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

1. Reattore Assemblea <ol><li> Disporre i componenti del reattore secondo lo schema di figura 1. <ol><li> Posizionare il reattore (R) su una piastra di miscelazione, aggiungere una ancoretta al reattore. Posizionare il serbatoio di riciclo (RT) accanto alla piastra di agitazione e reattore in modo che la porta effluente (superiore) del serbatoio è diretto verso il bordo del banco di laboratorio. </li><li> Posizionare il contenitore degli scarti (W) sotto la porta di effluente (superiore) del serbatoio di riciclo (RT). Posizionare il serbatoio di alimentazione (FT) accanto al serbatoio di riciclo (RT). Nota: Il serbatoio di alimentazione ha una capacità totale di 5 L. </li></ol></li><li> Fissare il reattore (R) contro il ribaltamento con uno stand di dimensioni appropriate e morsetto. Allo stesso modo, fissare il serbatoio di riciclo (RT) per impedire il movimento. </li><li> Inserire neoprene tubo della pompa peristaltica nel riciclo (Pompa A) e dei mangimi teste (pompa B) pompa. Fare riferimento alla tabella dei materiali per le specifiche tubi aggiuntivi. Installare le teste delle pompe sulle pompe con le viti Provided con gli accoppiatori. </li><li> Collegare il tubo di pompa A per le porte sul reattore e la vasca di riciclo. Inserire l&#39;estremità del tubo di pompa B nel serbatoio di alimentazione e il serbatoio di riciclo. Collegare la porta del reattore superiore al serbatoio di riciclo con la tubazione. Applicare fascette al tubo nei porti reattore. Nota: Le comunità fotosintetici possono beneficiare di illuminazione artificiale fornita da lampade. </li></ol> 2. Preparazione di soluzioni di riserva, Affluente / mangimi Solutions, e alghe Inoculant <ol><li> Preparare la soluzione minerale magazzino. Aggiungere quanto segue in un matraccio da 1 L con 500 ml di acqua deionizzata: 200 g di bicarbonato di sodio, 40 g di fosfato monobasico di potassio, 4 g di solfato di magnesio, 4 g di cloruro ferrico, 4 g di cloruro di calcio, 1 g di cloruro di rame, 1 g di cobalto cloruro esaidrato, 1 g di cloruro esaidrato di nichel, 1 g di solfato di zinco eptaidrato. Aggiungere ulteriori 400 ml di acqua deionizzata. Agitare con forza per favorire la dissoluzione dei sali. A seguito di dissoluzione di sali, aggiungere acqua deionizzata per portare il volume totale di soluzione per 1 L. </li><li> Preparare la soluzione di ammoniaca magazzino. In un matraccio tarato da 1 L, sciogliere 38.214 grammi di cloruro di ammonio in circa 900 ml di acqua deionizzata. Dopo la dissoluzione, aggiungere acqua deionizzata per portare il volume totale a 1.000 ml. Nota: 1 ml di soluzione madre diluita a 1 L rendimenti a 10 mg L-1 NH 4 + -N soluzione. </li><li> Preparare la soluzione di nitrato. In un matraccio tarato da 1 L, sciogliere 72,413 g di nitrato di potassio in circa 900 ml di acqua deionizzata. Dopo la dissoluzione, aggiungere acqua deionizzata per portare il volume totale a 1.000 ml. Nota: 1 ml di soluzione madre diluita a 1 L ottiene un 10 mg L-1 NO 3 - -N soluzione. </li><li> Preparare alimentazione soluzione / influente. Per fare una soluzione di alimentazione contenente 20 mg L -1 NH 4 + -N e 20 mg L -1 NO 3 - -N, diluire 2 ml di unasoluzione stock mmonia e 2 ml di nitrato di soluzione madre al volume totale 1 L. Prima della diluizione, aggiungere 0,5 ml soluzione minerale / L di soluzione compiuti. Preparare 5 L di influente in totale per avviare il reattore. </li><li> Preparare il inoculante alghe. <ol><li> Raccogliere un grande volume (almeno 10 L) di acqua da un corpo idrico alghe contenente come un ruscello o stagno. Lasciare che le alghe di stabilirsi lasciando i campioni di acqua indisturbato per 24 ore. </li><li> Decantare e scartare chiaro (non contenente alghe) acqua in cima dei campioni, lasciando una sospensione concentrata di alghe all&#39;interno dei contenitori. Unire la sospensione di alghe da tutti i campioni in un contenitore e ripetere la decantazione e gradini decantazione. </li><li> Misurare la biomassa all&#39;interno del campione concentrato. <ol><li> Essiccare un filtro di carta vuoto (0,45 micron MCE filtro a vuoto) e alluminio pesare barca O / N in un