• Journal of Microbiology and Biotechnology
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• v.1 no.2
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• pp.121-125
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• 1991

The culture conditions and nutritional requirements for enhanced mycelial growth of the ectomycorrhizal fungus P. tinctorius SMF were determined in flask scale experiments. Optimum culture conditions for growth of P. tinctorius SMF in a further modified Melin-Norkrans broth were as follows; temperature 25~$27^{\circ}C$, agitation 120 rpm, and pH 4.0. P. tinctorius SMF utilized various carbon sources including monosaccharides, disaccharides, and polysaccharides. D-Glucose and mannitol were respectively the first and second most suitable carbon sources for mycelial growth. With D-Glucose as the principal carbon source, supplementation of modified Melin-Norkrans (MMN) broth with Lysine (800 mg/l), Glutamic Acid (500 mg/l), or Proline (50 mg/l) enhanced mycelial yields 63%, 34%, and 22% respectively as compared to growth in medium lacking amino acids. ThiaminㆍHCl＋biotin＋pyridoxine supplementation also enhanced growth. As compared to mycelial growth in the MMN medium, growth of P. tinctorius SMF was enhanced 120% in MMN broth when the carbon/nitrogen ratio was 25/1 in citrate buffer at pH 4.5, and growth was 50% greater in MMN broth of carbon/nitrogen ratio with a 10/1~20/1 without using the buffer. Standard conditions established for growth of P. tinctorius SMF in MMN broth were 25~$27^{\circ}C$, agitation 120 rpm, buffered to pH 4.0 with citrate, in MMN medium containing 10 g/l D-glucose supplemented with 800 mg/l lysine. In this medium the carbon/nitrogen ratio was 20/1~25/1, and the maximal mycelial yield ($Y_{x/s}$ ) was 0.472 (4.72 mg/ml) after 7 days of incubation, as compared to 0.214 (2.14 mg/ml), when the fungus was grown in standard MMN broth.

• Journal of Microbiology and Biotechnology
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• v.18 no.12
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• pp.1938-1944
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• 2008

The procpb gene encoding human procarboxypeptidase B (proCPB, GeneBank access code AJ224866) was cloned and its Pichia expression plasmid, $pPIC9{\alpha}$/hproCPB (9.2 kb), was constructed, in which procpb was under the control of the AOXl promoter and connected to the downstream of the mating factor ${\alpha}$-1 ($MF{\alpha}1$) signal sequence. The plasmid was linearized by digestion with Sacl, and integrated into the genome of P. pastoris strain GS115. By culturing of Pichia transformant on methanol medium, the human proCPB was successfully expressed and secreted into the culture supernatant. Moreover, Western blot analysis of the extracellular proteins showed proCPB bands clearly at a molecular mass of 45 kDa, confirming the expression of proCPB with its right size. The CPB activity reached about 3.5 U/ml and 12.7 U/ml in the flask and fermentor batch cultures of Pichia transformant, respectively. No CPB enzyme activity was found in the intracellular fraction. When the fed-batch cultivation was performed with methanol and glycerol mixture as a feeding medium, the extracellular CPB activity was increased to 42.0 U/ml, which corresponds to a 3.3-fold higher level of CPB activity than that of batch culture. The $K_m$ and $k_{cat}$ values of recombinant human CPB enzyme for hippuryl-$_L$-Arg as a substrate were estimated to be 0.16 mM and $11.93\;sec^{-1}$, respectively.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.35-40
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• 2004

Rotavirus virus-like particle (VLP) composed of VP2, VP6, and VP7 was expressed in the Baculovirus Expression Vector System (BEVS). Sf9 cell, a host of the baculovirus, was cultured from a 0.5-1 spinner flask to the 50-1 bioreactor system. Sf9 cell was maintained at cell density between 3.0E＋05 and 3.0E＋06 cells/ml and grew up to 1.12E＋07 cells/ml in the bioreactor. Growth kinetics was compared under different culture systems and showed similar growth kinetics with 20.1-25.2 h of doubling time. Early exponentially growing cell culture was infected with three recombinant baculoviruses expressing VP2, VP6, and VP7 protein at 1.0, 2.0, and 0.2 moi, respectively. The expression of rotavirus proteins was confirmed by Western blot analysis and its three-layered virus-like structure was observed under an electron microscope. Rotavirus VLP was semipurified and immunized in ICR mice intramuscularly. Rotavirus-specific serum antibody was detected from 2 weeks after the immunization and lasted at least 21 weeks of the post-immunization, indicating its possible use as a vaccine candidate.

