Long-term Viability of Photosynthetic Cells Stacked in a Hydrogel Film within a Polydimethylsiloxane Microfluidic Device

유선각,배성재 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.4

Immobilizing cells while maintaining their longterm viability is important to utilize cells in biosensors and energy devices. In this study, we fabricated a hydrogel film of 10 μm thickness immobilizing photosynthetic cells, using a polydimethylsiloxane microfluidic device, and we monitored the viability of the cells for 30 days. Cell viability was measured by chronoamperometry using two electrodes located in the microfluidic device and was compared between hydrogel-immobilized and non-immobilized cells. The non-immobilized cells showed variation in viability. In contrast, the hydrogel-immobilized cells remained viable for 30 days. A simulation of the oxygen distribution changes by photosynthesis of the cells and mass transfer of cell culture nutrients (NaNO3) suggested that a proper environment for cell survival was effectively established inside the hydrogel. We successfully fabricated a photosynthetic cell-laden hydrogel with potential use in next-generation photosynthesisbased solar cells and sensors.

형질전환된 식물세포에서 고정화 방법을 통한 hCM-CSF의 생산성 증대 연구

노윤숙(Yun-Sook Noh),남형진(Hyung-Jin Nam),최홍열(Hong-Yeol Choi),탁사라(Sa-Ra Tak),김동일(Dong-Il Kim) 한국생물공학회 2015 KSBB Journal Vol.30 No.2

Plant cell immobilization can protect plant cells from shear forces and increase the stability of gene. An additional advantage of immobilization is the easiness for performing continuous culture with cell recycling. Therefore plant cell immobilization can overcome the limitations of plant cell applications. In addition, target protein should be selected from pharmaceutical proteins to get rid of low expression level problem. The enhanced production of human granulocyte-macrophage colony-stimulating factor (hGMCSF) was investigated in immobilized Nicotiana tabacum suspension cell cultures. When the cells were immobilized in polyurethane foam, specific production of hGM-CSF was higher than that in alginate bead immobilization. Optimum continuous culture condition was the addition of 60 g/L sucrose in growth media with exchanging media every 6 day. Under the same condition, specific hGM-CSF production was 7 times higher in a 500-mL spinner flask than that in 100-mL Erlenmeyer flasks. Therefore, development of an effective immobilization process would be possible when the advantage of easy cell recycling was used. Consequently, enhanced production of target proteins could be possible in immobilized continuous cultures when the advantages of immobilization were applied.

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>

Enhanced ε-Poly-L-lysine Production from Streptomyces ahygroscopicus by a Combination of Cell Immobilization and In Situ Adsorption

( Liu Sheng Rong ),( Qing Ping Wu ),( Ju Mei Zhang ),( Shu Ping Mo ),( Xiao Juan Yang ),( Chun Xiao ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.9

ε-Poly-L-lysine (ε-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of Llysine, which is used as a safe food preservative. The present study investigates the combined use of cell immobilization and in situ adsorption (ISA) to produce ε-PL in shaken flasks. Loofah sponge-immobilized Streptomyces ahygroscopicus GIM8 produced slightly more ε-PL than those immobilized on synthetic sponge, and sugarcane bagasse. Moreover, loofah sponge supported the maximum biomass. Hence, loofah sponge was chosen for cell immobilization. Meanwhile, the ion-exchange resin D152 was employed for ISA. The loofah sponge-immobilized cells produced 0.54 ± 0.1 g/l ε-PL, which significantly increased to 3.64 ± 0.32 g/l after combining with ISA through the addition of resin bags. The free cells with ISA using the dispersed resin yielded 2.73 ± 0.26 g/l of ε-PL, an increase from 0.82 ± 0.08 g/l. These data illustrate that the proposed combination method improved production most significantly compared with either immobilization or ISA only. Moreover, the immobilized cells could be repeatedly used and an ε-PL total amount of 8.05 ± 0.84 g/l was obtained. The proposed combination method offers promising perspectives for ε-PL production.

