Name: a novel bioreactor for high density cultivation of diverse microbial communities
Uploaded: 2015-12-25T13:00:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities at JoVE.com

The authors would like to acknowledge Aspen Walker at the University of Pennsylvania for her assistance in reactor maintenance and sample collection.

1. 원자로 조립 <ol><li> 도 1의 개략도에 따른 반응기 구성 요소를 정렬. <ol><li> , 혼합 접시에 반응기 (R)을 배치 반응기에 교반 막대를 추가합니다. 탱크의 유출 (위) 포트가 실험실 벤치의 가장자리쪽으로 향하도록 교반 판과 원자로 옆에있는 재활용 탱크 (RT)을 놓습니다. </li><li> 재활용 탱크 (RT)의 유출 (위) 포트 아래에있는 폐기물 용기 (W)를 놓습니다. 재활용 탱크 (RT) 옆에 공급 탱크 (FT)를 놓습니다. 주 : 저류조 5 L.의 총 용량을 갖는 </li></ol></li><li> 적절한 크기의 스탠드와 클램프와 팁에 대한 반응 (R)을 고정합니다. 마찬가지로, 이동을 방지하기 위해 재순환 탱크 (RT)를 고정. </li><li> 재활용 (펌프)에 네오프렌 연동 펌프 튜브를 삽입하고 (펌프 B) 펌프 헤드를 공급. 추가 배관 사양 재료 표를 참조하십시오. 나사 있습니다 provi와 펌프 드라이브에 펌프 헤드를 설치펌프 드라이브 공격 한. </li><li> 원자로 및 재활용 탱크의 포트에 펌프의 튜브를 연결합니다. 공급 탱크 및 재활용 탱크에 펌프 B의 튜브의 끝을 삽입합니다. 튜브와 재활용 탱크에 상부 원자로 포트를 연결합니다. 원자로 포트에서 튜브에 클램프를 적용합니다. 참고 : 광합성 지역 사회가 램프에 의해 제공되는 인공 조명 혜택을 누릴 수 있습니다. </li></ol> 주식 솔루션, 유입수 / 피드 솔루션 및 조류 접종 2. 준비 <ol><li> 미네랄 스톡 용액을 제조 하였다. 200g의 탄산 수​​소 나트륨, 40g 염기성 인산 칼륨, 4g 마그네슘, 황산 4g 염화철, 4g 염화칼슘, 1g의 염화 구리, 1g 코발트 : 탈 이온수 500 mL를 1 L 부피 플라스크에 다음을 추가 클로라이드 수화물, 1g, 염화 니켈 육수화물, 1g 황산 아연 수화물. 탈 이온수 추가 400 ML을 추가합니다. 소금의 용해를 촉진하기 위해 강제로 소용돌이. 다음 DISS염의 olution, 1 L.를 용액의 총 부피를 가지고 탈 이온수를 추가 </li><li> 암모니아 스톡 용액을 제조 하였다. 1 L 용량 플라스크에서, 탈 이온수 900 ml의 염화 암모늄 38.214 g을 용해. 용해 후, 1000 ml의 총 볼륨을 가져 탈 이온수를 추가합니다. 주 : 1 L로 희석 스톡 용액 1 ㎖를 10 mg을 L을 산출 -1 NH 4 + -N 용액. </li><li> 질산 스톡 용액을 제조 하였다. 1 L 용량 플라스크에서, 탈 이온수 900 ㎖ 중의 질산 칼륨 72.413 g을 용해. 용해 후, 1000 ml의 총 볼륨을 가져 탈 이온수를 추가합니다. 참고 : - -N 솔루션 1 L로 희석 원액 1 ml를 -1에서 3 10 밀리그램 (L)를 산출한다. </li><li> 공급 / 유입수 솔루션을 준비합니다. 2 ㎖를 희석, -N - 20 mg을 L를 포함하는 공급 용액 -1 NH 4 + -N 및 20 mg을 L -1 (3)을 만들려면mmonia 스톡 용액을 1 L의 총 용적에 질산 스톡 용액 2 ㎖. 전 희석, 0.5 ml의 미네랄 솔루션 / 솔루션의 L 만들어지고를 추가합니다. 원자로를 시작하는 데 총 유입수의 5 L를 준비합니다. </li><li> 조류 접종을 준비합니다. <ol><li> 이러한 스트림이나 연못 같이 조류 함유 수역에서 많은 양의 물 (적어도 10 L)를 수집한다. 조류는 24 시간 동안 방해받지 물 샘플을 남겨 정착하도록 허용합니다. </li><li> 샘플 병 내에 농축 조류 현탁액을 떠나는 따르다 및 샘플의 맨 클리어 (비 함유 조류) 물을 버린다. 