Earticle

Experiments for lignin peroxidase production have been conducted by aerobic fermentation of Phanerochaete chrysosporium under low shear rate and enriched oxygen environment. The result of flask cultures of white rot fungus indicated that high oxygen concentration and low shear force were essential for enhancement of lignin peroxidase production. Pentachlorophenol was readily degraded by lignin peroxidase produced in nutrient limited flask cultures. Polyurethane foam was fond to be an effective immobilization matrix of P. chrysosporium.

세계보건기구 (WHO)가 백신 생산에 권장하고 있는 표준세포 주인 Vero 세포에 약독화 일본뇌염바이러스인 SA14-14-2 ( (PDK)를 연속 계대배양을 통해 적응(adaptation)시켜, tIter가 107 pfu/mL을 넘는 SA-14-14-2(Vero)을 분리하였다 바이러스 배양 최적온도는 35℃이며, T -flask에서 배양된 바이러스의 최고 tIter는 감염 후 4일째에 4x107 pfu/mL로 관찰되었다. 또한 무혈청배지에서도 바이러스 증식이 활발하여 2% 혈청이 보충된 정우와 거의 비슷한 바이라스 tIter를 보였다. 바이러스 대량 배양을 위해 roller bottle culture와 미 립 담체 플 이용한 spinner flask culture 가능성에 대하여 고찰하였다 바이러스 감염을 위한 미립담체에서의 Vera cell monolayer는 초기 세포 농도 4ㅌ10 cells/mL로 접종하여 50 rpm에서 7일간 배양하여 얻을 수 있었다. 바이러스의 roller bottle 배양이 spinner flask 배양보다 바이러스 tIter변에서 2배 내지 3배 높 았고, 107 pfu/mL을 넘는 배양 기간도 하루 죄었다. 하지만 두 배양 방법 모두 T -flask 배양에서와 같이 무혈청 배지를 사용 하여도 바이라스 증식이 활발했고, 최고조의 tIter를 보이는 배 양기간은 감염 후 2일째로써 T -flask 배양에서 보다 2일 빨랐다. Roller bottle culture의 경우, 감염 후 3일부터 17일까지 2 일 간격으로 배양액을 무혈청 EMEM으로 100% 교체하면서 매 양을 지속한 결과 3일부터 9일까지 107 pfu/mL을 념는 tIter가 유지되는 것이 확인되어 바이러스의 multi-harvest가 가능한 것 로 고찰되었다. 상기의 결과는 생산성 면에서 매우 유리한 결 과로 제품의 생산 단가플 낮추고 작업 노력을 절감하는 기대 효 과가 클 것으로 예측된다.

An attenuated Japanese encephalitis virus (JEV) clone SA-14-14-2(Vero) was obtained through successive adaptation of a primary cell adapted strain, SA-14-14-2(PDK) to Vero cell, a continuous line of monkey kidney cells. The virus titer reached above the 107 plaque forming unit (pfu) per mL of culture supernatant with 3 passages in Vero cells and was maintained close to this level in the further passages. The optimum temperature for the virus growth was 35℃. Growth pattern of the virus indicated that optimum time for the virus harvest is 4 days post infection and the virus accomplished rapid initial propagation even in medium containing no serum supplement. The roller bottle (RB) system and the spinner flask (SF) system using micro-carrier (Cytodex-1) for the JEV cultivation were explored. When RB, SF, and T-flask culture system were compared, there was no significant difference in virus yield. Furthermore, the results indicated that virus could be harvested multiple times from 3 days to 9 days post infection; neither severe cytopathic effect (CPE) in the infected cells nor the decrease in the titer was observed on duration of 9 days

