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.原子炉アセンブリ<ol><li> 図1の概略図によれば、原子炉コンポーネントを配置します。 <ol><li>ミキシングプレート上リアクター（R）を配置し、反応器に攪拌棒を追加します。タンクの廃液（上）ポートは実験台の端に向けられているように、撹拌プレート、反応器の横にリサイクルタンク（RT）を配置します。 </li><li>リサイクルタンク（RT）の流出流（上）ポートの下に廃棄物容器（W）を配置します。リサイクルタンク（RT）の隣に供給タンク（FT）を配置します。 注：供給タンクは、5 Lの総容量があります</li></ol></li><li>適切なサイズのスタンドとクランプで転倒に対してリアクター（R）を固定します。同様に、移動を防止するために、リサイクルタンク（RT）で固定します。 </li><li>リサイクル（ポンプA）にネオプレン蠕動ポンプチューブを挿入し、（ポンプB）ポンプヘッドを養います。追加のチューブ仕様のための材料の表を参照してください。ネジ専らとポンプドライブにポンプヘッドを取り付けますポンプドライブでDED。 </li><li>原子炉のポートとリサイクルタンクにポンプAのチューブを接続します。供給タンクとリサイクルタンクにポンプBのチューブの端を挿入します。チューブとリサイクルタンクに上部原子炉ポートを接続します。原子炉ポートでチューブにクランプを適用します。 注意：光合成コミュニティはランプによって提供人工照明から利益を得ることができます。 </li></ol>ストックソリューションの流入/供給溶液、および藻類接種の調製<ol><li>ミネラル原液を準備します。 500mlの脱イオン水で1Lのメスフラスコに以下を加える：200gの炭酸水素ナトリウム、40グラムの一塩基性リン酸カリウム、4グラムの硫酸マグネシウム、4 gで塩化第二鉄、4グラムの塩化カルシウム、1gの塩化銅、1gのコバルト六水和物、1gの塩化ニッケル六水和物、1gの硫酸亜鉛七水和物。追加の脱イオン水400ミリリットルを追加します。塩の溶解を促進するために強制的に渦巻。以下のディス塩のolution 1にL.を溶液の総体積を脱イオン水を加えます</li><li>アンモニア原液を準備します。 1Lのメスフラスコに、約900 mlの脱イオン水の中での塩化アンモニウム38.214グラムを溶解します。溶解後、千ミリリットルの全容量のアップをもたらすために、脱イオン水を加えます。 注：1 Lに希釈したストック溶液1mlは、10mgのL -1 NH 4 + -N溶液が得られます。 </li><li>硝酸原液を準備します。 1Lのメスフラスコに、約900 mlの脱イオン水に硝酸カリウム72.413グラムを溶解します。溶解後、千ミリリットルの全容量のアップをもたらすために、脱イオン水を加えます。 注： - -N溶液1Lに希釈したストック溶液1mlは、10mgのL -1 NO 3が得られます 。 </li><li>フィード/流入溶液を準備します。 2mlの希-N - 20 mgのLを含む供給溶液-1 NH 4 + -Nと20mgのL -1 NO 3を作成するにはmmoniaストック溶液と1 Lの総体積に対する硝酸ストック溶液2ml。希釈する前に、行われて溶液0.5mlミネラル溶液/ Lを追加します。原子炉を起動して、合計で流入の5リットルを準備します。 </li><li>藻類の接種を準備します。 <ol><li>このような流れや池などの藻類含有水本体から大量の水（少なくとも10 L）を収集します。藻類は24時間静かに水試料を残すことによって解決することができます。 </li><li>サンプル瓶内に集中藻類懸濁液を残して、デカントし、サンプルの上部にクリア（非含有藻類）の水を捨てます。一つの容器にサンプルの全てから藻類サスペンションを結合し、沈降およびデカンテーションの手順を繰り返します。 </li><li>濃縮試料中のバイオマスを測定します。 <ol><li> COを測定し、室温で30分間デシケーター中で冷却した後103℃に設定されたオーブンでボートO / Nを秤量紙の真空フィルター（0.45μmのMCEの真空フィルター）とアルミニウムを乾燥フィルタの質量をmbined、ボートの重量を量ります。 </li><li>真空フィルタ20濃厚藻の懸濁液1ml及びフィルタを返すとO / Nを乾燥させるためにオーブンへのボートの重量を量ります。 </li><li>フィルタの合計質量を測定し、ボートの重量を量ります。濃縮試料中のバイオマス密度を計算します。 注：研究者が収集する必要があることを水試料の総容量は、ソース水域に依存します。 </li></ol></li></ol></li></ol> 3.シーディングと原子炉の起動<ol><li>反応器に供給溶液の750ミリリットルを追加します。供給溶液500ミリリットルとリサイクルタンクを埋めます。 </li><li>静かに反応器の底部近くに藻類の1.5グラムを含む接種サスペンションを追加するために、長いピペットを使用してください。次のステップに進む前に、目視でこれを確実に、接種材料は、反応器の底に沈殿することを許可します。 </li><li>細胞が定着した後、（10 revolutioをチューブクランプを取り外し、ゆっくりと流量にポンプAをオンにしますNS分-1 / 38ミリリットル分-1）。チューブに閉じ込められた空気は、反応器内に放出されます。 注：反応器への750ミリリットルの添加は、反応器を出るからポンプによって妨害任意のバイオマスを防ぐことができます。すべての空気が追い出されたことを確認するためにチューブを絞ります。 </li><li>この溶液を反応器に注入されると、徐々に再循環タンクに供給溶液を加えます。原子炉とリサイクルタンクの両方がトップポートを介して循環タンクを終了する能力と廃液が開始になるまで追加を続けます。 注：供給溶液の体積は、反応器に添加し接種の量に応じて変化する再循環タンクへ添加します。 </li><li>飼料タンクに残った供給溶液を注ぎます。 </li><li>分72.5ミリリットルのリサイクル流量を確立し、-1分19回転に循環ポンプ（ポンプA）を設定-1。藻類を守って反応器の底部からロフトを開始します。原子炉のグラデーションを使用して、藻類の双方向を決定omassゾーンの高さ。高さは、次のステップに進む前に一定であることを確認してください。 </li><li>非常に低速でミキシングプレートをオンにします。 1または2の設定が開始することが適切です。混合バーはさらに、バイオマスをロフティングを支援しますが、積極的な混合は、原子炉を残してリサイクルタンクを入力し、流​​出液中に残すために藻が発生します。原子炉（ 図2A）内の明確な藻類の境界を確立するために必要な設定で混合速度設定します。藻類バイオマスゾーンは高さが約10〜15 cmである必要があります。 </li><li>藻類プラグと原子炉流体との間の明確な境界を観察した後、供給ポンプを起動します。分1.5ミリリットルの流量を確立し、-1分25回転にポンプを設定-1。着信流入ストリームに起因する重力や位置ずれに原子炉流体出口に廃液ポートを確認します。 </li></ol> 4.サンプルの収集と分析<ol><li>サンプル収集活動のPRIを実行しまたは反応器システムのメンテナンスを実行します。毎日の流出と流入サンプルの20ミリリットルを収集します。リサイクルタンク内からの流出物のサンプルを収集します。供給タンクから直接流入サンプルを収集します。 </li><li>貯蔵前と分析に懸濁物質を除去するための真空フィルタサンプル。 </li><li>さらなる分析まで-20℃で流入、流出液のサンプルを保管してください。制限の凍結融解サイクルのサンプル数は、にさらされます。必要に応じて、サンプルは、サンプルの完全性を維持するためにアリコートに分割することができます。 </li><li>標準的な技術17を用いて硝酸塩、亜硝酸塩、アンモニアのサンプルの分析を行います。 注意：イオンクロマトグラフィー（IC）を利用著者らは、本明細書に提示される結果を生成します。仕様のための材料の表を参照してください。 </li></ol>