하나의 컨테이너에 모든 샘플에서 조류 서스펜션을 결합하고 정착과 경사 단계를 반복합니다. </li><li> 농축 된 시료 내에서 바이오 매스를 측정한다. <ol><li> 공동을 측정 실온에서 30 분 동안 데시 케이 터에서 냉각 한 후 103 ℃로 설정 한 오븐에서 보트 O / N 무게 종이 진공 필터 (0.45 μm의 MCE 진공 필터) 및 알루미늄을 건조필터의 질량을 mbined와 보트의 무게. </li><li> 진공 농축 조류 현탁액 20 ㎖ 필터와 필터를 반환 / N을 O를 건조 오븐 보트의 무게. </li><li> 필터의 결합 된 질량을 측정하고, 보트를 단다. 농축 된 시료 내에서 바이오 매스 밀도를 계산합니다. 참고 : 수사 소스 수역에 의존 할 것이다 수집해야 할 물 시료의 전량. </li></ol></li></ol></li></ol> 3. 시드 반응기 시작 <ol><li> 반응기에 공급 용액의 750 ML을 추가합니다. 공급 용액 500 mL를 재활용 탱크를 채우십시오. </li><li> 부드럽게 반응기의 하단 조류 1.5 g을 함유하는 접종 현탁액을 추가 긴 피펫을 사용한다. 접종원은 다음 단계로 진행하기 전에, 반응기의 바닥으로 침전 육안으로이를 보장 할 수있다. </li><li> 세포가 정착되면, (10 revolutio을 튜브 클램프를 제거하고 느린 유속으로 펌프를 켜십시오NS 최소 -1 / 38 ml의 분 -1). 튜브에 갇힌 공기는 반응기로 추방 될 것입니다. 주 : 750 ml의 반응기에의 첨가는 반응기로부터 방출로부터 펌프에 의해 방해되지 않도록 어떤 매스 것이다. 모든 공기가 배출되었는지 확인하기 위해 튜브를 짠다. </li><li> 용액을 반응기로 펌핑으로 점차적으로 재활용 탱크에 공급 용액을 추가합니다. 원자로 및 재활용 탱크 모두 용량과 상단 포트를 통해 재활용 탱크를 종료 유출 시작에 때까지 추가를 계속합니다. 주 : 공급 용액의 부피는 반응기에 첨가 접종의 부피에 따라 달라질 재순환 탱크에 첨가한다. </li><li> 공급 탱크에 남아있는 공급 용액을 붓는다. </li><li> 72.5 ml의 분의 재순환 유량을 설정, -1 인 min 19 회전에 재순환 펌프 (펌프)를 설정 -1. 조류는 반응기의 바닥에서 시작 로프트 관찰한다. 원자로에 그라데이션을 사용하면, 조류 BI를 결정omass 영역의 높이입니다. 높이가 다음 단계로 진행하기 전에 일정 있는지 확인합니다. </li><li> 매우 낮은 속도로 혼합 판을 켭니다; 1 또는 2의 설정을 시작하는 것이 적절하다. 혼합 바는 더 바이오 매스 로프트에 도움이 될 것입니다,하지만 공격적인 혼합, 반응을 남겨 재활용 탱크를 입력하고 유출에두고 조류가 발생합니다. 반응기 (도 2A) 내에 맑은 조류 경계를 설정하기 위해 필요한 설정에 속도 설정을 혼합; 조류 바이오 매스 구역은 높이 약 10~15cm해야한다. </li><li> 조류 플러그와 원자로 유체 사이에 명확한 경계를 관찰 한 후 공급 펌프를 시작합니다. 1.5 ml의 분의 유속을 확립, -1 인 min으로 25 회전 펌프를 설정 -1. 때문에 들어오는 유입 스트림에 의해 발생하는 중력과 변위 반응기 유체 출구에게 유출 포트를 관찰한다. </li></ol> 4. 샘플 수집 및 분석 <ol><li> 시료 채취 활동 PRI을 수행또는 반응기 시스템 유지 보수를 수행한다. 매일 유출과 유입 시료 20 ㎖를 수집합니다. 재활용 탱크 내에서 폐수 샘플을 수집합니다. 공급 탱크로부터 직접 유입수 샘플을 수집한다. </li><li> 진공 필터 샘플을 저장 및 분석에 앞서 부유 물질 제거합니다. </li><li> 추가 분석 할 때까지 -20 ° C에서 유입 및 유출 물 샘플을 저장합니다. 샘플을 실시 동결 해동 사이클의 수를 제한한다. 필요한 경우, 샘플은 샘플의 무결성을 유지하기 위해 분취 량으로 분할 될 수있다. </li><li> 표준 기술 (17)를 사용하여 질산염, 아질산염, 암모니아에 대한 시료 분석을 수행하십시오. 주 : 이온 크로마토 그래피 (IC) 제작자들이 이용 본원에 제시된 결과를 생성한다. 사양에 대한 자료 표를 참조하십시오. </li></ol>