양어장 순환수 속의 암모니아성 질소의 제거를 위한 Ba-alginate 와 Ca-alginate 반응기는 0.6시간의 수력학적 체류시간에서 51.0, 52.6 g NH3-N/m3/daty의 거의 유사한 암모니아성 집소의 제거속도릎 나타내었다. Ca-alginate를 이용한 합성양어장수 속의 암모니아성 짖소의 제거 실험에서 본 반응기의 암모니아성 짐소의 제거속도는 수력학적 체류시간이 0.3시간인 지점에서 82.0 g 로 NH3-N/m3/daty서 최고의 재거속도릎 나타냈으며 이때 암모니아 제거율은 48% 이었다. 수중의 암모니아의 농도가 2mg/L 정도의 범위에서는 반응기 내에 주입되는 공가량을 0.1 vvm 으로 공급하더라도 용존산소 농도를 7.0 - 5.6 g/m3로 유지 할 수 있었으므로 질화에 필요한 용존산소량을 충분히 유지할 수가 있는 것으로 나타났으며 pH 는 7.2 - 7.9의 범위플 유지할 수가 있어서 pH 변화에 따른 위 해 요소는 없는 것으로 나타났다.

Nitrifier consortium immobilized in Ca and Ba-alginate beads were packed into two bioreactors and the performances of bioreactors were evaluated for the removal of ammonia nitrogen from synthetic aquaculture water. The total ammonia nitrogen (TAN) concentration of the influent was continually kept about 2g TAN/㎥. At the HRT of 0.6hr, ammonia nitrogen removal rate of two bioreactors were about 52.6 and 51.0g TAN/㎥/day, respectively. At the respect of ammonia nitrogen removal, two bioreactor showed the similar abilities. The second trial with nitrifier consortium immobilized in Ca-alginate bead was carried out to evaluate the ammonia nitrogen removal rate for 35 days. The highest ammonia nitrogen removal rate was 82g TAN/ when HRT was about 0.3hr.

글루코스와 초산을 온라인 모니터링 하기위한 흐름주입분석기 술이 개발되었고 대장균발효공정에 이용되었다. 또한, Epoxy 고 분자 담체에 고정화된 GOD와 SOD 를 이용한 GOD-FIA와 SOD-FlA의 특성을 연구했다. 즉, FIA 의 조작온도, 운반용액 속의 첨 가제 (Triton, EDTA, natrium azid 등), 분석 시 료에 용 해된 신진대사물의 농도, pH, 초산측정시 사코진 농도 등에 따 라 고정화된 GOD와 SOD의 활성도, 즉 검출신호의 높이변화를 고찰했다. 최소배양액 과 복합배양액을 사용한 대장균 발효공정 에서 달루코스와 초산을 동시에 온라인 모니터링 하였으며 그결 과는 오프라인 분석결과와 잘 일치 하였다,

Flow-injection analysis (FIA) for on-line monitoring of glucose and acetate are described and employed in E.coli fermentation process. Glucose oxidase (GOD) for the detection of glucose is immobilized on epoxy polymer support, which is packed in a small cartirdge, and applied to a GOD-FIA system. The detection of acetate is based on the inhibition of acetate to the oxidation of sarcosine by sarcosine oxidase (SOD). SOD is also immobilized on epoxy polymer support and used for a SOD-FIA system. GOD-FIA system is characterized as well as SOD-FIA system by the investigation of the effects of pH, temperature and metabolites in samples on the peak height. GOD-FIA and SOD-FIA systems were also applied for on-line On-line measurements buy FIA measurements by FIA were in god agreement with off-line measurements.

Scutellaria baicalensis. G. 식물 세포의 현탁 배양에서 세포의 성장과 flavonoid glycosides 생산에 관한 구조적 성장 모텔 을 제안하였다 제안된 모델식은 현탁 배양의 형광을 측정함으 써 결정될 수 있는 세포 생존도와 세포 활동도 등플 세포의 생리학적 변수로서 고려하였다. 제안된 모델식에서는 세포의 상태에 따라 세포블 크게 생존 세포와 비생존 세포로 나누고 생존 세포를 분화 가능한 생존 세포와 비분화 생존 세포로 나누어 각 각의 모델을 구성하였다. 이 중 생존 세포 중량은 광섬유 형광 센서로 측정한 상대 형광 세가로부터 결정될 수 있었다. Flavonoid 배당체의 생산 속도는 분화 가능한 생존 세포와 바 분화 생존 세포에 의해 지배되며, 배양액의 삼투압에 의한 서l포 팽창과 세포내 생성불절의 방출은 비생존 세포에 의해 이루어진다고 가정하였다. 종속변수는 가칠농도(포도당), 세포 중량(건조 세포중량과 생체중량), 대사산물농도(flavone glycosides), 활동도와 생존도플 포함한다 Scutellaria baicalensis. G. 식물 세포 의 푹라스크 배양으로부터 모델과 실험결과가 잘 일치함을 알 수 있었다. 제시된 모델은 세포의 성장, flavone glycosides 및 중간매개체 합성을 예측할 수 있다.

