• Journal of Animal Science and Technology
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• v.65 no.2
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• pp.387-400
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• 2023

Ruminal protozoa, especially entodiniomorphs, engulf other members of the rumen microbiome in large numbers; and they release oligopeptides and amino acids, which can be fermented to ammonia and volatile fatty acids (VFAs) by amino acid-fermenting bacteria (AAFB). Studies using defaunated (protozoa-free) sheep have demonstrated that ruminal protozoa considerably increase intraruminal nitrogen recycling but decrease nitrogen utilization efficiency in ruminants. However, direct interactions between ruminal protozoa and AAFB have not been demonstrated because of their inability to establish axenic cultures of any ruminal protozoan. Thus, this study was performed to evaluate the interaction between Entodinium caudatum, which is the most predominant rumen ciliate species, and an AAFB consortium in terms of feed degradation and ammonia production along with the microbial population shift of select bacterial species (Prevotella ruminicola, Clostridium aminophilum, and Peptostreptococcus anaerobius). From an Ent. caudatum culture that had been maintained by daily feeding and transfers every 3 or 4 days, the bacteria and methanogens loosely associated with Ent. caudatum cells were removed by filtration and washing. An AAFB consortium was established by repeated transfers and enrichment with casamino acids as the sole substrate. The cultures of Ent. caudatum alone (Ec) and AAFB alone (AAFB) and the co-culture of Ent. caudatum and AAFB (Ec + AAFB) were set up in three replicates and incubated at 39℃ for 72 h. The digestibility of dry matter (DM) and fiber (NDF), VFA profiles, ammonia concentrations, pH, and microscopic counts of Ent. caudatum were compared among the three cultures. The co-culture of AAFB and Ent. caudatum enhanced DM degradation, VFA production, and Ent. caudatum cell counts; conversely, it decreased acetate: propionate ratio although the total bacterial abundance was similar between Ec and the Ec + AAFB co-culture after 24 h incubation. The ammonia production and relative abundance of C. aminophilum and P. anaerobius did not differ between AAFB alone and the Ec + AAFB co-culture. Our results indicate that Ent. caudatum and AAFB could have a mutualistic interaction that benefited each other, but their interactions were complex and might not increase ammoniagenesis. Further research should examine how such interactions affect the population dynamics of AAFB.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.179-187
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• 2011

This review describes the characteristics of various unculturable soil bacteria, successfully-cultivating examples of those bacteria, and the diverse factors to be considered for successful cultivation. Most importantly, the selection of proper media is very important because unculturable bacteria demand different types of nutrients at various concentrations of substrates, nitrogens and phosphorus. To develop a new medium to successfully culture unculturable bacteria from soil, molecular ecological studies should be combined together. The inoculum size on a plate is also important: less than 50 bacterial cells are recommended to be plated on a single culture plate. The environmental factors such as pH and salt concentration of the medium need to be adjusted as similar as possible to mimic the original soil environments, and the trial of the various temperatures and extended period of cultivation are better. Since one cannot simply tell about which one was unculturable among a great number of colonies grown on a newly developed medium, some suitable detection methods and fast identification methods are required. Many soil bacteria live with cooperation one another in their communities, so that enrichment such as coculture of using other bacterial metabolites and subsequent pure cultures can also guarantee successful cultivation of the previously uncultured bacteria in soil. Here, this review will discuss for the future perspectives to culture the unculturable soil bacteria.

• Korean Journal of Veterinary Service
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• v.22 no.3
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• pp.221-237
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• 1999

