Decolorization and Degradation of Reactive Azo Dyes by Fixed Bed Bioreactors Containing Immobilized Cells of Proteus vulgaris NCIM- 2027

Rijuta G. Saratale,Ganesh D. Saratale,Jo Shu Chang,Sanjay P. Govindwar 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.4

Immobilized cells of Proteus vulgaris NCIM 2027 completely decolorized C.I. Reactive Blue 172 (50mg/L) within 8 h along with a nearly 80% reduction in TOC and COD. The dye degradation efficiency of the immobilized cells was further improved by optimizing the physicochemical conditions, including agitation, temperature,pH, dye concentration, and biomass loading. Microbial toxicity study revealed the non-toxic nature of the degraded products. Repeated-batch decolorization was conducted to evaluate the reusability of the immobilized cells. The immobilized cells were used for continuous dye decolorization in a fixed bed bioreactor under different volumetric flow rates and dye feeding concentrations. In addition, the immobilized cells were applied to decolorize a mixture of seven reactive dyes in batch and continuous modes, resulting in efficient decolorization (in terms of ADMI value) and significant reduction in TOC and COD. This suggests the potential of using immobilized cells to treat dye-containing wastewater.

Rhodopseudomonas sphaeroides의 고정화균체에 의한 수소생산의 효율적 기질공급

김진상,홍용기,신일식,조학래,장동석 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.1

Rhodopseudomonas sphaeroides B6 세포를 agar gel 중에 고정했을 때, 수소생산을 위한 최적 agar 농도는 2%(w/v)였다. B6의 2% agar gel 고정화균체(300㎖ gel: 2.85 ㎎ dry cells/㎖)와 비고정세포(1ℓ culture: 0.87㎎ dry cells/㎖)에 있어서 초기의 최고 수소 생산활성은 각각 47.5 및 48.O ㎖/hr/culture로 거의 같았다. 그러나, 수소생산이 거의 정지된 배양후기에 lactate의 제한공급(10mmole)에 의한 비고정세포의 활성회복은 50% 이하로 감소되었지만, 고정화세포의 활성은 거의 초기상태로 회복되었다. B6의 고정화균체를 이용하여 12시간 주기의 광조사 조건으로, 매 1ℓ의 수소생산시 마다 그에 소비된 만큼의 기질에 상당하는 9.3mmole의 DL-lactate와 1.86 mmole의 L-glutamate 함유 기본배지를 주기적으로 공급한 결과, 228시간의 배양기간 동안 명암의 반복 조건에도 불구하고 평균 510 ㎖/day/300㎖ ge1(2.9 ㎎ dry cells/㎖)의 속도로 수소생산이 지속되었다. 이 결과는 광합성세균에 의한 수소생산에 있어서, 보다 효율적 기질공급과 가변적인 태양광 이용 조건에 대응되는 자동배양 시스템의 구성 가능성을 시사한다. The Photosynthetic bacterium, Rhodopseudomonas sphaeroides strain B6 was immobilized on agar gel. The optimum concentration of agar for hydrogen production was 2% (w/v). Maximum rates of hydrogen production by immobilized (300㎖ of gel; 2.85㎎ dry cells/㎖) and free cells (1ℓ culture; 0.87㎎ dry cells/㎖) were 47.5 and 48.0 ㎖/hr/culture, respectively. However, when both cultures were fed by 10mmoles of lactate as limited electron donor at the later period of incubation, the activity of hydrogen production by free cells was significantly decreased but, immobilized cells continued hydrogen production with almost the same initial rate. We examined hydrogen production by immobilized cells of strain B6 under periodic illumination for 12 hr-intervals. When the culture was periodically fed by basal medium containing 9.3 mmoles of DL-lactate and 1.86 mmoles of L-glutamate as consumed electron donor and nitrogen source, respectively, for every one liter of hydrogen produced, hydrogen was evolved continuously with the average rate of 510 ㎖/day/300 ㎖ gel (2.9 ㎎ dry cells/㎖) during the incubation time for 228 hr.

