• Journal of Microbiology and Biotechnology
• /
• v.21 no.2
• /
• pp.204-211
• /
• 2011

To yield high concentrations of protein expressed by genetically modified Escherichia coli, it is important that the bacterial strains are cultivated to high cell density in industrial bioprocesses. Since the expressed target protein is mostly accumulated inside the E. coli cells, the cellular product formation can be directly correlated to the bacterial biomass concentration. The typical way to determine this concentration is to sample offline. Such manual sampling, however, wastes time and is not efficient for acquiring direct feedback to control a fedbatch fermentation. An E. coli K12-derived strain was cultivated to high cell density in a pressurized stirred bioreactor on a pilot scale, by detecting biomass concentration online using a capacitance probe. This E. coli strain was grown in pure minimal medium using two carbon sources (glucose and glycerol). By applying exponential feeding profiles corresponding to a constant specific growth rate, the E. coli culture grew under carbon-limited conditions to minimize overflow metabolites. A high linearity was found between capacitance and biomass concentration, whereby up to 85 g/L dry cell weight was measured. To validate the viability of the culture, the oxygen transfer rate (OTR) was determined online, yielding maximum values of 0.69 mol/l/h and 0.98mol/l/h by using glucose and glycerol as carbon sources, respectively. Consequently, online monitoring of biomass using a capacitance probe provides direct and fast information about the viable E. coli biomass generated under aerobic fermentation conditions at elevated headspace pressures.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.2
• /
• pp.270-279
• /
• 2014

Microbial community shifts, associated with performance data, were investigated in an anaerobic batch digester treating high-solid food waste under mesophilic conditions using, a combination of molecular techniques and chemical analysis methods. The batch process was successfully operated with an organic removal efficiency of 44.5% associated with a biogas yield of 0.82 L/g $VS_{removal}$. Microbial community structures were examined by denaturing gel gradient electrophoresis. Clostridium and Symbiobacterium organisms were suggested to be mainly responsible for the organic matter catabolism in hydrolysis and acidogenesis reactions. The dynamics of archaeal and methanogenic populations were monitored using real-time PCR targeting 16S rRNA genes. Methanosarcina was the predominant methanogen, suggesting that the methanogenesis took place mainly via an aceticlastic pathway. Hydrogenotrophic methanogens were also supported in high-solid anaerobic digestion of food waste through syntrophism with syntrophic bacterium. Microbial community shifts showed good agreement with the performance parameters in anaerobic digestion, implying the possibility of diagnosing a high-solid anaerobic digestion process by monitoring microbial community shifts. On the other hand, the batch results could be relevant to the start-up period of a continuous system and could also provide useful information to set up a continuous operation.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.1
• /
• pp.101-111
• /
• 2017

The task of improving a fungal strain is highly time-consuming due to the requirement of a large number of flasks in order to obtain a library with enough diversity. In addition, fermentations (particularly those for fungal cells) are typically performed in high-volume (100-250 ml) shake-flasks. In this study, for large and rapid screening of itaconic acid (IA) high-yielding mutants of Aspergillus terreus, a miniaturized culture method was developed using 12-well and 24-well microtiter plates (MTPs, working volume = 1-2 ml). These miniaturized MTP fermentations were successful, only when highly filamentous forms were induced in the growth cultures. Under these conditions, loose-pelleted morphologies of optimum sizes (less than 0.5 mm in diameter) were casually induced in the MTP production cultures, which turned out to be the prerequisite for the active IA biosynthesis by the mutated strains in the miniaturized fermentations. Another crucial factor for successful MTP fermentation was to supply an optimal amount of dissolved oxygen into the fermentation broth through increasing the agitation speed (240 rpm) and reducing the working volume (1 ml) of each 24-well microtiter plate. Notably, almost identical fermentation physiologies resulted in the 250 ml shake-flasks, as well as in the 12-well and 24-well MTP cultures conducted under the respective optimum conditions, as expressed in terms of the distribution of IA productivity of each mutant. These results reveal that MTP cultures could be considered as viable alternatives for the labor-intensive shake-flask fermentations even for filamentous fungal cells, leading to the rapid development of IA high-yield mutant strains.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.6
• /
• pp.868-877
• /
• 2020

Bicarbonate has been considered as a better approach for supplying CO₂ to microalgae cells microenvironments than gas bubbling owing to cost-effectiveness and easy operation. However, the β-carotene production was too low in Dunaliella salina cultivated with bicarbonate in previous studies. Also, the difference in photosynthetic efficiency between these two carbon sources (bicarbonate and CO₂) has seldom been discussed. In this study, the culture conditions, including NaHCO₃, Ca²⁺, Mg²⁺ and microelement concentrations, were optimized when bicarbonate was used as carbon source. Under optimized condition, a maximum biomass concentration of 0.71 g/l and corresponding β-carotene content of 4.76% were obtained, with β-carotene yield of 32.0 mg/l, much higher than previous studies with NaHCO₃. Finally, these optimized conditions with bicarbonate were compared with CO₂ bubbling by online monitoring. There was a notable difference in F_v/F_m value between cultivations with bicarbonate and CO₂, but there was no difference in the F_v/F_m periodic changing patterns. This indicates that the high concentration of NaHCO₃ used in this study served as a stress factor for β-carotene accumulation, although high productivity of biomass was still obtained.