To investigate the specificity of rapid slide agglutination test for pullorum-gallinarum diseases and to obtain a basic data for avian salmonellosis control, salmonella isolation was peformed for the layer chickens positively reacted in pullonlm-typhoid agglutination test. The biochemical, serological and antimicrobial properties of the isolates were examined. The results obtained through this study were summarized as follows; 1. Of 2,384 chickens tested by the agglutination test, 606 chickens (25.4%) were positive reactors. 154 of 606 reactors and 49 of the non-reacting chickens were investigated for salmonella isolation, resulting in isolation of 68 strains of salmonellae from 27 chickens. 2. By organs, the isolation frequency from liver, cecum, spleen, ovary and gall bladder showed 8.9% (18 strains), 8.9% (18 strains), 7.4% (15 strains), 4.4% (9 strains) and 3.9% (8 strains), respectively. 3. By culture medium the combination of selenite broth and MacConkey agar revealed the highest isolation rate and the enrichment culture by delayed secondary enrichment culture method was found the most effective for salmonella isolation. 4. The serotypes of 68 salmonella isolates were identified as 3 strains of S pullorum, 24 strains of S gallinarum, 15 strains of S typhimurium, 8 strains of S enteritidis, 7 strains of S paratyphi A, 5 strains of S typhimurium and 6 strains of the other salmonellae. 5. The serotypes of 8 salmonella strains isolated from 49 chickens non-reacting in pullorum-typhoid agglutination test were identified as 3 strains of S typhimurium and 5 strains of S infantis. 6. When 24 chickens of which 68 strains of salmonellae isolated were examined by microplate agglutination test, the average antibody titer for pullorum antigen was $2^{5.25}$. The chickens at antibody titer between $2^3$ and $2^5$ showed the higher frequency of isolation as compared with the chickens at the other titers. 7. When salmonella isolates were tested the antimicrobial drug sensitivity by disk diffusion method, S paratyphi A were highly sensitive by 100% to ATM and GM, S typhimurium, by 88% to AM, CIP, IMP and TN, S infantis, by 100% to AM, CRO, ENR and PIP, S enteritidis,by 100% to IMP and PIP, S pullorum, by 100% to ATM, CRO, ENR and PIP and S gallinarum, by 92% to CRO, CIP and PIP.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.126-133
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• 1994

A Pseudomonas putida strain able to accumulate high amount of polyesters of medium-chain-length 3-hydroxyalkanoic acids ($PHA_{MCL)$) was isolated from soil in a landfill site using an enrichment technique. Culture condition of the isolated strain for polyester production in a one-step culture was optimized in a mineral-salts medium against pH and concentrations of ammonium sulfate, carbon source(e.g., octanoate), and phosphate. The optimal values for maximal cell growth and PHA accumulation were: pH; 7$\sim$8, $(NH_4)_2SO_4$; 8 mM, octanoate; 40 mM. The optimum temperature was in the range of $20\sim30^{\circ}C$, which was rather broader than in other bacteria. Cell growth was strongly inhibited by the phosphate limitation to less than 1 mM. An increase of phosphate concentration above 1 mM showed little effect on cell growth and polyester accumulation. When the strain was grown on octanoate under this optimized condition it produced 3.4 g dry biomass per liter and yielded 1.7 g PHA per liter amounting to 53 wt% of dry cells. The monomer units composing the polyester synthesized from octanoate were 3-hydroxyoctanoate (3HO), 3-hydroxycaproate (3HC), and 3-hydroxybutyrate (3HB) (85:13:2, mole ratio). Other low linear $C_3\simC_{10}$ monocarboxylic acids were also tested for polyester production.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.136-142
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• 1991

The selection of ethanol-tolerant strains was applied to enrichment culture of YPD broth medium containing various concentrations of ethanol. Isolates were identified to be Saccharomyces cerevisiae, the others as S. dairensis, S. exiguus, S. telluris, Saccharomycodes ludwigii, Schwanniomyces occidentalis var. occidentalis and Zygosaccharomyces florentinus. Among isolates S. cerevisiae YO-1 was screened as having the highest ethanol tolerance and produced 18% (v/v) ethanol after 4 days fermentation. The change of fatty-acyl residues represents that a progressive decrease in fatty-acyl unsaturation and a proportional increase in saturation in phospholipids of yeast cells during fermentation affected the yeast viability. Supplementation ethanol to the cultures led to an increase of unsaturated fatty-acyl residues, especially $C_{16}$ or $C_{18}$ residues, along with a decrease in the proportion of saturated residues in cellular phospholipids. Increasing the amount of soy flour led to an increase in the maximum number of viable yeast cells and ethanol production. It was possible in 4 days to reach 21% (v/v) ethanol by adding 4% soy flour as source of unsaturated fatty-acyl residues to the fermentation medium. Soy flour not only increased yeast population but also enhanced the physiological properties of yeast cells to be ethanol tolerant in the anaerobic culture.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.80-86
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• 1995

The bacterial strain WC-17 able to produce chitinase was isolated from soil using an enrichment technique. The isolated strain was identified as Acinetobacter sp. judging by their morphological and physiological characterisitics. The optimal culture conditions for the production of chitinase of Acinetobacter sp. WC -17 are 1.5% colloidal chitin and 1 % tryptone at $30^{\circ}C$ with pH 6.5. Since the enzyme was rapidly produced in a culture supplied with chitin, glucose, or N-acetylglucosamine but not with other polymers and monosaccharide, the enzyme was considered to be an inducible enzyme. Notably N- acetylglucosamine and glucose were found to be effective inducers at low concentrations but repressors at excessive concentrations. The cultural supernatant of Acinetobacter sp. WC-17 inhibited the growth of phytopathogenic fungi such as P.oryzae, R.solani, and F.solani. Among the phytopathogenic fungi tested, P.oryzae was the most sensitive. The conventional agar plate (PDA containing 1 % colloidal chitin) method also produced the same result.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.408-412
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• 2009