A structured kinetic model is proposed to describe cell growth and secondary metabolite, flavone glycosides, synthesis in batch suspension culture of Scutellaria baicalensis G. The model has been developed by representing the physiological state of cell described as the activity and viability which can be estimated based on the culture fluorescence. In the model, three type of cells are considered; active-viable, nonactive-viable and dead cells. Viable cell weight could be determined based on the relative fluorescence intensity. The flavone glycosides could be produced by both active-viable and non-active viable cells with a different production rate. And the model includes the cell expansion due to glucose concentration and death phase which accounts for the release of intracellular secondary metabolite into medium. Dependent variables include substrate concentration(glucose), cell mass(dry cell weight and fresh cell weight), product concentration(flavone glycosides), activity and viability. Satisfactory agreement between the model and experimental data is obtained from shake flask culture of Scutellaria baicalensis G. The proposed model can predict the cell growth and flavone glycosides synthesis as well as intermediate materials.

Optimal feeding strategies in bioreactor operation of Scutellaria baicalensis G. plant cell culture were investigated to maximize the production of flavone glycosides by using a structured kinetic model which can predict culture growth and flavone glycosides synthesis in a rigorous, quantitative manner. For the production of baicalin and wogonin-7-0-GA, the strategies for glucose feeding into Scutellaria baicalensis G. plant cell culture were proposed based on the model, which are a periodic fed-batch operation with maintenance of cell viability and of specific production rate respectively, and a perfusion operation with maintenance of specific production rate for baicalin and wogonin-7-0-GA. Simulation results showed that the highest volumetric concentration of flavone glycosides was obtained in a periodic fed-batch operation with maintenance of cell viability among all the suggested strategies. In the periodic fed-batch operations, the higher volumetric production of flavone glycosides was achieved compared with that in the perfusion operation. It can be concluded that a periodic fed-batch operation with maintenance of cell viability would be the optimal and practical operating strategy of Scutellaris baicalensis G. plant cell culture for the production of flavone glycosides.

Bacteriorhodopsin in the purple membrane (PM) of halobacteria has recently been attracting much attention to be used as a component of molecular electron device and optical computers. In order to increase the productivity of bacteriorhodopsin in high cell density cultures of Halobacterium halobium R1, an internal membrane cell-retention bioreactor system was employed. As a result, the production of cell mass at OD660 of 12 and of bacteriorhodopsin at 125-130 mg/L were obtained using the internal membrane bioreactor system at a dilution rate of 0.066 hr-1. The productivity achieved by the internal membrane system (0.7 mg/Lhr) was 3.5-fold higher than that obtained by the corresponding batch cultivations (0.2 mg/Lhr).

Cell recovery from high cell density broths of Alcaligenes eutrophus by pretreatment with aluminum-based coagulants such as aluminum sulfate, polyaluminum hydrooxide chloride silicate (PACS), and polyaluminum hydrooxide chloride (Hi-PAX) was carried out. Cells coagulated with coagulants could be successfully recovered above 95-99% by centrifugation or filtration. The optimum initial pH of fermentation broths for cell recovery was in the range of 10 to 12. Optimum coagulants dosage for cell recovery increased with increasing of cell concentrations (21-160 g/L). The optimum coagulant dosages to recover cells with more than 95% cell recovery by centrifugation for the cell concentrations ranged 21-160 g/L were as follows: aluminum sulfate, 416-1708 mg Al/L; PACS, 211-826 mg Al/L; Hi-PAX, 320-960 mg Al/L. At optimum conditions for the coagulation of cells, centrifugal forces for 95% of cell recovery were dependent on the cell concentration. The centrifugal forces at 82 g/L and 160 g/L of cell concentration were only 45g and 1600g, respectively.