Rhodocyclus gelatinosus KUP-74의 고정화균체로부터 ${\delta}-Aminolevulinate$의 연속생산

이소희,김현호,윤순규,임왕진,황세영,Lee, So-Hee,Kim, Hyun-Ho,Yun, Sun-Kyu,Lim, Wang-Jin,Hwang, Se-Young 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.1

Rhodocyclus gelatinosus KUP-74의 고농도 휴지균체로부터 ${\delta}-aminolevulinate$(ALA)를 연속적으로 생산하기 위한 최적조건을 검토하였다. 균체외 ALA양은 20 mg cells/ml의 균체농도까지는 농도 증가에 따른 rectangular hyperbola의 증가양식을 보였으나, 그 이상의 균체농도는 ALA 생산에 전혀 증가효과를 보이지 않았다. 20 mg cells/ml의 반응계에서, 균체외 ALA의 효과적 생산을 위한 첨가물질의 최저농도는 levulinate 4 mM과 L-glutamate 5 mM로 나타났으며, 배양 3시간후 ALA 양의 최대치를 보였다. 한편, Ca-alginate를 이용한 포괄법으로 고정화시킨 균체로부터 효과적으로 ALA를 생산하기 위한 첨가물질의 최적농도는 levulinate 6 mM과 L-glutamate 10 mM로 나타났으며, 배양 6시간째에 균체외 ALA 양의 최대치를 보였다. 또한, 이 조건 하에서 고정화균체와 고농도의 휴지균체에 의한 ALA의 연속생산성을 검토한 결과, 이들 균체에 의한 50% ALA 생산성은 각각 185시간과 100시간에서 나타났으며, 생산된 균체외 ALA의 회수를 위해서는 균체를 고정화시키는 방법이 효과적이었다. An optimal condition for the continuous production of ${\delta}-aminolevulinate$(ALA) was investigated using high concentrated resting cells of Rhodocyclus gelatinosus KUP-74. The increase of the amount of extracellular ALA versus the concentration of resting cells showed rectangular hyperbolic pattern until 20 mg cells/ml, but no further increase in the ALA amount by increasing its concentration was occurred. The highest yield of the extracellular ALA was observed after 3 hr of incubation of 1 ml reaction system containing 20 mg cells, 4 mM levulinate and 5 mM L-glutamate. On the other hand, the immobilized cells prepared by Ca-alginate inclusive method needed to incubate for 6 hr with 6 mM levulinate and 10 mM L-glutamate to give maximal yield of the extracellular ALA. In addition, under these conditions the resulted continuous productivities of the ALA by immobilized cells and highly concentrated resting cells were appeared 50 percent decreases after incubations for 185 hr and 100 hr, respectively, and the method of the cells to be immobilized was more efficient to recover the extracellular ALA produced.