A study on the screening and isolation of microorganism was performed for the removal of main malodor, such as ammonia, produced from the livestock farm. The main malodor components in livestock farm are ammonia, volatile fatty acids, sulfur compounds and trimethylamine. Damages to man and livestock were originated from malodors mainly due to ammonia, and thus ammonia reduction experiments were performed. Sludge of sewage treatment plant was inoculated in the sesame dregs culture, from which ammonia gas was produced. An aerobically grown, pure cultured isolated from the 10th enrichment culture was analyzed by 16S rRNA sequencing and identified as Alcaligenes sp. NS-1. This strain NS-1 precultured in the sesame dregs was found to remove ammonia gas with an efficiency of approximately 99-100% at an average concentration of 40 ppmv of ammonia gas. When the strain NS-1 sprayed to pig excrements, the removal efficiency at an average concentration of 100 ppmv of ammonia was approximately 60% after 16 hr.

• Korean Journal of Microbiology
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• v.39 no.2
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• pp.102-107
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• 2003

With the enrichment culture technique, bacterial strains which degrade diesel oil were isolated from soil contaminated with diesel oil. One of the isolates named GENECO 1 showed the highest activity for emulsification of diesel oil as well as the highest growth rate. This strain, GENECO 1, was identified as a Pseudomonas sp. based on its biochemical, physiological characteristics and 16S rDNA sequences. The optimal cultural conditions for cell growth and oil emulsifying activity of its culture were as follow; $30^{\circ}C$ for temperature, 7.0 for pH. Diesel oil degradation was analysed by the gas chromatography. More than 95% of 1% treated diesel oil were converted into a form no longer extractable by mixed organic solvents after 96 hours incubation.

• Journal of Microbiology and Biotechnology
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• v.7 no.1
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• pp.43-48
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• 1997

Three strains capable of degrading a chlorophenol were isolated by selective enrichment from soils contaminated with industrial wastewater. A Pseudomonas solanacearum TCP114 could use 2,4,6-trichlorophenol (TCP) as sole carbon and energy source, while two strains of Pseudomonas testosteroni CPW301 and Arthrobacter ureafaciens CPR706 could use 4-CP. All isolates also grew well on phenol. The degradation of one component by a pure strain was strongly affected by the presence of other compounds in the medium, CPW301 and CPR706 entirely lost the ability to degrade 4-CP and phenol in the presence of TCP. TCP114 also lost the ability to degrade phenol when 4-CP was added to the culture medium. These restrictions on the degradability could be overcome by employing defined mixed cultures (TCP114 and one strain of 4-CP degrading strains). All three components were successfully degraded by defined mixed cultures through their cooperative activities. It was also demonstrated that defined mixed cultures could be immobilized by using calcium alginate for the semi-continuous degradation of the three component mixture. Immobilization could not only accelerate the degradation rate, but also allowed the reuse of the cell mass several times without loss of the cells' degrading capabilities.

• Microbiology and Biotechnology Letters
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• v.51 no.1
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• pp.69-82
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• 2023

Nitrilases are a hydrolase group of enzymes that catalyzes nitrile compounds and produce industrially important organic acids. The current objective is to optimize nitrilase production using statistical methods assisted with artificial intelligence (AI) tool from novel nitrile degrading isolate. A nitrile hydrolyzing bacteria Bacillus subtilis AGAB-2 (GenBank Ascension number- MW857547) was isolated from industrial effluent waste through an enrichment culture technique. The culture conditions were optimized by creating an orthogonal design with 7 variables to investigate the effect of the significant factors on nitrilase activity. On the basis of obtained data, an AI-driven support vector machine was used for the fitted regression, which yielded new sets of predicted responses with zero mean error and reduced root mean square error. The results of the above global optimization were regarded as the theoretical optimal function conditions. Nitrilase activity of 9832 ± 15.3 U/ml was obtained under optimized conditions, which is a 5.3-fold increase in compared to unoptimized (1822 ± 18.42 U/ml). The statistical optimization method involving Plackett Burman Design and Response surface methodology in combination with an AI tool created a better response prediction model with a significant improvement in enzyme production.