Earticle

Effective Microorganisms (EM), a fermented medium developed by Professor Higa at the University of the Ryukyus, is a mixed culture containing dozens of microorganisms which are beneficial to nature including people, animals, plants and many microbial species in environment. EM is known to contain more than 80 kinds of anaerobic or aerobic microbes including photosynthetic bacteria, lactic acid bacteria, yeast, actinomycetes, fungi and so on, with yeast, lactic acid bacteria and photosynthetic bacteria as the main species of EM. Antioxidant effect generated by the concert of complex coexistence and coprosperity among these microbes is considered to be the main source of EM benefits. Currently, EM is earning an increasing attention with applications in agriculture, forestry, animal husbandry, fisheries, environment and medicine among others. At the same time, however, a quantitative interpretation of EM system based on a mixed culture model needs efforts from biochemical engineers for efficient production and further promotion of EM. In this paper, we describe the functions of major microbes in EM and current researches and applications of EM in agriculture, forestry, animal husbandry, fisheries, environment and medicine.

Earlier in Korea Kimchi was made in every family and every province has own taste and specialties. These days almost of the Kimchis are manufactured. We collected variable Kimchis, which were made for private use and isolated microorganisms. Some interesting micobial cells were identified and studied for its application as food and drinks. One of them was identified as Lactobacillus sakei KJ123. This strain is known as producing interesting aromatic components during Sakei fermentation like Kimchi in variable conditions. We tried to develop a health beverage with fermentation process. The Cucurbita maxima has been known as a traditional healthy food and variable positive effects on the human body were already reported. In this study we tried to develop a production process for a healthy fermented drink on this substrate with strains originated from Kimchi. Many kinds of lacctobacilli species existed in the fermented food cannot survive in the acidic conditions like human stomach. So we selected resisting strains in this conditions. The survival rate of Lactobacillus sakei cells in the artificial gastric juice and bile acid and other physiological characteristics at the variable conditions have been tested. After fermentation process some sensory tests on the product with panels were tried.

Studies were made to improve the stability of astaxanthin which has application limitations caused by light and thermal stability problems in spite of its strong anti-oxidant property. Astaxanthin was extracted from Haematococcus pluvialis with supercritical carbon dioxide. Liposomal encapsulation of astaxanthin to improve the stability was made with high pressure homogenizer. The narrow size distribution was observed with astaxanthin liposomes. Tests on light and thermal stabilities resulted that the liposormal encapsulation improved the stability of astaxanthin for cosmeceutical purposes.

Human cytotoxic T-lymphocyte antigen 4- immunoglobulin (hCTLA4Ig), an immunosuppressive agent, was expressed in transgenic rice cells using RAmy3D promoter and RAmy1A signal peptide for the inducible production and secretion into culture media by sugar depletion. In this study, sodium butyrate was used as a small molecular enhancer (SME) to enhance the production of hCTLA4Ig in transgenic rice cell suspension cultures. When 1 mM sodium butyrate was added in sugar-free media, relative viability was not reduced, while the productivity was improved 1.3-fold. In addition, by supplementing 87 mM sodium pyruvate as an alternative energy source during the production phase, death rate of the cells was decreased. When sodium pyruvate was not added, most cells became dead at day 6. However, by adding sodium pyruvate, 18% of viability can be maintained until day 10 and the production of hCTLA4Ig was enhanced 1.4-fold. When the combination of sodium pyruvate and sodium butyrate at optimum concentrations was added, the highest viability and hCTLA4Ig production could be obtained. The highest level of hCTLA4Ig reached up to 35 mg/L at day 10.

Bacillus subtilis natto producing high level of a fibrinolytic enzyme was selected and Ultra NattokinaseⓇ was manufactured by fermentation and purification. It was performed the evaluation of the antithrombotic effect of Ultra NattokinaseⓇ (20,000 FU/g) with rat blood plasma. The maximum aggregation (inhibition ratio) was 71% (0%), 69% (2.8%), 62% (12.7%), 16% (77.5%) and 9% (87.3%), respectively, in the order of 0, 5, 10, 50 and 100 mg/mL of Ultra NattokinaseⓇ solutions. Ultra NattokinaseⓇ had antithrombotic effect, which was associated with the suppression of collageninduced platelet aggregation. Ultra NattokinaseⓇ in the topic of the FDP (fibrinogen degradation products) in blood coagulation tests showed a significant increasing trend. And based on the daily record of meal 39 people of ITT (what ?) group consisted with 19 people of NP (what ?) group and 20 people of PN (what ?) group except four people, two people who took vitamin K affecting the experiment and two people who took alcohol, finding to be taken Ultra NattokinaseⓇ showed an increase in the FDP value after four weeks. In addition, FDP value of 41 people of ITT group except two people having metabolic syndrome was increased by Ultra NattokinaseⓇ.