• Journal of Life Science
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• v.19 no.11
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• pp.1617-1622
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• 2009

Optium conditions for the production of mycelia and extracellular polysaccharide (EXPS) from submerged mycelial culture of Inonotus obliquus and their immunomodulating activities were investigated. The optmium production of mycelia and EXPS from I. obliquus was observed in mushroom complete medium (MCM). The optimum pH, temperature, and agitation speed for the production of mycelia and EXPS were 5.5, $25^{\circ}C$, and 150 rpm, respectively. The culture period for maximum production of mycelia (10.89 g/l) and EXPS (1.25 g/l) in shake flask cultivation was 11 days. The anticomplementary activity of intracellular polysaccharide (INPS) and EXPS form I. obliquus increased in a dose-dependent manner. Lysosomal enzyme activity of EXPS and INPS increased by 2.0- and 2.2-fold at $100{\mu}g/ml$ concentration, respectively, compared to the control group.

• Microbiology and Biotechnology Letters
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• v.5 no.1
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• pp.24-28
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• 1977

For the production of microbial cells from cellulosie materials by cellulore-assimilating bacteria, Cellulomonas flavigena GFB 24-1, isolated by authors, utilization of this organism on some microbiological properties was investigated. The results of these studies were summarized at follows; 1. When the organism was incubated in the growth medium at pH 7.0 for 50 hours, its growth was the most effective and the level of excreted total protein in the menstruum increased continuously during the stationary phase of cell growth. 2. The optimal enzyme activity was observed in the pH region of 5 to 7 and culture period of 40 to 50 hours. 3. The microbial degestibility of cellulosic wastes such as sawdust, rice hull, rice straw, peanut hull and used newspaper was less than 30％, whereas that of cellulose powder was 47.1％ and rice straw was digested 77％ by NaOH treatment. 4. Bacterial cells incubated in the growth medium were increased up to 8％ of sustrate concentration and showed a decrease on further concentration. 5. The production of microbial cells from NaOH treated rice straw was obtained 10.6mg/ml of culture medium.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1670-1679
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• 2020

The substantial use of fungal enzymes to degrade lignocellulosic plant biomass has widely been attributed to the extensive requirement of powerful enzyme-producing fungal strains. In this study, a two-step screening procedure for finding cellulolytic fungi, involving a miniaturized culture method with shake-flask fermentation, was proposed and demonstrated. We isolated 297 fungal strains from several cellulose-containing samples found in two different locations in Thailand. By using this screening strategy, we then selected 9 fungal strains based on their potential for cellulase production. Through sequence-based identification of these fungal isolates, 4 species in 4 genera were identified: Aspergillus terreus (3 strains: AG466, AG438 and AG499), Penicillium oxalicum (4 strains: AG452, AG496, AG498 and AG559), Talaromyces siamensis (1 strain: AG548) and Trichoderma afroharzianum (1 strain: AG500). After examining their lignocellulose degradation capacity, our data showed that P. oxalicum AG452 exhibited the highest glucose yield after saccharification of pretreated sugarcane trash, cassava pulp and coffee silverskin. In addition, Ta. siamensis AG548 produced the highest glucose yield after hydrolysis of pretreated sugarcane bagasse. Our study demonstrated that the proposed two-step screening strategy can be further applied for discovering potential cellulolytic fungi isolated from various environmental samples. Meanwhile, the fungal strains isolated in this study will prove useful in the bioconversion of agricultural lignocellulosic residues into valuable biotechnological products.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.2
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• pp.252-258
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• 1998

The strain producing biosurfactant was isolated from soil smples. The isolated strain was identified as the genus Nocardia through its morphological, cultural and physiolgical characteristics. A high concentration of the biosurfactant by Nocardia sp. L-417 was obtained after 4 days of cultivation in the culture medium containing 3% n-hexadecane, 0.1% $NaNO_3$, 0.02% $K_2HOP_4$, 0.01% $H_2PO_4$, 0.01% $MgSO_4$.$7H_2O$, 0.01% $CaCl_2$, 0.02% yeast extract, and 0.02% tryptone. The optimum pH and temperature for biosurfactant production were pH 6.0 and $30^{\circ}C$, respectively. Furthermore, most biosurfactans were produced during the exponential growth phase, and this fact indicated that the biosurfactans production was growth-associated. The biosurfactant showed the good emulsification activities on various emulsifying substrates such as bunker A, paraffin, corn oil which are used widely in industries.