forno che è stato impostato a 103 ° C Dopo raffreddamento in essiccatore per 30 minuti a RT misurare la combined massa del filtro e pesare barca. </li><li> Filtro a vuoto 20 ml di sospensione concentrata di alghe e restituire il filtro e pesare barca a forno ad asciugare O / N. </li><li> Misurare la massa combinata del filtro e pesare barca. Calcolare la densità della biomassa nel campione concentrato. Nota: Il volume totale di campione che gli investigatori dovranno raccogliere dipenderà corpo acqua di fonte. </li></ol></li></ol></li></ol> 3. Semina e avvio il reattore <ol><li> Aggiungere 750 ml di soluzione di alimentazione al reattore. Riempire il serbatoio di riciclo con 500 ml di soluzione di alimentazione. </li><li> Utilizzare una lunga pipetta per aggiungere delicatamente una sospensione inoculare contenente 1,5 g di alghe vicino al fondo del reattore. Consentire l&#39;inoculo di depositarsi sul fondo del reattore, garantisce questo mediante osservazione visiva, prima di procedere alla fase successiva. </li><li> Una volta che le cellule si sono stabiliti, rimuovere i fermagli del tubo e accendere Pompa A per una portata lenta (10 Revolutions min -1/38 ml min -1). Aria intrappolata nel tubo sarà espulso nel reattore. Nota: L&#39;aggiunta di 750 ml al reattore impedirà qualsiasi biomassa disturbati dalla pompa in uscita dal reattore. Spremere il tubo per assicurare che tutta l&#39;aria è stata espulsa. </li><li> Aggiungere gradualmente soluzione di alimentazione al serbatoio di riciclo come la soluzione viene pompata nel reattore. Continuare l&#39;aggiunta fino sia il reattore e il serbatoio di riciclo sono a capacità e inizia effluenti uscire serbatoio riciclo tramite la porta superiore. Nota: Il volume di soluzione di carica da aggiungere al serbatoio di riciclo varierà con il volume del inoculante aggiunta al reattore. </li><li> Versare la soluzione di alimentazione rimanente nel serbatoio di alimentazione. </li><li> Impostare la pompa di ricircolo (Pompa A) a 19 giri min -1, che stabilisce un flusso di riciclo del 72,5 ml min -1. Osservare le alghe cominciano a soppalco dal fondo del reattore. Utilizzando le gradazioni sul reattore, determinare il bi alghealtezza zona omass. Assicurarsi che l&#39;altezza è costante prima di procedere alla fase successiva. </li><li> Accendere la piastra di miscelazione a velocità molto bassa; un ambiente di 1 o 2 è opportuno iniziare. La barra di miscelazione aiuterà a loft ulteriore biomassa, ma miscelazione aggressivo causerà alghe per lasciare il reattore, inserire il serbatoio di riciclo, e lasciare in effluenti. Impostare miscelazione velocità ad un ambiente necessario per stabilire un confine netto alghe all&#39;interno del reattore (Figura 2A); zona biomassa algale dovrebbe essere di circa 10-15 cm di altezza. </li><li> Avviare la pompa di alimentazione dopo aver osservato un chiaro confine tra la spina algale e il fluido del reattore. Impostare la pompa 25 giri min -1, che stabilisce un flusso di 1,5 ml min -1. Osservare l&#39;uscita del fluido del reattore porto effluente dovuta alla gravità e spostamento causato dal flusso entrante in entrata. </li></ol> 4. Raccolta e analisi <ol><li> Svolgere attività di raccolta dei campioni prio per eseguire la manutenzione del sistema reattore. Raccogliere 20 ml di campioni di effluenti e influenti quotidiano. Raccogliere campioni effluenti all&#39;interno della vasca di riciclo. Raccogliere campioni influenti direttamente dal serbatoio di alimentazione. </li><li> Campioni del filtro di vuoto per rimuovere i solidi prima di archiviazione e l&#39;analisi sospesi. </li><li> Conservare influente ed effluente campioni a -20 ° C fino ad ulteriore analisi. Limitare il numero di cicli di scongelamento congelare i campioni sono sottoposti. Se necessario, i campioni possono essere suddivisi in aliquote per mantenere l&#39;integrità del campione. </li><li> Effettuare analisi campione per nitrati, nitriti, ammoniaca e utilizzando tecniche standard 17. Nota: Gli autori hanno utilizzato cromatografia ionica (IC) per produrre i risultati presentati nel presente documento. Fare riferimento alla tabella dei materiali per la specifica. </li></ol>

