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

1. Assemblée Réacteur <ol><li> Disposer les composants du réacteur selon le schéma de la figure 1. <ol><li> Placez le réacteur (R) sur une plaque de mélange, ajouter une barre d&#39;agitation dans le réacteur. Placez le réservoir de recyclage (RT) à côté de la plaque d&#39;agitation et le réacteur de telle sorte que l&#39;orifice d&#39;effluent (en haut) du réservoir est dirigée vers le bord de la table de laboratoire. </li><li> Placer le récipient de déchets (W) au-dessous de l&#39;orifice d&#39;effluent (en haut) de la cuve de recyclage (RT). Placez le réservoir d&#39;alimentation (FT) à côté du réservoir de recyclage (RT). Remarque: Le réservoir d&#39;alimentation a une capacité totale de 5 L. </li></ol></li><li> Fixez le réacteur (R) contre le basculement avec un stand et le collier de taille appropriée. De même, fixer le réservoir de recyclage (RT) pour empêcher le mouvement. </li><li> Insérez néoprène tubes de pompe péristaltique dans le recyclage (Pompe A) et nourrir têtes (Pompe B) de la pompe. Reportez-vous à la table des matières pour les spécifications de tubes supplémentaires. Installez les têtes de pompe sur les entraînements de pompe avec les vis dispoded avec les entraînements de pompe. </li><li> Connectez le tuyau de la pompe A aux ports sur le réacteur et le réservoir de recyclage. Insérez l&#39;extrémité de la tubulure de la pompe B dans le réservoir d&#39;alimentation et le réservoir de recyclage. Connectez le port du réacteur haut vers le réservoir de recyclage avec des tubes. Appliquer brides à la tubulure au niveau des ports du réacteur. Remarque: les communautés photosynthétiques peuvent bénéficier de l&#39;éclairage artificiel fourni par des lampes. </li></ol> 2. Préparation des solutions mères, Solutions Influent / d&#39;alimentation, et les algues Inoculant <ol><li> Préparer la solution minérale de stock. Ajouter ce qui suit à un 1 L fiole jaugée de 500 ml d&#39;eau déminéralisée: 200 g de bicarbonate de sodium, 40 g de phosphate monobasique de potassium, le sulfate de magnésium 4 g, 4 g de chlorure ferrique, 4 g de chlorure de calcium, 1 g de chlorure de cuivre, 1 g de cobalt chlorure hexahydraté, 1 g de chlorure de nickel hexahydraté, 1 g de sulfate de zinc heptahydraté. Ajouter un 400 ml supplémentaires d&#39;eau déminéralisée. Agiter vigoureusement pour encourager la dissolution de sels. Diss suivantsolution de sels, ajouter de l&#39;eau désionisée pour porter le volume total de solution à 1 L. </li><li> Préparer la solution d&#39;ammoniac stock. Dans une fiole jaugée de 1 litre, dissoudre 38.214 grammes de chlorure d&#39;ammonium dans environ 900 ml d&#39;eau désionisée. Après dissolution, ajouter de l&#39;eau désionisée pour porter le volume total à 1000 ml. Note: 1 ml de solution d&#39;achat d&#39;actions dilué à 1 L donne un 10 mg L -1 NH 4 + -N solution. </li><li> Préparer la solution de nitrate de stock. Dans une fiole jaugée de 1 litre, on dissout 72,413 g de nitrate de potassium dans environ 900 ml d&#39;eau désionisée. Après dissolution, ajouter de l&#39;eau désionisée pour porter le volume total à 1000 ml. Note: 1 ml de solution d&#39;achat d&#39;actions dilué à 1 L donne un 10 mg L -1 NO 3 - -N solution. </li><li> Préparer la solution d&#39;alimentation / affluent. Pour faire une solution d&#39;alimentation contenant 20 mg L -1 NH 4 + -N et 20 mg L -1 NO 3 - -N, diluer 2 ml d&#39;unemmonia solution de stock et 2 ml de solution de nitrate d&#39;achat d&#39;actions à 1 L volume total. Avant la dilution, ajouter 0,5 ml de solution minérale / L de solution réalisés. Préparer 5 litres d&#39;affluent au total pour démarrer le réacteur. </li><li> Préparer l&#39;inoculant des algues. <ol><li> Recueillir un grand volume (au moins 10 L) d&#39;eau à partir d&#39;un corps de l&#39;eau contenant des algues comme un ruisseau ou un étang. Laisser les algues à régler en laissant les échantillons d&#39;eau au repos pendant 24 heures. </li><li> Décanter et jeter le (non-algues contenant) l&#39;eau claire au sommet des échantillons, laissant une suspension d&#39;algues concentrée dans les bouteilles d&#39;échantillon. Combinez la suspension d&#39;algues de tous les échantillons dans un récipient et répéter la décantation et étapes de décantation. </li><li> Mesurer la biomasse au sein de l&#39;échantillon concentré. <ol><li> Séchez un filtre à vide de papier (filtre à vide de 0,45 um MCE) et d&#39;aluminium peser bateau O / N dans un four qui a été réglé à 103 ° C Après refroidissement dans un dessiccateur pendant 30 min à température ambiante mesurer la combined masse du filtre et peser bateau. </li><li> On filtre sous vide de 20 ml de concentré suspension d&#39;algues et retourner le filtre et peser bateau au four pour sécher O / N. </li><li> Mesurer la masse combinée du filtre et peser bateau. Calculer la densité de la biomasse à l&#39;intérieur de l&#39;échantillon concentré. Remarque: Le volume total d&#39;échantillon d&#39;eau que les enquêteurs devront recueillir dépendra de la masse d&#39;eau de la source. </li></ol></li></ol></li></ol> 3. L&#39;ensemencement et démarrage du réacteur <ol><li> Ajouter 750 ml de solution d&#39;alimentation dans le réacteur. Remplir le réservoir de recyclage avec 500 ml de solution d&#39;alimentation. </li><li> Utiliser une pipette pour ajouter longue doucement inoculer une suspension contenant 1,5 g d&#39;algues près du fond du réacteur. Laisser l&#39;inoculum se déposer au fond du réacteur, assurer ce par observation visuelle, avant de passer à l&#39;étape suivante. </li><li> Une fois que les cellules se sont installés, retirez les colliers de serrage et tourner sur la pompe A à un débit lent (10 revolutions min -1/38 ml min -1). L&#39;air emprisonné dans le tube sera expulsé dans le réacteur. Remarque: L&#39;addition de 750 ml dans le réacteur permet d&#39;éviter toute biomasse perturbé par la pompe à partir de la sortie du réacteur. Presser le tube pour assurer que tout l&#39;air a été expulsé. </li><li> Ajouter graduellement la solution d&#39;alimentation vers le réservoir de recyclage en tant que la solution est pompée dans le réacteur. Continuer l&#39;addition jusqu&#39;à ce que à la fois le réacteur et le réservoir de recyclage sont à pleine capacité et commence effluents pour sortir de la cuve de recyclage via le port haut. Remarque: Le volume de la solution d&#39;alimentation pour être ajouté à la cuve de recyclage varie avec le volume de l&#39;inoculum ajouté dans le réacteur. </li><li> Verser la solution d&#39;alimentation restant dans le réservoir d&#39;alimentation. </li><li> Régler la pompe de recyclage (Pompe A) à 19 tours min -1, établissant un débit de recyclage de 72,5 ml min -1. Observer les algues grenier commencent à partir du fond du réacteur. En utilisant les graduations sur le réacteur, déterminer le bi d&#39;alguesomass hauteur de la zone. Assurez-vous que la hauteur est constante avant de passer à l&#39;étape suivante. </li><li> Tournez sur la plaque de mélange à très basse vitesse; une valeur de 1 ou 2 est approprié de commencer. La barre de mélange aidera à lofting biomasse plus loin, mais mélange agressif provoquer des algues de quitter le réacteur, entrer dans le réservoir de recyclage, et laisser dans l&#39;effluent. Réglez la vitesse de mélange à un réglage nécessaire d&#39;établir une limite d&#39;algues clairement l&#39;intérieur du réacteur (figure 2A); la zone de biomasse d&#39;algues doit être d&#39;environ 10 à 15 cm de hauteur. </li><li> Démarrer la pompe d&#39;alimentation après avoir observé une frontière claire entre la prise d&#39;algues et le fluide du réacteur. Régler la pompe à 25 tours min -1, en ​​établissant un débit de 1,5 ml min -1. Observer la sortie de fluide de l&#39;orifice réacteur effluent due à la gravité et du déplacement provoqué par le courant d&#39;arrivée entrant. </li></ol> 4. Collecte et analyse des échantillons <ol><li> Mener des activités de collecte de l&#39;échantillon priou pour de la maintenance du système de réacteur. Recueillir 20 ml d&#39;échantillons d&#39;effluents et influents quotidienne. Prélever des échantillons d&#39;effluents de l&#39;intérieur du réservoir de recyclage. Prélever des échantillons de l&#39;influent directement à partir du réservoir d&#39;alimentation. </li><li> Des échantillons de filtre sous vide pour éliminer les solides avant le stockage et l&#39;analyse suspendus. </li><li> Stocker l&#39;affluent et les échantillons d&#39;effluents à -20 ° C jusqu&#39;à une analyse plus approfondie. Limiter le nombre de cycles de gel-dégel échantillons sont soumis. Si nécessaire, des échantillons peuvent être divisés en aliquotes pour maintenir l&#39;intégrité de l&#39;échantillon. </li><li> Procéder à une analyse de l&#39;échantillon de nitrate, nitrite, l&#39;ammoniac et utilisant des techniques standard 17. Remarque: Les auteurs utilisés chromatographie ionique (IC) pour produire les résultats présentés ici. Reportez-vous à la table des matières pour la spécification. </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.