Preparation of Corncob Grits as a Carrier for Immobilizing Yeast Cells for Ethanol Production

( Sang Eun Lee ),( Choon Geun Lee ),( Do Hyung Kang ),( Hyeon Yong Lee ),( Kyung Hwan Jung ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.12

In this study, DEAE-corncobs [delignified corncob grits derivatized with 2-(diethylamino)ethyl chloride hydrochloride (DEAE·HCl)] were prepared as a carrier to immobilize yeast (Saccharomyces cerevisiae) for ethanol production. The immobilized yeast cell reactor produced ethanol under optimized DEAE·HCl derivatization and adsorption conditions between yeast cells and the DEAE-corncobs. When delignified corncob grit (3.0 g) was derivatized with 0.5M DEAE·HCl, the yeast cell suspension (OD600 = 3.0) was adsorbed at >90% of the initial cell OD600. This amount of adsorbed yeast cells was estimated to be 5.36 mg-dry cells/g-DEAE corncobs. The Qmax (the maximum cell adsorption by the carrier) of the DEAE-corncobs was estimated to be 25.1 (mg/g), based on a Languir model biosorption isotherm experiment. When we conducted a batch culture with medium recycling using the immobilized yeast cells, the yeast cells on DEAE-corncobs produced ethanol gradually, according to glucose consumption, without cells detaching from the DEAE-corncobs. We observed under electron microscopy that the yeast cells grew on the surface and in the holes of the DEAEcorncobs. In a future study, DEAE-corncobs and the immobilized yeast cell reactor system will contribute to bioethanol production from biomass hydrolysates.

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.

Immobilization of the Hyperthermophilic Archaeon Thermococcus onnurineus Using Amine-coated Silica Material for H₂ Production

배승섭,나정걸,이성목,강성균,이현숙,이정현,김태완,Bae, Seung Seob,Na, Jeong Geol,Lee, Sung-Mok,Kang, Sung Gyun,Lee, Hyun Sook,Lee, Jung-Hyun,Kim, Tae Wan The Korean Society for Microbiology and Biotechnol 2015 한국미생물·생명공학회지 Vol.43 No.3

초고온성 고세균 Thermococcus onnurineus NA1은 개미산, 일산화탄소, 또는 전분 등을 이용해서 수소를 생산하는 것으로 알려져 있다. 본 연구에서는 T. onnurineus NA1의 고정화 세포를 이용한 수소생산을 고찰하였다. 고정화 실험결과, T. onnurineus NA1은 표면에 아민기가 코팅된 규조토 담체에 정전기적 인력에 의해 효과적으로 고정화되었고, 1 g의 담체에 고정화 될 수 있는 최대 세포의 양은 71.7 mg-dcw로 확인되었다. 고정화 세포를 이용한 세 번의 반복회분식 배양을 통해 개미산으로부터 수소생산 특성을 고찰하였고, 그 결과 배양이 반복됨에 따라 고정화 세포 농도의 증가에 기인하여 초기수소생산속도가 2.3 에서 4.0 mmol l⁻¹ h⁻¹로 상당량 증가됨이 관찰되었다. 따라서, T. onnurineus NA1의 고정화세포 시스템은 수소생산을 위한 좋은 대안이 될 수 있을 것으로 사료된다. 본 연구는 초고온성 고세균의 고정화세포를 수소생산에 적용한 첫 번째 사례이다. Previously we reported that the hyperthermophilic archaeon, Thermococcus onnurineus NA1 is capable of producing hydrogen (H₂) from formate, CO or starch. In this study, we describe the immobilization of T. onnurineus NA1 as an alternative means of H₂ production. Amine-coated silica particles were effective in immobilizing T. onnurineus NA1 by electrostatic interaction, showing a maximum cell adsorption capacity of 71.7 mg-dried cells per g of particle. In three cycles of repeated-batch cultivation using sodium formate as the sole energy source, immobilized cells showed reproducible H₂ production with a considerable increase in the initial production rate from 2.3 to 4.0 mmol l⁻¹ h⁻¹, mainly due to the increase in the immobilized cell concentration as the batch culture was repeated. Thus, the immobilized-cell system of T. onnurineus NA1 was demonstrated to be feasible for H₂ production. This study is the first example of immobilized cells of hyperthermophilic archaea being used for the production of H₂.