• KSBB Journal
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• v.11 no.6
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• pp.636-641
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• 1996

The production of extracellular mannitol by a new mannitol-producing bacterium, Leuconostoc sp. KY-002 was studied in shake flask cultures. The new isolate has a capability of utilizing fructose and sucrose for mannitol formation. Maximum mannitol production was obtained with fructose as the sole carbon source. Under the optimal culture conditions, within 70 hours of incubation, a final concentration of 26 g/L of mannitol from 50 g/L fructose was obtained with an indicated yield of 52% based on fructose consumed. However, higher concentrations of fructose ranging from 100 to 250 g/L could not effectively be transformed to mannitol due to a lack of osmotolerance. The strain produced no other polyols such as glycerol and sorbitol as by-products. Yeast extract was the best nitrogen source and high levels of inorganic phosphate up to 10 g/L promoted mannitol formation. Any mineral ions and salts did not play important role in both cell growth and mannitol production. Nicotinic acid enhanced mannitol production by 16%. The optimum culture temperature and initial pH were $35^{\circ}C$ and 6, respectively.

• Mycobiology
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• v.35 no.2
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• pp.54-61
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• 2007

Two isolates of Tricholoma matsutake T-008 and T-034, preserved in Entomopathogenic Fungal Culture Collection (EFCC) of Korea, were used in the present study. The isolates had 100% Bootstrap homology with Tricholoma matsutake U62964 and T. matsutake AB188557 and AF309538 preserved in Gene Bank of NCBI. Mycelial growth of T. matsutake was highest in TMM and MYA at $25^{\circ}C$. The highest dry wt. of mycelium was obtained after 65 days of culture, when 6 mycelial discs were inoculated in 100 ml of broth in 250 ml shaking flask. Mycelial mats were observed in clumped condition at the inoculation sites of pine forest after two weeks of inoculation. After 5 months of inoculation, mycelia mats were observed growing inside soil and walls of a few inoculation sites, while mycelial mats growth up to $5{\sim}8$ cm were observed in the roots of pine tree after 6 months. The survival rate of the inoculum was about 40% of the total inoculation sites. The survival rate was found below 20% when the mycelium was inoculated in the summer. The reasons for low survival rates of the mycelium were mainly due to dry season and the soil-borne small animals such as earthworm and mole. After one year of inoculation, no external difference was observed between the artificially inoculated mycelia and the naturally existing mycelia of T. matsutake. The present study showed that fruiting bodies of T. matsutake could be produced by artificial inoculation under the appropriate environmental conditions.

• KSBB Journal
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• v.10 no.5
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• pp.533-539
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• 1995

An autolytic system based on a thermally inducible phage lambda, λHL1, has been applied for the recovery of poly(3-hydroxybutyrate) [PHB] from a recombinant Escherichia coli XL1-Blue, harbouring a plasmid (pSYL105) containing the Alcaligenes eutrophus PHB biosynthesis genes. The lytic capability ofλHL1 was evaluated in flask culture for both lysogens, XL1-Blue (λHL1) and XL1-Blue (λHL1, pSYL105). When the optical density of culture at 600nm(OD600) reached 0.2, cell lysis was induced by increasing the temperature from $30^{\circ}C$ to $42^{\circ}C$. Most cells of XL1-Blue ($\lambda$HL1) were lysed by the autolytic system in an hour after the thermal induction, while the lytic efficiency was slightly lower for XLl-Blue (λHL1, pSYL105). The existence of pSYL105 in cells seemed to inhibit, to some extent, the lytic capability of λHL1 even at low PHB content. The lylic efficiency remarkably decreased as the induction was delayed to allow PHB accumulation. When a chemical induction using 2% (v/v) chloroform was introduced after an hours of thermal induction, we could obtain a good lytic efficiency.