• Journal of Microbiology and Biotechnology
• /
• v.23 no.7
• /
• pp.913-922
• /
• 2013

An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The ${\beta}$-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) ${\beta}$-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to ${\beta}$-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant ${\beta}$-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at $55^{\circ}C$ and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by $FeSO_4$ (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a ${\beta}$-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.11
• /
• pp.1891-1907
• /
• 2016

Yeasts that are present in marine environments have evolved to survive hostile environments that are characterized by high exogenous salt content, high concentrations of inhibitory compounds, and low soluble carbon and nitrogen levels. Therefore, yeasts isolated from marine environments could have interesting characteristics for industrial applications. However, the application of marine yeast in research or industry is currently very limited owing to the lack of a suitable isolation method. Current methods for isolation suffer from fungal interference and/or low number of yeast isolates. In this paper, an efficient and non-laborious isolation method has been developed and successfully isolated large numbers of yeasts without bacterial or fungal growth. The new method includes a three-cycle enrichment step followed by an isolation step and a confirmation step. Using this method, 116 marine yeast strains were isolated from 14 marine samples collected in the UK, Egypt, and the USA. These strains were further evaluated for the utilization of fermentable sugars (glucose, xylose, mannitol, and galactose) using a phenotypic microarray assay. Seventeen strains with higher sugar utilization capacity than the reference terrestrial yeast Saccharomyces cerevisiae NCYC 2592 were selected for identification by sequencing of the ITS and D1/D2 domains. These strains belonged to six species: S. cerevisiae, Candida tropicalis, Candida viswanathii, Wickerhamomyces anomalus, Candida glabrata, and Pichia kudriavzevii. The ability of these strains for improved sugar utilization using seawater-based media was confirmed and, therefore, they could potentially be utilized in fermentations using marine biomass in seawater media, particularly for the production of bioethanol and other biochemical products.

• Korean Chemical Engineering Research
• /
• v.49 no.4
• /
• pp.470-474
• /
• 2011

Rice straw is the main grain straw and is produced in large quantities every year in Korea. Pretreatment of lignocellulosic biomass using soaking process was carried out mild conditions at atmospheric pressure and temperature of $60^{\circ}C$. We found enzymatic hydrolysis condition of pretreated biomass. In case of a rice straw, compared with previous lignocellulosic biomass, we found that hydrolysis time was a shorter than others. Hydrolysis of SAA-treated rice straw has shown conversion rate was higher at $50^{\circ}C$. Hydrolysis was ended between 40~48 hour. Glucose conversion rate was higher when enzyme loading is 65 FPU/ml and 32 CbU/ml. When substrate concentration was 5%(w/v), it was that conversion rate was 83.8% after hydrolysis for 72 hr. In simultaneous saccharification and fermentation(SSF) experiment about SAA-treated rice straw, ethanol productive yield was highest from $40^{\circ}C$. The yield of that time was 33.05% from 48 hour.

• Korean Chemical Engineering Research
• /
• v.53 no.4
• /
• pp.468-471
• /
• 2015

Microalgae have the advantages of being able to utilize the solar energy and culturing at a low cost. In particular, microalgae have a great potential in the production of biodiesel due to the high lipid content. Lipids produced from microalgae are converted to fatty acid methyl ester (FAME) by trans-esterification reaction and FAME is called a biodiesel in general. In addition, microalgae can also be utilized as a substrate for ethanol fermentation after saccharification reaction. In this study, three types of microalgae (Nanochloris, Dunaliella tertiolecta, Tetraselmis) were cultured and their lipid contents were compared. In addition, the effects of lighting period on the growth rate and lipid content were studied. Finally, the amounts of glucose produced from each saccharified microalgae were investigated. As a result, we demonstrated that D. tertiolecta has 43.6% higher lipid content and 22% higher glucose conversion than two others.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.12
• /
• pp.1681-1691
• /
• 2012

This work was conducted to evaluate the effect of dilute sodium hydroxide (NaOH) on barley straw at boiling temperature and fractionation of its biomass components into lignin, hemicellulose, and reducing sugars. To this end, various concentrations of NaOH (0.5% to 2%) were applied for pretreatment of barley straw at $105^{\circ}C$ for 10 min. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and Fourier transform infrared (FTIR) spectroscopy studies revealed that 2% NaOH-pretreated barley straw exposed cellulose fibers on which surface granules were abolished due to comprehensive removal of lignin and hemicellulose. The X-ray diffractometer (XRD) result showed that the crystalline index was increased with increased concentration of NaOH and found a maximum 71.5% for 2% NaOH-pretreated sample. The maximum removal of lignin and hemicellulose was 84.8% and 79.5% from 2% NaOH-pretreated liquor, respectively. Reducing sugar yield was 86.5% from 2% NaOH-pretreated sample using an enzyme dose containing 20 FPU of cellulase, 40 IU of ${\beta}$-glucosidase, and 4 FXU of xylanase/g substrate. The results of this study suggest that it is possible to produce the bioethanol precursor from barley straw using 2% NaOH at boiling temperature.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.12
• /
• pp.1673-1680
• /
• 2012