고정상세포분리기의 개발 및 Cyclosporin A 생산을 위한 고정화 연속배양공정에의 적용

이태호,박성관,장용근,전계택 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

균사형성 미생물인 고정상곰팡이의 연속배양공정에 필수적인 효율적인 고정상세포분리기를 개발하고 이를 실제로 면역억제제인 cyclosporin A(CyA) 연속생산공정에 적용하였다. 점성이 큰 고분자인 carboxymethyl cellulose 용액을 이용한 실험에서 개발된 고정상 세포분리기는 분리능이 우수하여 고점도·고유속의 경우에도 고정성담체가 거의 유실되지 않았다. 이는 고정상세포분리기내에 설치된 공기유출관과 분리관의 역할에 기인하는 것으로 보인다. 또한 실제 곰팡이 세포를 이용한 고정상 연속배양(IPRS)의 경우 고정상세포분리기를 설치함으로써 담체밀도의 감소와 배양액내의 고농도(10g/l) 현탁 곰팡이 세포의 존재에도 불구하고 안정한 조업가능성을 보였다. 고정상 연속배양의 높은 희석속도에도 불구하고 균체 및 CyA의 생산성이 높게 나타났으며, 담체가 반응기 밖으로 거의 유출되지 않은 점으로 보아 고정상세포분리기의 우수한 성능 및 조업안정성을 확인할 수 있었다. We have developed an efficient immobilized cell separator for continuous operation of immobilized fungal cell cultures, and applied this separator to actual fermentation process for the production of cyclosporin A (CyA), a powerful immunosuppressant. In the experiments employing highly viscous polymer (carboxymethyl cellulose) solution, the decantor showed good separating performances at high solution viscosites and fast dilution rates. Air duct and cylindrical separator installed inside the decantor turned out to play key roles for the efficient separation of the immobilized cells. By installing the decantor in an immobilized perfusion reactor system (IPRS), continuous immobilized culture was stably carried out even at high dilution rate for a long period, leading to high productivities of free cells and CyA. Almost no immobilized biomass existed in effuluent stream of the IPRS, demonstrating the effectiveness of the decantor system for a long-term continuous fermentation. It was noteworthy that we could obtain these results despite of the unfavorable fermentation conditions, i.e., reduced density of the biosupports caused by overgrowth of cells inside the bead particles and existence of high density of suspended fungal ells (10 g/l) in the fermentation broth.

Kinetic study of acetaldehyde conversion to ethanol by free and CNT-immobilized baker’s yeast in a gas-phase packed bed reactor

Khadijeh Sherafatmand,Shohreh Fatemi,Zeinab Salehi 한국공업화학회 2015 Journal of Industrial and Engineering Chemistry Vol.30 No.-

Catalytic activity of the whole cell baker’s yeast (Saccharomyces cerevisiae) was studied for convertingacetaldehyde (AC) to ethanol (ET) in a gas-phase packed bed reactor. The baker’s yeast was considered atfree and immobilized conditions, to convert AC as an air pollutant to the less harmful product of ET. Theinfluence of operating conditions, such as inlet AC concentration, cells’ water content and temperature,was studied on the cells’ activity. The best conditions from the point of ET productivity and cell stabilitywas detected at temperature of 318 K and water content of 0.23 0.02 g water/g dry cell, using free cells. The cell immobilization was performed by doping on the surface of multiwalled carbon nanotube (CNT)under a mild centrifugal force. The activity of CNT-immobilized cells towards ET formation was improved by6% and the stability of the cells was improved by 43% respectively in comparison with the free cells’ activityand stability.

Specific capture, recovery and culture of cancer cells using oriented antibody-modified polystyrene chips coated with agarose film

Jeong, Jiyun,Lee, Yeolin,Yoo, Yeongeun,Lee, Myung Kyu Elsevier 2018 Colloids and surfaces. B, Biointerfaces Vol.162 No.-

<P><B>Abstract</B></P> <P>Agarose gel can be used for three dimensional (3D) cell culture because it prevents cell attachment. The dried agarose film coated on a culture plate also protected cell attachment and allowed 3D growth of cancer cells. We developed an efficient method for agarose film coating on an oxygen-plasma treated micropost polystyrene chip prepared by an injection molding process. The agarose film was modified to maleimide or Ni-NTA groups for covalent or cleavable attachment of photoactivatable Fc-specific antibody binding proteins (PFcBPs) via their N-terminal cysteine residues or 6xHis tag, respectively. The antibodies photocrosslinked onto the PFcBP-modified chips specifically captured the target cells without nonspecific binding, and the captured cells grew 3D modes on the chips. The captured cells on the cleavable antibody-modified chips were easily recovered by treatment of commercial trypsin-EDTA solution. Under fluidic conditions using an antibody-modified micropost chip, the cells were mainly captured on the micropost walls of the chip rather than on the bottom of it. The presented method will also be applicable for immobilization of oriented antibodies on various microfluidic chips with different structures.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We developed a method for agarose film coating on a 3D micropost chip. </LI> <LI> Covalent or cleavable antibody-modified chip was fabricated by film modification. </LI> <LI> Cells were specifically and efficiently captured on the antibody-modified chips. </LI> <LI> Cells captured on the cleavable chips were recovered by trypsin-EDTA treatment. </LI> <LI> Cells were predominantly captured on the micropost walls under fluidic conditions. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Photobacterium phosphoreum의 생체발광 유지도에 관한 연구

