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>

<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.

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 border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Aeration stone</td>
			<td style="width:96px;">Alita</td>
			<td style="width:119px;">AS-3015C</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Aerator</td>
			<td style="width:96px;">Top Fin</td>
			<td style="width:119px;">Air-1000</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Ammonium chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">A9434</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Anion analysis column</td>
			<td style="width:96px;">Shodex</td>
			<td style="width:119px;">IC SI-52 4E</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Beaker (600 mL)</td>
			<td style="width:96px;">Corning Pyrex</td>
			<td style="width:119px;">1000-600</td>
			<td style="width:259px;">Used as mixing vessel (MV). Addition of hose barbs at the bottom and 500 mL levels. Outside diameter of hose barbs 3/8&#34;.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Calcium chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">C5670</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Cation analysis column</td>
			<td style="width:96px;">Shodex</td>
			<td style="width:119px;">IC YS-50</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Cobalt chloride hexahydrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">C8661</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Copper chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">222011</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Ferric chloride</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">157740</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Filter (vacuum)</td>
			<td style="width:96px;">Fisherbrand</td>
			<td style="width:119px;">09-719-2E</td>
			<td style="width:259px;">0.45 um membrane filter, MCE, 47 mm diameter</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:213px;">Graduated cylinder (1000 mL)</td>
			<td style="width:96px;">Corning Pyrex</td>
			<td style="width:119px;">3025-1L</td>
			<td style="width:259px;">Used as reactor vessel (R). Addition of hose barbs at bottom, 500 mL, and 1 L levels. Outside diameter of hose barbs 3/8&#34;.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">HPLC/IC</td>
			<td style="width:96px;">Shimadzu</td>
			<td style="width:119px;">Prominence</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Magnesium sulfate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">M2643</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Masterflex L/S variable speed drive</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">07553-50</td>
			<td style="width:259px;">Drive for recycle and feed pumps (2 needed)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Nickel chloride hexahydrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">N6136</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Potassium nitrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">P8291</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">(Monobasic) Potassium phosphate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">P5655</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump head</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">07018-20</td>
			<td style="width:259px;">Recycle pump head</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump head</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">07013-20</td>
			<td style="width:259px;">Feed pump head</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump tubing</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">6404-18</td>
			<td style="width:259px;">Recycle pump tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Pump tubing</td>
			<td style="width:96px;">Masterflex</td>
			<td style="width:119px;">6404-13</td>
			<td style="width:259px;">Feed pump tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sodium bicarbonate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Zinc sulfate heptahydrate</td>
			<td style="width:96px;">Sigma Aldrich</td>
			<td style="width:119px;">Z0251</td>
			<td style="width:259px;"></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 ...

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

Research

JoVE Journal

Bioengineering

16.9K 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

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度勾配多層重合（DGMP）：マルチコンパートメントを作成するための新規な技術、組織工学のためのカスタマイズ可能な足場

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

生物工学

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

組織工学のための二軸メカニカルローディングバイオリアクターの設計

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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 ...

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 ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

新規声帯バイオリアクターの構築とキャラクタリゼーション

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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 ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

動力学的成長試験のための温度、光およびpHモニタリングを有するベンチスケール藻類光合成バイオリアクターの構築およびセットアップ

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

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 ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

シュードモナスシリンゲpvに対する耐性のハイスループット同定苗フラッドアッセイを用いたトマトのトマト

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

ハイスループットマイクロ流体装置を用いた動的環境条件の生成

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...