To investigate the feasibility of the microbial process for removal of heavy metals from the high solid content sludge, the effect of sludge concentration on the solubilization of heavy metals by an iron oxidizing bacterium Thiolbacillus ferrooxidans was examined. With increasing the sludge concentration, the removal efficiency of heavy metals and the oxidation rate of iron were inhibited. Especially, when the sludge concentration is over 5% (w/v), the activity of T. ferrooxidans was remarkably inhibited. This inhibition is considered to occur due to the dissolved inhibitory materials such as organic compounds, heavy metals, and others which were extracted from the sludge during incubation period. In conclusion, the microbial process by T. ferrooxidans is only effectively used in ranges of 1.3 to 4.0% (w/v) sludge concentration.

Production of inulo-oligosaccharides from inulin was carried out with the maximum yield of 94% using a purified endoinulinase from Aspergillus ficcum. The optimum reaction temperature and pH were 55-60 and pH 5.5-6.0, respectively. The Michaelis constant and maximum reaction velocity of the endoinuinase for inulin were 13.27 g/L and 0.13 g/Lmin at 55, pH 5.5, respectively. Inulin source had no significant effect on oligosaccharide yield and product composition, although initial production rate differed according to inulin origins. The reaction pH was a critical factor in inulo-oligosaccharide production because considerable free monosaccharides were released, decreasing oligosaccharide yield at low pH conditions. An empirical relationship describing the reaction performance was developed from kinetic data: the time to reach maximum oligosaccharide yield (tw) as a function of initial concentration of inulin (lo) and enzyme (Eo); i.e., log tM = -1.025 log Eo - 0.011 l0 + 2.655.

The effects of various step-fortifications of the initial medium with amino acids, glucose, and vitamines on the growth and metabolism of a hybridoma cell line in batch cultures were quantified. Comparisons between the metabolic rates of the various cultivations were made for the exponential growth phase. Fortification of the basal medium resulted in higher cell densities through a prolonged growth phase, but the maximum specific growth rate was not affected. The uptake rate of glutamine increased with the addition of amino acids but did not change upon the addition of glucose or vitamines. The specific glucose consumption decreased slightly with the addition of amino acids but increased production of lactate and {{{{ { NH}`_{4 } ^{ +} }}}}. A reciprocal relationship between the yields of {{{{ { NH}`_{4 } ^{+ } }}}} and lactate indicated a joint regulation of glycolysis and glutaminolysis.

Human Erythropoietin (EPO) gene is cloned in quail fibrosarcoma cell, QT35. Because molecular weight of EPO is similar to that of serum albumin, cell culture with serum containing medium makes purification of EPO very difficult. Using fractional factorial study, we have developed serum free medium for the recombinant QT35 cell lines, QT N4D4 and QT SY-IMP, which have cloned EPO with glutamine synthetase (GS) gene amplification system and with puromycin selective marker, respectively. Among the seven frequently used medium components, fibronectin, BSA, and EGF were the most important for EPO production. However, sufficient fibronectin supplement to the medium did not make any good attachment of QT35 to culture plate over 3 days. Therefore, to maximize EPO production, we attempted a medium-shift at confluence from serum containing medium to serum free medium(QT SFM6). Using the medium-shift protocol with QT SFM6, nearly the same productivity of EPO was achieved comparing with that without medium-shift. This result was true in both QT35 cell lines in three types of culture, i.e. T flask, microcarrier and roller bottle cultures.