<ol>
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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.

Questa sezione inizia con una discussione di variazioni di protocollo necessari per affrontare eventuali questioni operative, nonché utilizzando diverse comunità microbiche. La forza di questo disegno reattore saranno discussi, tra cui la possibilità di governare il controllo del flusso di ossigeno e la formazione di fiocchi alta densità all&#39;interno del reattore. Saranno inoltre menzionate le sfide attuali e le possibili vie di studio. Protocollo sfumature e variazioni <br /...

Reflui urbani è comunemente trattati con processo a fanghi attivi per ridurre i solidi sospesi (SS), richiesta di ossigeno biologica (BOD), azoto organico e inorganico, e fosforo 5,6. Il processo a fanghi attivi, un mezzo di depurazione secondario, comporta l&#39;ossidazione del carbonio organico in una vasca di aerazione riempito con un liquido misto di refluo e riciclati microrganismi eterotrofi (comunemente indicato come fanghi attivi) 5-7. Il liquido misto quindi entra in una relativamente grande chiarificatore (decantatore) dove il fango si deposita per facilitare la raccolta, sia di essere smaltiti o riciclati al serbatoio di aerazione, mentre, acque reflue trattate chiarificata può continuare a trattamento terziario o disinfezione prima di essere rilasciato in acque riceventi 5-7. Separazione efficiente di acque reflue trattate e solidi (fanghi) nel chiarificatore secondario è essenziale per il corretto funzionamento di un erasistema di trattamento tewater, come ogni fanghi attivi continuare al di là dei chiarificatori aumenterà la BOD e SS nell&#39;effluente 5-8. Un certo numero di processi biologici alternative esistono per trattamento secondario delle acque reflue, che riducono o eliminano la necessità di grandi serbatoi chiarificatori, compresi crescita divisoria (biofilm) reattori, bioreattori a membrana (MBR), e reattori fanghi granulari. Nei reattori biofilm, la formazione di biofilm, in cui i microrganismi naturalmente aggregato e collegare come uno strato su una superficie solida, permette di ritenzione biomassa e accumulo senza la necessità di un serbatoio chiarire. Reattori biofilm possono essere classificati in tre tipi: i reattori a letto ricco, reattori a letto fluido, e la rotazione contattori biologici. Reattori a letto pranzo, come un percolatori e torri biologiche, utilizzano un stazionario superficie solida crescita 5,6. Reattori a letto fluidizzato (FBRs) dipendono dalla attacco dei microrganismi di particelle,come sabbia, carbone attivo granulare (GAC), o di perle di vetro, che sono tenuti in sospensione da un alto tasso di flusso verso l&#39;alto 9,10. Rotazione reattori biologici dipendono biofilm formati sulla media associati ad un albero rotante che consentono il biofilm essere alternativamente esposte all&#39;aria e il liquido viene trattata 5,6. MBR utilizzano unità di filtrazione a membrana, sia all&#39;interno del bioreattore (configurazione sommerso) o esternamente tramite ricircolo (configurazione side-stream) 5,11. Le membrane servono per ottenere una buona separazione della biomassa e particelle solide dal liquido trattato 11,12. Reattori fango granulare sono reattori flusso verso l&#39;alto in cui la formazione di granuli estremamente densi e ben sedimentazione di microrganismi si verifica quando sono esposti a elevato flusso verso l&#39;alto dell&#39;aria superficiale velocità vettoriale 13. Come ulteriore alternativa al processo a fanghi attivi, un reattore upflow romanzo, ora chiamato un bioreattore ad alta densità (HDBR), era designed e costruito da vendite e Shieh (2006) per studiare la rimozione COD da fanghi attivi dai flussi di rifiuti sintetici in condizioni di scarsa F / M che sono noti per causare la formazione di cattiva decantazione fanghi (cioè, carica fanghi) 1,7,14. Il sistema utilizzato HDBR modificato reattori a letto fluido che in genere consistono di un reattore flusso verso l&#39;alto e un serbatoio di riciclo esterno. Reattori a letto fluido sono in genere azionati con corrente di riciclo Portate abbastanza alto per mantenere il substrato di crescita biofilm sospeso ma bassi abbastanza in modo che il substrato biofilm coperte viene mantenuta. A differenza di reattori a letto fluido, il HDBR descritto nelle vendite e Shieh (2006) ha usato le portate del flusso di riciclaggio relativamente bassi che, insieme con aerazione esterna, impedito interruzione della zona di biomassa formata all&#39;interno del reattore 1. Studi successivi hanno dimostrato la capacità di questo progetto del reattore di trattare con successo una serie di flussi di azoto utilizzando nitrificanti / batteri denitrificanti 3,4. In tutto stalloneies la formazione di una stabile, zona biomassa densa all&#39;interno HDBR eliminato la necessità di un processo di flocculazione / sedimentazione esterno 1-4. Come riportiamo qui, l&#39;uso del HDBR crescere culture dense è stato anche testato in una configurazione photobioreactor (PBR) alla coltura delle alghe. Discutiamo i vantaggi e gli svantaggi di questo sistema reattore romanzo per la coltivazione delle alghe e le sue potenzialità per superare un grande ostacolo nella commercializzazione di biocarburanti algali associati alla raccolta di biomassa (ad esempio, una buona separazione solido-liquido 15,16). Il protocollo che segue illustra i passaggi necessari per assemblare, avvio, campione, e mantenere un HDBR con alghe come la comunità microbica di interesse. Saranno inoltre menzionate variazioni del protocollo di avvio e il funzionamento delle culture eterotrofi e nitrificazione / denitrificanti. Infine, saranno evidenziati i vantaggi generali, gli svantaggi e le incognite di questo disegno romanzo reattore.