This study was investigated to improve the saccharification yield of Ulva pertusa Kjellman by the high pressure homogenization process. It was found that the high pressure homogenization pretreatment effectively destructed the cell wall structures only by using water. The high pressure homogenization process was operated under various conditions such as 10000, 20000 or 30000 psi　with different recycling numbers. The optimal condition was determined as 30000 psi and 2 pass of recycling numbers and the sugar conversion yields were 16.02 (%, w/w) of glucose and 14.70 (%,w/w) of xylose, respectively. In the case of enzymatic　treating the hydrolyzates with 5 FPU/glucan of celullase and 100 units/mL of amyloglucosidase, 65.8% of carbohydrates was converted into glucose. Using the hydrolysates of Ulva pertusa Kjellman, 48.7% of ethanol was obtained in the culture S.cerevisiae. These results showed that the high pressure homogenization process could efficiently hydrolyze the marine resource by using only water for bioethanol production.

Microbial fuel cell (MFC) wes enriched using sludge in wastewater treatment. The microbial community of activated sludge and enriched MFC were analyzed by FISH (fluorescent in situ hybridization) and 16S rDNA sequencing. Bacteroidetes group were pre-dominant in activated sludge by FISH. α group, γ group and Acintobacter group were dominant and they were similar to distribution. The average value of 10 peak of MFC is 0.44C. When MFC wase enriched by sludge, γ-Proteobacteria, Plantomycetes group increased 70% and 60%, respectively. In results of 16S rDNA sequencing, Sphiringomonas sp. was comprised in α proteobacteria and Enterobacter sp., Klebsiella sp., Acinetobacter sp., Bacillus sp. were comprised in γ proteobacteria and Chryseobacterium sp. was comprised in Flavobacteria were isolated from sludge.

The conventional monitoring system for water pollution measurement is quite simple and independent and it has a lot of draw backs such as cost, installation, etc. So, in this paper, we have measured the water pollution system through a bacterial species, photobacterium phosphoreum. A novel integrated monitoring system technology has been developed which could easily dictate and analyze the major water pollutants and its surrounding environment in an accurate way. The system constitutes of bionic technology, information technology and environmental engineering technology. As a result, integrated monitoring system can observe the water pollution and various water environment of the whole country. Also, through the sensors of USN, Zigbee, RFID and middle ware, which can provide service and construct service platform, a properly standardized plan with remarkable service platform has been established through this investigation.

Because beverage waste contains a lot of sugar, it can be used as a valuable resource for energy. But beverage waste is discharged through the water treatment. To prevent the waste of the energy resource, we produced bioethanol by using beverage waste in this study. In order to produce bioethanol, we added distillers stillage and NaOH for fermentation condition (nutrients and pH adjustment). As a results, ethanol concentration was 5.92 vol%. In contrast, ethanol concentration of blank (not added nutrients) was low and fermentation rate was very slow. Because components of the distillers stillage help the yeast growth, fermentation yield and rate was improved. Finally, we operated distillation and dehydration process by using fermented mash and produced fuel bioethanol (more than 99.5 wt%). We think that this results may provide useful information with application of commercial ethanol production using beverage waste.

Alginate bead has been supplemented with various polymers to control permeability and to enhance mechanical strength. In this report, fibroin-reinforced alginate hydrogel was prepared, in which spatial localization of fibroin molecules was investigated. Confocal laser scanning microscopy revealed that fibroin molecules formed a fibrous network in the alginate-fibroin beads, which was expected to enhance mechanical strength as same as in many composite materials. Uniaxial compression test showed that fibroin-reinforced alginate beads had increased mechanical strength only after methanol treatment that caused β-sheet formation among fibroin molecules. Simultaneous curing and dialysis of alginate beads were carried out to remove excesscalcium but to retain fibroin in the dialysis chamber, which fabricated beads without internal fibrous fluorescent stains. Fibroin molecules were only found beneath the surface of the beads. The fibroin-diffused shell was further processed to form a thick wall after drying or was mobilizedto the centre of the bead by methanol treatment. Accordingly, the structure analyses provide processing methods of fibroin to form a wall or center clumps, which could be applied to design controlled delivery device.