In this study, DEAE-corncobs [delignified corncob grits derivatized with 2-(diethylamino)ethyl chloride hydrochloride ($DEAE{\cdot}HCl$)] were prepared as a carrier to immobilize yeast (Saccharomyces cerevisiae) for ethanol production. The immobilized yeast cell reactor produced ethanol under optimized $DEAE{\cdot}HCl$ derivatization and adsorption conditions between yeast cells and the DEAE-corncobs. When delignified corncob grit (3.0 g) was derivatized with 0.5M $DEAE{\cdot}HCl$, the yeast cell suspension ($OD_{600}$ = 3.0) was adsorbed at >90% of the initial cell $OD_{600}$. This amount of adsorbed yeast cells was estimated to be 5.36 mg-dry cells/g-DEAE corncobs. The $Q_{max}$ (the maximum cell adsorption by the carrier) of the DEAE-corncobs was estimated to be 25.1 (mg/g), based on a Languir model biosorption isotherm experiment. When we conducted a batch culture with medium recycling using the immobilized yeast cells, the yeast cells on DEAE-corncobs produced ethanol gradually, according to glucose consumption, without cells detaching from the DEAE-corncobs. We observed under electron microscopy that the yeast cells grew on the surface and in the holes of the DEAE-corncobs. In a future study, DEAE-corncobs and the immobilized yeast cell reactor system will contribute to bioethanol production from biomass hydrolysates.

• Microbiology and Biotechnology Letters
• /
• v.42 no.2
• /
• pp.202-206
• /
• 2014

A response surface method based on a central composite design experiment was used to determine the optimum conditions for pretreatment of persimmon peel. It was mathematically predicted that the maximum amount of reducing sugars would be obtained at an $H_2SO_4$ concentration of 1.77% (w/v) and a heat treatment time of 26.4 min. A reducing sugar concentration of 63.23 g/l was obtained under the optimum pretreatment conditions determined by RSM. Under anaerobic growth conditions, Saccharomyces cerevisiae NK28 produced 15.52 g/l of ethanol with a yield of 0.34 g ethanol/g glucose from pretreated persimmon peel, which corresponded to 14% and 26% enhancements in ethanol productivity and ethanol yield, respectively, compared with those obtained in aerobic growth conditions. This study suggests that persimmon peel might be a useful substrate for bioethanol production by yeast fermentation.

• Journal of the Korea Organic Resources Recycling Association
• /
• v.19 no.3
• /
• pp.79-89
• /
• 2011

The wasted corn stalk from Gangwon province is composed of 44.6 % glucan, 19.0 % xylan, 23.8 % lignin, 4.5 % ash and 8.1 % others. Statistical analysis, full factorial design, revealed that temperature was the most influential factor in the dilute sulfuric acid pretreatment and that the influence of temperature on xylose yield was 3.5 and 3.2 times higher than those of treatment time and acid concentration, respectively. Temperature was also the most influential factor for glucose yield in the pretreatment but it was less than 5 % throughout the pretreatment. Although minor sugar yield was observed when microwave or ultrasonication was solely introduced as a pretreatment method, the complex method incorporating microwave or ultrasonication into dilute sulfuric acid pretreatment enhanced sugar yield significantly. In particular, xylose yield was doubled when microwave and dilute sulfuric acid treatment was sequentially applied. The optimization of pretreatment and enzymatic hydrolysis as well as the investigation on the complex pretreatment in detail are left for further study.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.4
• /
• pp.625-632
• /
• 2019

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The $pGMF{\alpha}-HGN$ plasmid harboring AGAH71 and AGAG1 genes encoding ${\beta}-agarase$ and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro-L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae $2805/pGMF{\alpha}-HGN$ strain was cultivated into YP-containing agarose medium at $40^{\circ}C$ for 48 h (for saccharification) and then $30^{\circ}C$ for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.2
• /
• pp.244-255
• /
• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.