overexpressing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from E. coli. Phosphoenolpyruvate carboxylase (ppc) and phosphoenolpyruvate carboxykinase (pck) were artificially expressed in E. coli and all experiments of fermentation using mutant E. coli were performed under anaerobic condition. The ppc overexpressing strain showed about 1.5~2-fold higher than wild type strain and pck overexpressing strain was observed the increased production of succinic acid by 1.3 times compared to wild type strain.

The actinomycetes strain J392 was found to exhibit broad spectrum of antimicrobial activity towards various pathogenic microorganism. The 16S rRNA sequence revealed that the strain was closely related to Streptomyces lanatus with 98.2% homology. Based on its genetic, morphological and biochemical characteristic the strain J392 was confirmed as belonging to the genus Streptomyces and we named as Streptomyces sp. J392. The production of antibiotic from this strain was found to be most favorable when cultured for 2 days at 28°C and 180rpm in shaking incubator, using glucose and peptone as respective carbon and nitrogen sources. The antibiotic was extracted using ethyl acetate (1:1 v/v) and partially purified using both column and thin layer chromatography techniques. Two antibiotic compounds based on its different solubility on ethyl acetate and methanol, were identified. Antimicrobial activity of the compound against different pathogenic strains was assessed using the minimum inhibitory concentrations (MICs). It was found to be active against different strains, including MRSA 693E, MRSA (4-5), MRSA (5-3), MRSA (5-3), VRSA, Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 1928), Staphylococcus aureus (KCTC 1928) and Mycobacterium smegmatis (ATCC 9341). These result suggest that J392s possessing potential antibacterial agent.

Consolidated bioprocessing (CBP) of cellulosic biomass to ethanol combines the steps including cellulose production, saccharification, and fermentation into one step. Although this process is considered as the most efficient way to reduce the production cost of ethanol from cellulosic biomass, the microorganisms enabling the CBP are still under development. We develop cellulolytic yeast strains for ethanol production by imitating the cellulosome complex system of the cellulolytic bacterium, Clostridium thermocellum. The protein that mediates the assembly of the C. thermocellum cellulosome is the scaffoldin component that contains cellulose-binding module (CBM) and cohesin domains which interact with the enzyme-borne dockerin domains. Here, a modified scaffoldin including CBM and cohesin domains is fused to Aga2 and displayed on the surface of Saccharomyces cerevisiae. Various cellulases including dockerin domain are secreted from separate yeast cells and assembled to the scaffoldin successfully by using mixed yeast cell culture. This result represents the possible reconstitution of cellulosome structure and production of cellulosic ethanol without additional cellulase supply.

Cellulosomes are cellulolytic complexes formed by highly specific interaction between one of the cohesin modules present in the scaffolding protein and a dockerin module of the catalytic components. Cellulosomal enzymes produced by C. cellulovorans grown on different carbon sources, such as Avicel, xylan, and AXP [Avicel : xylan : pectin (3:1:1)], were separated by two-dimensional electrophoresis. Using fluorescently labeled cohesin domains as described, the cohesin domains, which differ by about 58.3% in their primary structures, showed different binding profiles to the cellulosomal subunits. Fluorescence intensities of the fluorescently labeled cohesin-dockerin interactions involving cohesin6 and cohesin9 with EngY and XynB were similar, whereas EngE and EngH showed different intensities (by 1.5- to 6-fold). An enzyme-linked interaction assay showed that EngE bound preferentially to the cohesin6 comparing to cohesin9, while XynB and ManA preferences were similar. In conclusion, cohesin6 and cohesin9 showed that different binding pattern with the cellulosomal subunits under different culture conditions, it suggested cohesin-dockerin interactions occurred in a non-random fashion.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible thermoplastic that can be synthesized in many microorganisms from almost all genera of the microbial kingdom. PHAs are usually accumulated within cells when growth is limited by nutrients such as nitrogen, oxygen, and other essential elements while in the presence of excess carbon. PHAs have attracted much industrial attention because of their potential use as biodegradable thermoplastic. Volatile fatty acids (VFAs) are produced by anaerobic treatment of food wastes and can be utilized as inexpensive substrates for PHA synthesis. In this study, several Ralstonia eutropha strains were grown on the mixture of VFAs (acetic, propionic and butyric acid) as carbon and energy source for growth and PHA synthesis. R. eutropha KCTC 2658 accumulated PHAs up to 50% of dry cell weight from total 5 g/L of VFAs (acetic acid : propionic acid : butyric acid = 1:2:2). The consumption of VFAs was determined using HPLC. Batch cultures were carried out to monitor cell growth, substrate consumption, and PHA accumulation.

Dengue hemorrhage fever is one of the most serious and recurrent epidemics in tropical countries. Hitherto there has not been a commercially available vaccine against this virus. This is because there are four similar but not identical serotypes and an effective vaccine must be able to neutralize all serotypes simultaneously. Recently a potential neutralizing epitope, namely the domain III of envelop protein, has been identified and several proof-of-concepts have been established for this particular antigen. In this study we attempt to develop an oral vaccine against the serotype 2 by expressing this antigen in fusion with Heat Labile Toxin (LTB) protein from E.coli in Saccharomyces cerevisiae. The protein was expressed in both secretory form and intracellular form. Success of the expression was determined by Western blotting and GM1 ELISA. Optimization for protein expression was also attempted. Finally the product is intended to be tested on mice to determine the efficacy of this vaccine against dengue virus serotype 2.