In this study, a EXGA gene code for exo-β-1,3- glucanase from Aspergillus oryzae was overexpressed and secretory produced in Saccharomyces cerevisiae. To overexpress the β-1,3-glucanase, pGInu-exgA and pAInu-exgA plasmids having GAL10 and ADH1 promoter, respectively, and exoinulinase signal sequence (Inu s.s) were constructed and introduced in S. cerevisiae SEY2102 and 2805. The recombinant β-1,3- glucanase was successfully expressed and secreted into the medium and the β-1,3-glucanase activity in 2102/pGInu-exgA and 2102/pAInu-exgA strain were 5.01 unit/mL and 4.09 unit/mL, respectively. In the 2805/pGInu-exgA and 2805/pAInu-exgA strain, the β-1,3-glucanase activity showed 3.23 unit/mL and 3.22 unit/mL, respectively. Secretory efficiency in each strain reached 95% to 98%. Subsequently, the recombinant β-1,3-glucanase was used for ethanol production. Ethanol productivity in 2102/pAInu-exgA strain was 0.83 g/L when pre-treated Laminaria japonica which has initial reducing sugar of 1.4 g/L was used as substrate. It is assumed that the polysaccharides of Laminaria japonica was effectively saccharified by recombinant β-1,3-glucanase, resulting in increase of ethanol productivity. These results suggested that recombinant β-1,3- glucanase was efficiently overexpressed and secreted in S. cerevisiae SEY2102 as host strain by using ADH1 promoter-Inu s.s system.

These studies were conducted to investigate the alcohol degradation effects of the extract of herbal composition (DTS20) containing Viscum album L., Lycium chinense L., Inonotus obliquus and Acanthopanax senticosus H., on the alcohol administered mice. To investigate anti-hangover effect, alcohol and alcohol dehydrogensae (ADH) concentration of blood were measured after oral administration of ethanol. The administration of DTS20 (200-500 mg/kg) had beneficial actions toward alcohol degradation in acute alcohol treated mice model. The oral administration of DTS20 showed decreased gastric mucous membrane damage produced in ethanol treated mice. In addition, intraperitoneal administration of DTS20 showed anti-inflammatory effects in inhibition tests of vascular permeability produced by acetic acid. DTS20 also reduced the concentration of nitric oxide (NO), tumor necrosis factor (TNF)-α in macrophages that were activated by LPS. These results demonstrate that DTS20 possesses potential to stimulate the alcohol degradation and inhibit the inflammatory effects in mice.

1,2-propanediol (1,2-PD) is a commodity chemical that is currently produced from petrochemical derivatives. Saccharomyces cerevisiae is well characterized and a successful industrial microorganism to enable the improvement of the 1,2-propanediol production by metabolic engineering. A recombinant S. cerevisiae M3G3 was used to produce 1,2-propanediol. S. cerevisiae M3G3 is the diploid strain that contains 3 copies of mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase). S. cerevisiae M3G3 was cultivated at various culture conditions by changing culture temperature, glucose concentration, and inducer concentration. Also the effect of induction time was studied to optimize the production of 1,2-propanediol. Batch and fed-batch cultivation of S. cerevisiae M3G3 was performed by using a 5 L jar fermenter. The highest concentration of 1,2-propanediol in batch cultivation was 0.86 g/L and it was further improved to 1.33 g/L in fed-batch cultivation.

In this study, inorganic flocculant with biodegradable polymer flocculant was usedfor microalgae harvest. The aim of this study was to optimize the concentration of inorganic flocculant, the concentration of biodegradable polymer flocculant and reaction volume for decreasing the amounts of flocculant and obtaining the suitable pH range for seawater by response surface methodology. The flocculation of three marine microalgae, Chlorella ellipsoidea, Dunaliella bardawil, and Dunaliella tertiolecta, using inorganic flocculants and biodegradable polymer flocculants was investigated. The results indicated that the optimal flocculant quantity showed 0.1 g/L of ferric chloride, 7.5 g/L of chitosan on Chlorella ellipsoidea. In the case of Dunaliella bardawil, the optimal flocculant quantity showed amount of ferric sulfate more than 0.12 g/L and chitosan more than 0.75 g/L. In the case of Dunaliella tertiolecta, the optimal flocculant quantity showed 1.0 g/L of sodium aluminate, 0.75 g/L of chitosan.