김현숙,정성제,전억한 한국산업미생물학회 2000 한국미생물·생명공학회지 Vol.28 No.2

P. phosphoreum의 생존과 생체발광도는 온도에 의해 많은 영향을 받는다. 냉동 저장한 세포의 경우 glycerol의 보호작용으로 세포농도와 생균수는 측정기간 동안 일정하게 유지된 반면 생체발광도는 glycerol 첨가 직후 급속히 감소하였으며, 저장 이후에도 감소된 생체발광도가 활성화되지 못하였다. 최적 생육온도인 20℃의 경우 저장 초기 세포가 성장함에 따라 세포수의 증가를 보였으나 일정 시간 이후 세포 분해 현상으로 인하여 생균수 및 세포 집락수의 감소를 나타내었으며, 생체발광도는 저장 3일 이후 소멸되었다. 이와는 대조적으로 4℃에 저장한 세포의 생체발광도는 저장 10일 동안 지속되어 가장 높은 생체발광 유지도를 나타내었으나 장기간 저온 저장으로 인하여 세포가 VBNC 상태에 돌입됨에 따라 총균수와 생균수는 일정한 반면 저장 10일 이후 세포 집락수의 급격한 감소를 나타내었으며, 저장 20일 이후 간균에서 구균으로 세포 형태상의 변화를 나타내었다. 이에 따라 세포 저장 시 접종원의 농도를 달리하여 VBNC 상태와 생체발광도의 관련성을 조사한 결과 VBNC 세포가 증가할수록 생체발광도의 감소를 나타내었다. 따라서 VBNC 세포를 감소시키기 위하여 세포를 고정화하여 저장한 결과 별도의 활성제 없이 실온에서 다시 활성화되어 고정화하지 않은 세포에 비해 2.3배 높은 생체발광 유지도를 나타내었으며, 저온저장에 따른 platebility 소실과 세포 응축현상이 나타나지 않았다. 이러한 결과는 세포의 고정화 방법을 이용하여 4℃에서도 세포의 생존 및 생체발광 유지도를 향상시킬 수 있으며, 동결 건조법의 단점을 보완해 줄 것으로 생각된다. The object of this work is to improve the maintenance of bioluminescence from stored Photobacterium phosphoreum in a view of developing continuous monitoring system for pollutants. The long-term experiments were performed to determine the effect of storage temperature and immobilization on the maintenance of bioluminescence and viability of P. phosphoreum. A naturally luminescent bacterium, P. phosphoreum was starved in 2.5% NaCl solution at 20℃, 4℃, -20℃ and -70℃ for 30 days. In vivo luminescence was measured by luminometry, and total cell concentrations and concentrations of culturable and viable cells were determined by acridine orange staining, dilution plate counting, and direct viable counting, respectively. The bioluminescence emission from cells stored at 4℃ was maintained up to 10 days while those with starved cells at other temperature ranges decreased to background level within 3 days. In terms of viability of cells, concentrations of cells stored at 20℃ were rapidly decreased as a result of cell lysis, leading to a drop in culturable and viable counts while cells stored at 4℃ was shown viable but nonculturable state during starvation. With immobilized cells on strontium alginate, the bioluminescence showed higher maintenance than free cells and decreased with count number of nonculturable cells.