The fermentation conditions for the maximal production of the useful polysaccharides(water soluble polysaccharide and cell bound polysaccharide) from marine bacterium Zoogloea sp.(KCCM 10036) were investigated with a 5 L jar fermentor. The maximal production of these polysaccharides was obtained under the conditions of initial pH 7.8, 30℃, 400 rpm of agitation speed, 2 Wm of aeration rate, 10%(w/v) of inoculum size and 2.5%(w/v) of glucose substrate and 10.38 g/L of total polysaccharide was produced. Apparent viscosity of the culture broth was increased with the production of these polysaccharides and the maximum value was reached to 22,500 cp.

A novel process was developed to simultaneously produce invertase and yeast extract from baker's yeast using ultrafiltration (UF) and microfiltration (MF) membrane processing. After the extraction of invertase under the optimal condition obtained in this study, invertase was separated from yeast cells using a hollow fiber membrane with a pore size of 0.1um . The resulting permeate containing invertase was concentrated using a hollow fiber membrane with a nominal molecular weight cut-off of 30 kDa. The yeast cell and permeate solutions, which were obtained after MF and UF membrane processing, respectively, were mixed together, and the autolysis was performed at 50 ℃in the presence of 5% (w/v) ethanol and 1% (w/v) NaCl. As a result, the yeast extract and invertase could be simultaneously produced from baker's yeast by this novel process.

The dilute-acid pretreatment/hydrolysis of hemicellulose in oak wood using a percolation reactor was investigated. The experimental conditions ranged 160∼180℃ and 0.05∼0.2 wt.% sulfuric acid. XMG(xylan+mannan+galactan) recovery was higher when sulfuric acid was used as leaching solvent than water. Also it was important for high XMG recovery to keep leaching temperature higher after reaction. XMG recovery was decreased as the size of wood chips was increased. At an optimum condition (reaction condition= 170℃, 0.1% sulfuric acid, 1ml/min, 10min, leaching condition=0.1% sulfuric acid, 2mL/min, 20 min), the product yield and the sugar concentration were about 92% and 2.7%, respectively.

Agar degrading enzyme-agarase-was purified from the culture fluid of Cytophaga so/ ACLJ-18, by acetone precipitation, DEAE-Cellulose, Sephadex G-100 and CM-Sephadex C25 column chromatographies. The molecular weight of purified agarase was estimated to be 24,700 dalton by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for agarase activity were 7.0 and 40℃, respectively. this agarase was stable in the pH range of 6.5 - 8.0 and 40℃, and required 0.35M NaCl for optimum activity. And this agarase was inhibited by metal ions such as Ba2+, Cu2+, Co2+, Mn2+, Hg2+, Zn2+, and showed specificity on agar.

The methylotrophic yeast, Pichia pastoris, is known to be a potential host to offer many advantages for production of recombinant proteins. Fermentation parameters were optimized to enhance the heterologous B-galactosidase productivity with P. pastoris. Optimum concentration of methanol, used as inducer, was observed to be 8 g/L and the extent of repression of AOX1 promoter by glycerol was lower than by glucose. The degradation of the gene product B-galactosidase by protease was inhibited as the pH increased from 5 to 8 and the yeast extract(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%) as nitrogen source increased expression level 4 times higher compared to yeast nitrogen base(1%). Induction method, in which methanol is just added to fermentation medium without centrifugation, was found to be as much effective as the one with centrifugation.

Erythritol is a four-carbon sugar alcohol with a low calorific value and non-cariogenicity. Erythritol is a new functional sweetener which can be used as sugar alternative. Erytheitol dose not cause discomfort such as diarrhoea and flatulence upon ingestion. The purpose of this study is to develope a novel process of erythritol economically in a large scale. To obtain a high erythritol producer, we have screened 3500 colonies from molasses, honey and honey combs. We have selected 40 erythritol-producing microorganisms, one of which yields 140g/L erythritol in 40% glucose medium. We have tested this strain in 5L fermentor to examine the fermentation characteristics. Results of fermentation show that the erythritol production was about 1.4g/Lhr in 400g/L glucose media with a 42% conversion. Further improvements require mutation for a higher producer, process optimization to reduce glycerol, and suppression of excessive foaming.