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M., Eveillard, D., Tonon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+metabolic+approach+to+study+algal-bacterial+interactions+in+changing+environments.">A metabolic approach to study algal-bacterial interactions in changing environments.</a> Mol Ecol. 23 (7), 1656-1660 (2014).</li><li>Watanabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiotic+association+in+Chlorella+culture.">Symbiotic association in Chlorella culture.</a> FEMS Microbiol Ecol. 51 (2), 187-196 (2005).</li><li>Burke, C., Thomas, T., Lewis, M., Steinberg, P., Kjelleberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition,+uniqueness+and+variability+of+the+epiphytic+bacterial+community+of+the+green+alga+Ulva+australis.">Composition, uniqueness and variability of the epiphytic bacterial community of the green alga Ulva australis.</a> ISME J. 5 (4), 590-600 (2011).</li><li>Krohn-Molt, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenome+survey+of+a+multispecies+and+alga-associated+biofilm+revealed+key+elements+of+bacterial-algal+interactions+in+photobioreactors.">Metagenome survey of a multispecies and alga-associated biofilm revealed key elements of bacterial-algal interactions in photobioreactors.</a> Appl Environ Microbiol. 79 (20), 6196-6206 (2013).</li><li>Cooper, E. D., Bentlage, B., Gibbons, T. R., Bachvaroff, T. R., Delwiche, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metatranscriptome+profiling+of+a+harmful+algal+bloom.">Metatranscriptome profiling of a harmful algal bloom.</a> Harmful Algae. 37, 75-83 (2014).</li></ol>

The authors have nothing to disclose and declare that they have no competing financial interests.

이 섹션에서는 가능한 운영 문제뿐만 아니라 다른 미생물 군집을 사용하여 해결하는 데 필요한 프로토콜의 변화에​​ 대한 논의와 함께 시작됩니다. 이 반응기 설계의 장점은 산소 유량의 제어 및 반응기 내의 고밀도 플록의 형성을 제어하는​​ 기능을 포함하여, 설명한다. 현재 도전과 조사 가능한 도로도 언급됩니다. 프로토콜 뉘앙스의 변화 문화의 다른 유...