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M., Defoirdt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+microalgal-bacterial+interactions+for+aquaculture.">Significance of microalgal-bacterial interactions for aquaculture.</a> Reviews in Aquaculture. 6 (1), 48-61 (2014).</li><li>Dittami, S. 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.

このセクションでは、可能な運用上の問題に対処するだけでなく、異なる微生物群集を使用するために必要なプロトコルの変化の議論で開始します。この反応器の設計の長所は、反応器内の酸素フラックス及び高密度フロックの形成制御を管理する能力を含めて、説明されます。現在の課題や調査の可能な手段も言及されます。 プロトコルニュアンスとバリエーショ...

地方自治体の廃水は、一般的に浮遊物質（SS）、生物学的酸素要求量（BOD）、有機および無機窒素、及びリン含量5,6を低減するために、活性汚泥プロセスで処理されます。活性汚泥法、二次廃水処理の手段は、入ってくる廃水リサイクル従属栄養微生物の混合液を充填した曝気槽中の有機炭素の酸化を伴う5-7（一般的に、活性汚泥と呼ばれます）。混合液は、その後明らかにし、下水処理水が中に放出される前に、三次処理や消毒に続けることができますが、汚泥は、曝気槽に配置されたか、再循環することのいずれかに、より簡単に収集するために落ち着く比較的大きな清澄（沈殿槽）に入り、受信水域5-7。二次清澄における下水処理水と固形物（汚泥）の効率的な分離があったの適切な機能のために不可欠ですtewater処理システム、任意の活性汚泥が排出物5-8の BODとSSを増加させる清澄を超えて継続しました。 代替生物学的プロセスの数が減少または（バイオフィルム）成長添付リアクター、メンブレンバイオリアクター（MBRの）、および粒状汚泥リアクターを含む大規模な明確タンクの必要性を排除する、廃水の二次処理のために存在します。バイオフィルムリアクターでは、骨材自然微生物および固体表面上の層として添付しているバイオフィルムの形成は、明確にタンクを必要とせずに、バイオマスの保存・蓄積することができます。バイオリアクターは、3種類に分類することができる：充填床反応器、流動床反応器、および生物接触器を回転させます。このようしたたるフィルタおよび生物学的塔として充填床反応器は、固定固体成長表面5,6を利用します。流動床反応器（高速炉）は、粒子への微生物の付着に依存しますこのような高い上昇流速度9,10によって懸濁液中に保持される砂、粒状活性炭（GAC）、またはガラスビーズ、など。回転の生物学的反応器は、バイオフィルムが交互に空気と5,6を治療されている液体にさらされることを可能にする回転軸に取り付けられたメディア上に形成されたバイオフィルムによって異なります。 MBRのは、バイオリアクター（水没設定）内または外部再循環（サイドストリーム構成）5,11のいずれかを介して、膜ろ過ユニットを使用しています。膜は、処理液11,12からバイオマスおよび固体粒子の良好な分離を達成するのに役立ちます。グラニュール汚泥反応器は、それらが高い空塔上昇流速度13にさらされているときに、微生物の非常に緻密でよくセトリング顆粒の形成が発生する上昇流反応装置です。 活性汚泥プロセスの別の代替として、今高密度バイオリアクター（HDBR）と呼ばれる新規な上向流反応器システムは、designeましたDと貧しい沈降スラッジの発生を引き起こすことが知られている低F / Mの条件で合成廃棄物の流れ（ すなわち 、増量汚泥）1,7,14から活性汚泥によりCOD除去を研究するために販売および（2006）Shiehによって建てられました。利用HDBRシステムは、典型的には、上向流反応器および外部循環タンクから成る流動床反応器を修正しました。バイオフィルムで覆われた基板が保持されるように、流動床反応器は、典型的には、十分に懸濁し、バイオフィルムの成長基体を維持するのに十分高いが、低再循環流の流量で作動されます。流動床反応器とは異なり、HDBRはセールスに記載されており、（2006）Shiehは、外部通気とともに、 反応器1内に形成されたバイオマスゾーンの破壊を防止し、比較的低い再循環流の流速を使用していました。その後の研究では、正常に硝化/脱窒細菌に3,4を用いて、窒素フラックスの範囲を治療するために、この反応器設計の能力を実証しています。すべてのスタッドでHDBR内で安定、高密度のバイオマスゾーンの形成が外部凝集/沈降プロセス1-4の必要性を排除IES。 私たちがここで報告したように、高密度の文化を成長させるHDBRの使用はまた、藻類の培養のための光バイオリアクター（PBR）の構成でテストされています。私たちは、藻類栽培とバイオマス収穫（ すなわち 、良好な固液分離15,16）に関連した藻類バイオ燃料の実用化の大きなハードルを克服するために、その可能性のためにこの新規原子炉システムの利点と欠点について説明します。以下のプロトコルは、起動時からのサンプル、組み立て、および関心の微生物群集としての藻類とHDBRを維持するために必要な手順を説明します。従属と硝化/脱窒文化の起動と操作プロトコルの変化も言及されます。最後に、一般的な利点、欠点、およびこの新規原子炉の設計の未知数が強調表示されます。

<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 mgの-NL -1で供給物中の全窒 ​​素含有量を維持しながらHDBRは、流入アンモニアと硝酸塩濃度のいくつかの比率の上に藻類を育成するために使用されました。流入、流出液のサンプルを毎日採取しました。バイオマス濃度試料を各条件の開始時と終了時に採取しました。反応器は、条件を変更した後に定常状態の平衡に到達するのに平均3-5日かかりました。流入条件の広い範囲にわ...

Watch this Scientific Journal Video about A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities at JoVE.com

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.

多様な微生物群集の高密度栽培のための新しいバイオリアクター

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

17.0K ビュー.  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.

ビデオ: 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 ...

生物工学

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.

ベンチトップリアクターの操作

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.

腐食性煙道ガスを用いたベンチスケール光バイオリアクターにおける微細藻類栽培とバイオマス定量

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

環境学

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.

使い捨て空気圧バイオリアクターシステムを使用して、哺乳動物細胞の培養

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

環境微生物培養のためのIn Situ模擬嫌気性法

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.

著者スポットライト:In Situ Culturingによる未知の環境における陸生嫌気性微生物の謎の解明

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."

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

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.

自動 Microbioreactor 技術を用いた異種タンパク質生産の最適化のための汎用プロトコル

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.

高スループットの自動モデル IgG1 CHO 細胞での生産のための Microbioreactor システム

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.

中国ハムスター卵巣細胞培養におけるハイスループット自動マイクロバイオリアクターを用いたプロセス最適化

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

多様な微生物群集の高密度栽培のための新しいバイオリアクター

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

ベンチトップリアクターの操作

腐食性煙道ガスを用いたベンチスケール光バイオリアクターにおける微細藻類栽培とバイオマス定量

使い捨て空気圧バイオリアクターシステムを使用して、哺乳動物細胞の培養

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

自動 Microbioreactor 技術を用いた異種タンパク質生産の最適化のための汎用プロトコル

高スループットの自動モデル IgG1 CHO 細胞での生産のための Microbioreactor システム

中国ハムスター卵巣細胞培養におけるハイスループット自動マイクロバイオリアクターを用いたプロセス最適化