Mycelia of Phellinus linteus, a medicinal mushroom mycelia, was successfully cultivated on solid- and liquid-state substrate using whey permeate as an alternative substrate and the optimal growth conditions was determined using response surface analysis. The growth rates to simultaneous variation in whey permeate concentration (45-65 g lactose/L), pH (4-6), and temperature (20-30 oC) on two types media was evaluated. Two equations were developed to describe the differences the growth conditions for maximum mycelial growth depending on the variables. For solid-state cultivation (SSC), the optimal conditions were 63.7 g lactose/L, pH 4.6, and 26.5 oC. On the other hand, the optimal conditions were 66.4 g lactose/L, pH 5.2, and 29.7 oC on liquid-state cultivation (LSC). Temperature had significant effect on the mycelial growth in both culture conditions at 1% a-level. pH only had a significant effect on mycelial growth in SSC, and whey permeate concentration only had a significant effect in LSC. Therefore, our results suggest a novel bioconversion method from cheese-process waste to valuable mycelia.

The root of Sciadopitys verticillata could be a good candidate for natural preservatives because of antiseptic properties. To study the possibility of Sciadopitys verticillata as natural preservatives for cosmetics, anti-microorganismic activities were tested. Root shell of Sciadopitys verticillata were extracted with various solvents including water and ethanol. Various anti microorganismic tests were evaluated against Staphylococcus aureus, Pseudomonas aerginosa, Escherichia coli and Candida albicans by paper disc diffusion method and minimal inhibitory concentration. The ethanol extract of Sciadopitys Verticillata root shell showed strong antibacterial activity against gram positive S. aureus and gram negative E.coli. The root shell of Sciadopitys verticillata showed low cytotoxicity by MTT assay with fibroblast.

Surfactant for improved oil recovery (IOR) has been for many years, particularly in the 1970’ and 1980’ when the technology was put on a sound scientific basis. Unfortunately, the economic reality of the process performance in field trials has precluded widespread deployment of this technology. Many surfactants have been evaluated for their ability to recover incremental oil and this study is focus on Polysorbate 20 as candidate for this IOR application. This laboratory study determined the characteristics of Polysorbate 20 surfactant formulations, in particular for their capability to create low interfacial tensions (IFT) with n-alkane hydrocarbons. Formulations explored included a wide range of sorbitan-based surfactants as co-surfactants with this Polysorbate 20 surfactant. Some combination of Polysorbate 20 with co-surfactant did exhibit low interfacial tension (IFT) values of 0.01 dyne/m or less versus n-octane. Laboratory testing did confirm the useful properties that the IFT for these Polysoarbate 20 formulations can be largely independent of both salinity and temperature. Preliminary studies also suggest Polysorbate 20 will have only modest adsorption onto kaolinite clay and crushed sandstone.

In this study, Saccharomyces cerevisiae (TYEGLAC3-1) was used for heterologous production of laccase3 (lac3). A new laccase, laccase3 is of interest because it is induced specifically by the presence of tannic acid but not by other commonly known fungal laccase inducers such as ferulic acid and 2,5-xylidine, which are structurally related to lignin. Thus, producing laccase3 on an industrial scale may be useful for processing plant materials with high contents of tannic acid. Statical experimental methods were used to optimize the media components for mass production of laccase3 (lac3) by Saccharomyces cerevisiae (TYEGLAC3-1). Basic medium for both of the growth and the production is composed of glucose, casamino acid, yeast nitrogen base without amino acids, tryptophan and adenine. The production medium composition as carbon and nitrogen sources selected by one factor at a time method was galactose and glutamic acid, respectively. Full factorial design (FFD) was used to find the effect of each factor on the response variable, as well as the effects of interactions between factors on the response variable. Based on these results, the steepest ascent method (SAM) and response surface methodology (RSM) were applied to determine the optimal concentrations to produce laccase3. The obtained optimal concentrations of the production medium were (g/L): galactose 19.16, glutamic acid 5 and yeast nitrogen base without amino acid 10.46. It was found that the laccase3 activity of the optimal condition medium (277.04 mU/mL) was about 13.6 times higher than that to obtain from the basic medium (20.50 mU/mL). Finally, fermentation conditions were examined for the laccase3 production from TYEGLAC3-1. The effect of temperature, pH, rpm and vvm on the laccase3 production of TYEGLAC3-1 was also studied in a batch fermenter by using optimized production medium. Laccase3 by batch culture was 1157.04 mU/mL at 25℃, pH 3.5, 100 rpm and 1 vvm.

The main object of this research is to study physiological analysis of various organisms by Physiological Activating Compounds (PACs) extracted from Rhodobacter sphaeroides. In order to investigate the effects of the physiological activation, four sequential steps were employed to accomplish this: i) optimal growth conditions of R. sphaeroides under the differential organic acids, various light intensity, and wavelength, ii) effect of physiological activation of HeLa on PACs supplemented condition, iii) impact of PACs on the viability, growth and reproduction of Daphnia magna, and iv) improvement of biohydrogen production from constructed recombinant Escherichia coli by PACs.In this study, we provided the increased physiological activation of organisms and fermentation efficiency of microorganism through PACs from purple nonsulfur photosynthetic bacteria, R. sphaeroides.