하수는 일반적 부유 고형물 (SS), 생물학적 산소 요구량 (BOD), 유기 및 무기 질소 및 인의 함유량 5,6-을 줄이기 위해 활성 슬러지 공정으로 처리한다. 활성 오니 법, 이차 폐수 처리의 수단은, 수신 된 폐수와 미생물 재순환 영양 혼합액 가득 폭기조 내의 유기 탄소의 산화를 수반 5-7 (통상적으로 활성 슬러지로 언급). 혼합액은 슬러지가 쉽게 수집 침전 비교적 큰 정화기 (침전조)에 입사 배치 또는 정화, 폐수 처리가 급 치료 또는 소독을 계속할 수있는 반면에 방출되기 전에, 다시 폭기조로 재순환 될 어느 수신 물 5-7. 차 침전지에서 처리 된 폐수와 고형물 (슬러지)의 효율적인 분리했다의 적절한 기능을 위해 필수적이다tewater 처리 시스템, 어떤 활성 슬러지가 유출 5-8에서 BOD와 SS를 증가 정화기를 넘어 계속있다. 다른 생물학적 과정의 수를 감소 시키거나 부착 성장 (생물막) 반응기의 멤브레인 생물 반응기 (MBR에) 및 입상 슬러지 반응기 명확히 포함 큰 탱크의 필요성을 제거하는 폐수 처리의 보조를 위해 존재한다. 생물막 반응기, 천연 골재 및 미생물 고체 표면 상에 층으로서 부착 된 바이오 필름의 형성에서, 명확히 탱크를 필요로하지 않고 바이오 매스 보유 및 축적을 허용한다. 생물막 반응기는 세 가지 유형으로 분류 될 수있다 : 충전 층 반응기, 유동층 반응기, 및 생물학적 접촉자를 회전. 이러한 살수 필터 및 생물학적 타워로 충전 층 반응기는, 고정 견고한 성장 표면 5,6를 사용한다. 유동층 반응기 (FBRs)는 입자 미생물의 부착에 따라 달라집니다모래와 같은 입상 탄소 (GAC), 또는 높은 상향 유량 9,10 의해 현탁액에 유지되는 유리 비드를 활성화. 회전 생물 반응기는 생물막 있도록 회전축에 부착 된 용지 상에 형성된 바이오 필름을 교대로 공기에 노출되도록 상기 액체 5,6 치료할에 의존한다. MBR에는 생물 반응기 (잠수 구성) 내부 또는 외부 재순환 (측면 스트림 구성) 5,11를 통해 중, 막 여과 장치를 사용합니다. 멤브레인 (11, 12)에서 처리 된 액체 매스와 고체 입자의 양호한 분리를 달성하는 역할을한다. 입상 슬러지 반응기는 미생물이 매우 치밀하고 잘 정착 과립의 형성은 이들이 높은 표면 상향 공기에 노출 될 때 발생한다 (13)를 상향 유동 반응기의 속도이다. 활성 오니 법, 신규 상향 유동 반응기 시스템에 대한 또 다른 대안으로서, 현재 고밀도 생물 반응기 (HDBR)라고 고안했다D와 가난한 침전 슬러지의 형성을 일으키는 것으로 알려져 있습니다 낮은 F / M 조건에서 합성 폐기물 (즉, 벌킹 슬러지) 1,7,14에서 활성 슬러지에 의한 COD 제거를 공부 판매 (2006) Shieh에 의해 만들어진. 이용 HDBR 시스템은 일반적으로 상향 유동 반응기 외부의 재순환 탱크로 구성 유동층 반응기를 변형. 생물막 덮인 기판이 유지되도록 유동층 반응기는 일반적으로 충분한 정지 생물막 성장 하층을 유지하기에 충분히 높지만 낮은 재순환 스트림 흐름 속도로 동작한다. 유동층 반응기와 달리 HDBR 매출액 및 설명 (2006)은 외부 Shieh 통기와 함께, 반응기 (1)의 내부에 형성된 매스 존의 중단을 방지, 비교적 낮은 재순환 스트림의 유속을 사용 하였다. 후속 연구는 성공적 박테리아 3,4- 탈질 / 질산화 된 질소를 이용하여 플럭스의 범위를 치료하는 반응기 설계의 능력을 증명 하였다. 모든 스터드에서HDBR 내의 안정한 고밀도 매스 존의 형성은 외부 응집 / 침전 공정 1-4에 대한 필요성을 제거 이거 야. 우리가 여기 보고서로, 고밀도 문화를 성장 HDBR의​​ 사용은 또한 조류의 배양을위한 광 생물 반응기 (PBR) 구성에서 테스트되었습니다. 우리는 혜택과 조류 재배에이 소설 반응기 시스템의 단점과 바이오 매스 수확 (즉, 좋은 고체 - 액체 분리 15, 16)과 관련된 조류 바이오 연료의 상용화에 큰 장애물을 극복하기위한 가능성을 논의한다. 다음 프로토콜은 시작부터 샘플, 조립, 관심있는 미생물 군집으로 조류와 HDBR을 유지하는 데 필요한 단계를 설명합니다. 종속 영양 및 질산화 / 탈질 문화의 시작 및 동작 프로토콜의 변화도 언급 될 것입니다. 마지막으로, 일반적인 장점, 단점,이 새로운 원자로 설계의 미지수가 강조 표시됩니다.