Optimization of Citric Acid Production by Immobilized Cells of Novel Yeast Isolates

( Abd El-latif Hesham ),( Yasser S. Mostafa ),( Laila Essa Omar Alsharqi ) 한국균학회 2020 Mycobiology Vol.48 No.2

Citric acid is a commercially valuable organic acid widely used in food, pharmaceutical, and beverage industries. In this study, 260 yeast strains were isolated from soil, bread, juices, and fruits wastes and preliminarily screened using bromocresol green agar plates for their ability to produce organic acids. Overall, 251 yeast isolates showed positive results, with yellow halos surrounding the colonies. Citric acid production by 20 promising isolates was evaluated using both free and immobilized cell techniques. Results showed that citric acid production by immobilized cells (30-40 g/L) was greater than that of freely suspended cells (8-19 g/L). Of the 20 isolates, two (KKU-L42 and KKU-L53) were selected for further analysis based on their citric acid production levels. Immobilized KKU-L42 cells had a higher citric acid production rate (62.5%), while immobilized KKU-L53 cells showed an ~52.2% increase in citric acid production compared with free cells. The two isolates were accurately identified by amplification and sequence analysis of the 26S rRNA gene D1/D2 domain, with GenBank-based sequence comparison confirming that isolates KKU-L42 and KKU-L53 were Candida tropicalis and Pichia kluyveri, respectively. Several factors, including fermentation period, pH, temperature, and carbon and nitrogen source, were optimized for enhanced production of citric acid by both isolates. Maximum production was achieved at fermentation period of 5 days at pH 5.0 with glucose as a carbon source by both isolates. The optimum incubation temperature for citric acid production by C. tropicalis was 32 ℃, with NH₄Cl the best nitrogen source, while maximum citric acid by P. kluyveri was observed at 27 ℃ with (NH₄)₂ SO₄ as the nitrogen source. Citric acid production was maintained for about four repeated batches over a period of 20 days. Our results suggest that apple and banana wastes are potential sources of novel yeast strains; C. tropicalis and P. kluyveri which could be used for commercial citric acid production.

Repeated-batch Culture of Immobilized Gibberella fujikuroi B9 for Gibberellic Acid Production: An Optimization Study

Kim, Chang-Joon,Lee, Sang-Jong,Chang, Yong-Keun,Chun, Gie-Taek,Jeong, Yeon-Ho,Kim, Sung-Bae The Korean Society for Biotechnology and Bioengine 2006 Biotechnology and Bioprocess Engineering Vol.11 No.6

The performance of immobilized fungal cells on celite beads for the production of gibberrelic acid was investigated in flasks and 7-L stirred-tank reactor. Repeated incubations of immobilized fungal cells increased cell concentrations and volumetric productivity. The maximum volumetric productivity obtained in the immobilized-cell culture was 3-fold greater than that in suspended-cell culture. The concentration of cotton seed flour (CSF), among the various nutrients supplied, most significantly influenced productivity and operational stability. Notably, insoluble components in CSF were found to be essential for production. CSF at 6 g/L with 60 g/L glucose was found to be optimal for gibberellic acid production and stable operation by preventing excessive cell growth.

Immobilized Laminin-derived Peptide Can Enhance Expression of Stemness Markers in Mesenchymal Stem Cells

Nafiseh Tavakolpoor Saleh,Alireza Naderi Sohi,Elaheh Esmaeili,Somayeh Karami,Fatemeh Soleimanifar,Nikoo Nasoohi 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.6

Due to number of reasons such as ease of isolation and their broad differentiation capability, adult human mesenchymal stem cells (MSCs) are widely considered as of the most promising cells for regenerative medicine and tissue engineering. Nevertheless quick decrease in expression of transcription factors associated with stemness and self-renewal during ex vivo expansion of MSCs is an impediment against their therapeutic applications. Since the influence of extra cellular matrix (ECM) proteins on the fate of stem cells is well documented, the culture of MSCs on ECM-derived synthetic biomolecules is worth investigating. In the present study, a lamininderived peptide, YIGSR was covalently immobilized on the chitosan film surface using carbodiimide chemistry and confirmed by fluorometry. The results obtained from surface characterization by atomic force microscopy (AFM) and contact angle measurement, showed no significant difference in topological features and hydrophilicity after peptide immobilization. Employment of these surfaces for culture of human adipose-derived mesenchymal stem cells demonstrated that the immobilized YIGSR peptide has a favorable effect on adhesion and maintaining viability of the MSCs as well as on the expression of stemness markers (Nanog, Oct-4, and Sox-2) in these cells.