In this study, two different extraction techniques, organic solvent extraction and supercritical carbon dioxide (SCCO2) extraction, were employed to evaluate the extraction efficiency of oil from Chlorella vulgaris. In the organic solvent extraction, the effects of various organic solvent on the extraction yield were investigated. The SCCO2 extraction was carried out while varying such operating parameters as temperature, pressure, SCCO2 flow rate, and cosolvent. About 4.9 wt% of oil was extracted from ground Chrollera vulgaris for 18 h when dichloromethane/methanol (2：1, v/v) was used as an extraction solvent. The oil yield of the SCCO2 extraction was found to be very low (0.53 wt%) and to increase up to about 0.86 wt% with the addition of cosolvent.

In this study, the canadian peat moss extract was purified by a supercritical CO2 using three different conditions and assessed its biological activities. Peat moss was extracted by acid-alkaline extraction method (sample 1) and purified by a supercritical CO2 at 40℃ under pressure of 100 bar (sample 2), 120 bar (sample 3) or 150 bar (sample 4). We evaluated the antioxidant activities of the samples by 1,1- diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging, Fe2+/ ascorbate (FTC) and 2-thiobarbituric acid (TBA) methods. The antioxidant activities were examined by comparing the results with that of ascorbic acid as a positive control. Sample 3 showed relatively higher DPPH radical-scavenging activities than other samples. The antioxidant activity by FIC method exhibited similar results as the DPPH radicalscavenging activities. On the other hand, sample 2 showed higher antioxidant activity measured by TBA method of all. The whitening effects of the samples were examined using mushroom tyrosinase and B16F10 melanoma cells. Sample 3 exhibited overall significant whitening effects, however, other samples showed relatively lower effects. These results suggest that the peat moss extract purified by a supercritical CO2 could be used as a cosmetic ingredient for the anti-aging and whitening effects.

Twenty six microorganisms were isolated from soil and horse manure samples from in Iowa, U.S. Microorganisms were cultivated and screened by using plate count agar (PCA) at 35℃ containing 1% (w/v) oat spelt xylan instead of glucose. The xylanase activities of bacterial strains were analyzed by measuring the concentration of reducing sugar by DNS method. All isolated strains were characterized as the rod form and gram positive strains. Among the isolated strains, the HM6 strains gave the highest xylanase activity. This strain was identified as Bacillus pumilus HM6 by 16S rDNA sequence, morphological and biochemical analysis. Optimal culture temperature and initial medium pH for B. pumilus HM6 were 30-35℃ and pH 6-7, respectively. The maximum xylanase activity of 6879 IU/mL was obtained after growth of HM6 with 1% (w/v) oat spelt xylan at 35℃ for 6 days. Studies on enzymatic properties showed that the optimum conditions for the highest xylanase activity were 60℃ and pH 8.0. In addition, xylanase activity was stable over 2 hours at 50℃, whereas activity decreased after 30 min at 70℃.

Nowadays biodiesel (fatty acid methyl ester, FAME) has been becoming an important issue as a desired alternative of energy products because of non-toxic, biodegradable properties, and lower exhaust emissions. During esterification of fatty acids or transesterification of oils and fats with short chain alcohols by the alkali-catalyzed methanolysis, FAME and unrefined glycerol are generated. Quantification of glycerol as a by-product is important because of a determinant of biodiesel quality. However, the glycerol analysis by gas chromatography (GC) method has laborious works with sample preparation, long time and cost of sample analysis. Thus, there is a need to analyze glycerol more simply. Herein we demonstrate that the colorimetric assay for glycerol analysis conducted by UV-vis spectrophotometer at the wavelength 617 nm whose peak is maximum intensity of malachite green, resulting in the red-shift occurred proportionally as a function of glycerol amount. Thus, it is considered the solvent media for malachite green fading for biodiesel production: (1) water, (2) MeOH, and (3) EtOH. The resulting findings show that the peak intensity at 617 nm in glycerol-malachite green mixture had a relationship between glycerol concentration and degree of peak shift as increase in pure glycerol concentration approximately at pH 7.0. However, when it was measured the unrefined glycerol concentration by diluting and adjusting with water to buffer (pH 7.0), it was not observed the absorption peak at 617 nm because of impurities and OH ions. In case of glycerol from biodiesel production factories, glycerol concentration could be successfully measured.