<table><tbody><tr><td>Aeration stone</td><td>Alita</td><td>AS-3015C</td><td></td></tr><tr><td>Aerator</td><td>Top Fin</td><td>Air-1000</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Anion analysis column</td><td>Shodex</td><td>IC SI-52 4E</td><td></td></tr><tr><td>Beaker (600 ml)</td><td>Corning Pyrex</td><td>1000-600</td><td>Used as mixing vessel (MV). Addition of hose barbs at the bottom and 500 ml levels. Outside diameter of hose barbs 3/8&quot;.</td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>C5670</td><td></td></tr><tr><td>Cation analysis column</td><td>Shodex</td><td>IC YS-50</td><td></td></tr><tr><td>Cobalt chloride hexahydrate</td><td>Sigma Aldrich</td><td>C8661</td><td></td></tr><tr><td>Copper chloride</td><td>Sigma Aldrich</td><td>222011</td><td></td></tr><tr><td>Ferric chloride</td><td>Sigma Aldrich</td><td>157740</td><td></td></tr><tr><td>Filter (vacuum)</td><td>Fisherbrand</td><td>09-719-2E</td><td>0.45 &amp;#956;m membrane filter, MCE, 47&amp;nbsp;mm diameter</td></tr><tr><td>Graduated cylinder (1,000 ml)</td><td>Corning Pyrex</td><td>3025-1L</td><td>Used as reactor vessel (R). Addition of hose barbs at bottom, 500 ml, and 1 L levels. Outside diameter of hose barbs 3/8&quot;.</td></tr><tr><td>HPLC/IC</td><td>Shimadzu</td><td>Prominence</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Masterflex L/S variable speed drive</td><td>Masterflex</td><td>07553-50</td><td>Drive for recycle and feed pumps (2 needed)</td></tr><tr><td>Nickel chloride hexahydrate</td><td>Sigma Aldrich</td><td>N6136</td><td></td></tr><tr><td>Potassium nitrate</td><td>Sigma Aldrich</td><td>P8291</td><td></td></tr><tr><td>(Monobasic) Potassium phosphate</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Pump head</td><td>Masterflex</td><td>07018-20</td><td>Recycle pump head</td></tr><tr><td>Pump head</td><td>Masterflex</td><td>07013-20</td><td>Feed pump head</td></tr><tr><td>Pump tubing</td><td>Masterflex</td><td>6404-18</td><td>Recycle pump tubing</td></tr><tr><td>Pump tubing</td><td>Masterflex</td><td>6404-13</td><td>Feed pump tubing</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Sigma Aldrich</td><td>Z0251</td><td></td></tr></tbody></table>

a novel bioreactor for high density cultivation of diverse microbial communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

40 mg의 -nl -1 피드 총 질소 함량을 유지하면서 HDBR는 유입 암모니아와 질산의 농도의 비율을 통해 여러 조류를 배양하기 위해 사용되었다. 유입 및 유출 물 샘플을 매일 찍은; 생물량 밀도 샘플은 각 조건의 시작과 끝에서 촬영 하였다. 반응기 조건을 변경 한 후 정상 상태 평형에 도달하기 위해 평균 3~5일 취했다. 유입수 조건의 넓은 범위에 걸쳐 뚜렷한 매스 영역은 이전 연구를 (도 2...

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A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

다양한 미생물 커뮤니티의 고밀도 배양위한 새로운 생물 반응기

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

a-novel-bioreactor-for-high-density-cultivation-diverse-microbial

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

17.0K 조회수.  Drexel University. A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

비디오: A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

연구

생체공학

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

벤치 탑 생물 반응기의 운전

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

부식성 연도 가스가 있는 벤치 스케일 광생물반응기에서 미세조류 재배 및 바이오매스 정량화

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...

환경과학

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

단일 사용 공압 생물 반응기 시스템을 사용하여 포유 동물 세포의 배양

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

환경 미생물 배양을 위한 현장 시뮬레이션 혐기성 방법

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

저자 스포트라이트: In Situ Culturing을 통해 미지의 환경에서 육상 혐기성 미생물의 미스터리 풀기

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

환경적으로 획득한 혐기성 미생물을 배양하기 위한 In Situ Informed Simulated Medium 형식 세트

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

자동화 된 Microbioreactor 기술을 사용 하 여 분리 단백질 생산의 최적화를 위한 일반 프로토콜

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

높은 처리량의 사용 모델 IgG1 조 세포에서의 생산을 위한 Microbioreactor 시스템 자동화

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

중국 햄스터 난소 세포 재배에서 고처리량 자동화 마이크로 바이오리액터를 사용한 공정 최적화

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

다양한 미생물 커뮤니티의 고밀도 배양위한 새로운 생물 반응기

요약

더 많은 비디오 탐색

이 비디오의 챕터

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

벤치 탑 생물 반응기의 운전

부식성 연도 가스가 있는 벤치 스케일 광생물반응기에서 미세조류 재배 및 바이오매스 정량화

단일 사용 공압 생물 반응기 시스템을 사용하여 포유 동물 세포의 배양

환경적으로 획득한 혐기성 미생물을 배양하기 위한 In Situ Informed Simulated Medium 형식 세트

환경적으로 획득한 혐기성 미생물을 배양하기 위한 In Situ Informed Simulated Medium 형식 세트

환경적으로 획득한 혐기성 미생물을 배양하기 위한 In Situ Informed Simulated Medium 형식 세트

자동화 된 Microbioreactor 기술을 사용 하 여 분리 단백질 생산의 최적화를 위한 일반 프로토콜

높은 처리량의 사용 모델 IgG1 조 세포에서의 생산을 위한 Microbioreactor 시스템 자동화

중국 햄스터 난소 세포 재배에서 고처리량 자동화 마이크로 바이오리액터를 사용한 공정 최적화