<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.

Il HDBR stato usato per coltivare le alghe in diversi rapporti di concentrazione influente ammoniaca e nitrati, pur mantenendo un contenuto di azoto totale nel mangime a 40 mg -NL -1. Influente e campioni effluenti sono stati presi tutti i giorni; campioni di densità di biomassa sono stati prelevati all&#39;inizio e alla fine di ogni condizione. Il reattore sono voluti in media 3-5 giorni per raggiungere l&#39;equilibrio di stato stazionario dopo sono state modificate le condizioni. Su una vasta gamma di con...

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.

Un bioreattore Novel per l&#39;alta densità di coltivazione diverse comunità microbiche

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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Bioengineering

16.8K Views.  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.

Video: 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

Gradiente di densità multistrato polimerizzazione (DGMP): una tecnica innovativa per la creazione di comparti multipli, Ponteggi personalizzabili per l&#39;ingegneria tissutale

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

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Bioingegneria

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.

Progettazione di un biassiale Carico meccanico bioreattore per l&#39;ingegneria tissutale

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.

Il funzionamento di un bioreattore da banco

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.

Costruzione e caratterizzazione di un Fold bioreattore Novel Vocal

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.

La coltivazione di cellule di mammifero utilizzando un singolo-uso del sistema pneumatico Bioreattore

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.

Costruzione e messa a punto di un biotestattore fotosintetico di alghe a bilanciere a banco con monitoraggio della temperatura, della luce e del pH per test di crescita cinetica

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.

Ottimizzazione dei processi mediante microreattori automatizzati ad alta velocità effettiva nella coltivazione delle cellule ovaio dell'ammasso cinese

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.

Identificazione ad alto contenuto di velocità effettiva della resistenza a Pseudomonas syringae pv. Pomodoro nel pomodoro utilizzando 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. ...

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.

Generazione di condizioni ambientali dinamiche utilizzando un dispositivo microfluidico ad alta produttività

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