• Microbiology and Biotechnology Letters
• /
• v.41 no.2
• /
• pp.236-241
• /
• 2013

Pilocarpine is an imidazole alkaloid, found exclusively in the Pilocarpus genus, with huge pharmaceutical importance. In order to extract pilocarpine from Pilocarpus jaborandi, environmentally friendly enzyme-assisted extraction was applied. Viscozyme$^{(R)}$ L, a commercially available enzyme cocktail, was used for the study. The conditions for extraction were optimized on the basis of substrates, enzymes, temperatures and pHs. Optimum conditions for extraction with the highest yield were 30 h reaction of 100 mg substance at $45^{\circ}C$ in 40 ml of 50 mM acetic acid, pH 4. A 10% enzyme concentration was found to be the best for extraction. Total pilocarpine content after extraction was analyzed by HPLC. The total pilocarpine content ($1.14{\mu}g/mg$) obtained from Viscozyme$^{(R)}$ L treatment was 3.08-fold greater than those of the control treatment ($0.37{\mu}g/mg$).

• Applied Biological Chemistry
• /
• v.31 no.4
• /
• pp.376-381
• /
• 1988

The optimal reaction conditions were investigated to produce L-phenylalanine from transcinnamic acid and ammonia by Rhodotorula glutinis IFO 0559. The highest amount of L-phenylalanine was produced when the reaction mixture containing 200mM of traps-cinnamic acid, 4M of $NH_4OH$, 250mM of $(NH_4)_2SO_4$, 0.005% of cetylpyridinium chloride (pH 10.5) and 50mg/ml of dry cell was used. Among the nonaqueous organic solvents, petroleum ether was the most effective on the production of L-phenylalanine. The optimal concentration of petroleum ether in the reaction mixture was 50%. Under the optimal conditions, 21.1g/l of L-phenylalanine was produced in 12hr, and the yield was 63.9% based on transcinnamic acid.

• Microbiology and Biotechnology Letters
• /
• v.30 no.3
• /
• pp.253-257
• /
• 2002

The time-dependent changes of red pigments production in solid-state plant scale fermentor using barley cultured with Monascus sp., instead of rice which was traditionally used, were investigated in this study. A steady increase in the yield of red pigments in barley occurred between the 3rd and 6th days. The optimized conditions (inoculation volume = 6∼8%, initial pH = 6, air supply = 0.6∼0.8 m) promoted the production of red pigments. Short-time steaming of barley (< 20 min) decreased fungal growth and pigments production due to the insufficient gelatiniza-tion. The optical density of the red pigments under the optimized conditions was 120 at 500 ]nm per gram of barley. In addition, the metabolites from the fermented barley with Monucus sp. showed antibacterial effects against Escherichia coli and Salmonella typhimurium. Barley was shown to be one of the best grain sources for solid-state fermentation with Monascu sp., fur obtaining natural pigments and also functional food materials.

• Microbiology and Biotechnology Letters
• /
• v.14 no.6
• /
• pp.461-466
• /
• 1986

A highly cyclodextrin glucanotransferase producing strain of Bacillus sp. was isolated from soil, and basic studies on the characteristics of the strain and its enzyme, conditions for the enzyme production, and the enzyme utilization were carried out. The isolated strain was aerobic, motile, endospore-forming and rod-shaped bacterium. Optimum pH and temperature for the enzyme action were 6.0 and 45$^{\circ}C$, and the enzyme was stable within 5$0^{\circ}C$, and between pH 6.0 and 10.0. The highest yield of the enzyme was obtained using the medium containing 2% corn starch as a carbon source, and 5% corn steep liquor, 0.1% urea and 0.25% ammonium sulfate as nitrogen sources. The fermentation conditions for the enzyme production in a jar fermentor were cetermined to be 3$0^{\circ}C$, 200rpm, 0.6vvm and 60hr cultural period. Stevioside transglycosylation catalyzed by this enzyme was identified by high performance liquid chromatography.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.1
• /
• pp.1-7
• /
• 2000

Bioconversion of fumarate to succinate was anaerobically conduced in a synthetic medium containing glycerol as a hydrogen donor and fumarate as a hydrogen acceptor. We investigated the effects of pH, carbon and nitrogen sources, conversion substrate, and other culture conditions on the production of succinate using a nwely isoloated Enterococcus facalis PKY1. Addition of a variety of carbonates to the medium significantly increasd the rates of production of succinate. The production of succinate and cell growth were relatively satisfactory in the pH range of 7.0-7.6. By using glycerol as a hydrogen donor, high purity succinate was produced with few byproducts. Yeast extract as a sole nitrogen source was the most effective for producing succinalte. As a result, the optimum condition of biconversion was obtained at a medium containing 20g/I glycerol, 50 g/l fumarate, 15 g/l yeast extract, 10 g/l $K_2HPO_4$, 1 g/I NaCl, 50ppm $MgCl_2{\cdot}6H_2O$, 10ppm $FeSo_4{\cdot}7H_2O$, and 5 g/I $Na_2CO_3$ at pH 7.0-7.6. Under the optimum condition, a succinate concentration of 153 g/I was produced in 36 h. The total volumetric production rate and the molar yield of succinate were 4.3 g/l/h and 85%, respectively.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.11
• /
• pp.1850-1858
• /
• 2018

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase ($Dac_{ph}$) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature $40^{\circ}C$, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.