포괄담체에 의한 Zymomonas mobilis 균체의 고정화

한면수,정동효 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.4

세균을 고정화시켜 에탄올을 생산하기 위하여 4종류의 Zymomonas mobilis를 사용하였다. 이들 중 glucose와 sucrose로부터의 에탄올생성능이 가장 우수한 것으로 Zymomonas mobilis KCTC 1534를 선별하였다. 선별된 균주의 고정화 특성을 살펴본 결과 발효 최적온도와 pH는 각각 33℃ 와 5.0으로 고정화 균체나 유리 균체에 대하여 큰 차이가 없었다. 그러나 최대 발효 가능온도는 유리 균체가 37℃인데 비하여 alginate, κ-carageenan, agar에 고정화했을 때 각각 43, 37, 40℃로서 균체를 고정화할 경우 유리균체에서 보다 높은 온도에서도 발효가 가능함을 확인하였다. Alginate 고정화 균체는 2% alginate 농도에 건조균체량 11.74 g/ℓ가 최적이었다. 고정화 균체의 활성화는 10% glucose 농도의 생산배지에서 20∼25시간을 요하였고. 4℃에 보관할 경우 calcium chloride 2%를 가한 증균배지에서 약 4주간은 전균체량의 90% 이상이, 10주간에는 80% 이상의 균체가 생존하였다. The immobilization characteristics of Zymomonas mobilis for ethanol production were examined. Four different strains of Zymomonas mobilis have been used for ethanol production. Among those, Zymomonas mobilis KCTC 1534 has been selected as the best strain for the highest ethanol productivity from glucose and sucrose. The optimum temperature and pH of the selected strain for ethanol production were 37℃ and 5.0 respectively for both free and immobilized cells. When the cells were immobilized by the gel entrapment method, the immobilized cells could produce ethanol at a little higher temperature than free cells. Calcium alginate was selected as the best gel for immobilizing cells. The immobilized cells could maintain the viability of 80% in 10 weeks storage at 4℃ in the medium with 2% calcium chloride. 20∼25 hours of preincubation in 10% glucose solution was required for the activation of immobilized cells entrapped within calcium alginate gel.

Immobilization of the Hyperthermophilic Archaeon Thermococcus onnurineus Using Amine-coated Silica Material for H2 Production

( Seungseob Bae ),( Jeonggeol Na ),( Sungmok Lee ),( Sunggyun Kang ),( Hyunsook Lee ),( Junghyun Lee ),( Taewan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2015 한국미생물·생명공학회지 Vol.43 No.3

Previously we reported that the hyperthermophilic archaeon, Thermococcus onnurineus NA1 is capable of producing hydrogen (H2) from formate, CO or starch. In this study, we describe the immobilization of T. onnurineus NA1 as an alternative means of H2 production. Amine-coated silica particles were effective in immobilizing T. onnurineus NA1 by electrostatic interaction, showing a maximum cell adsorption capacity of 71.7 mg-dried cells per g of particle. In three cycles of repeated-batch cultivation using sodium formate as the sole energy source, immobilized cells showed reproducible H2 production with a considerable increase in the initial production rate from 2.3 to 4.0 mmol l-1 h-1, mainly due to the increase in the immobilized cell concentration as the batch culture was repeated. Thus, the immobilized-cell system of T. onnurineus NA1 was demonstrated to be feasible for H2 production. This study is the first example of immobilized cells of hyperthermophilic archaea being used for the production of H2.

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.