Cette section commence par une discussion des variations de protocole nécessaires pour régler des problèmes opérationnels possibles ainsi que l&#39;aide de différentes communautés microbiennes. Les points forts de ce modèle de réacteur seront discutés, y compris la capacité de gouverner le contrôle des flux d&#39;oxygène et la formation de flocs de haute densité dans le réacteur. Les défis actuels et les possibilités d&#39;investigation seront également mentionnés. Nuan...

Les eaux usées municipales est généralement traitée avec des procédés à boues activées afin de réduire les matières en suspension (MES), la demande biologique en oxygène (DBO), de l&#39;azote organique et inorganique, et la teneur en phosphore de 5,6. Le procédé de boues activées, un moyen de traitement secondaire des eaux usées, entraîne l&#39;oxydation du carbone organique dans un bassin d&#39;aération rempli d&#39;une liqueur mixte des eaux usées entrant et micro-organisme hétérotrophe recyclé (communément appelé boues activées) 5-7. La liqueur mixte entre alors dans une relativement grande clarificateur (de bassin de décantation) où la boue se dépose pour la collecte plus facile, soit être éliminés ou recyclés dans le bassin d&#39;aération, tandis que le, les eaux usées traitées clarifié peut continuer à un traitement tertiaire ou la désinfection avant d&#39;être libéré en eaux réceptrices 5-7. La séparation efficace des eaux usées traitées et des solides (boues) dans le décanteur secondaire est essentiel pour le bon fonctionnement d&#39;un étéSystème de traitement des tewater, tout comme la boue activée en continuant au-delà des clarificateurs augmentera la DBO et SS dans l&#39;effluent 5-8. Un certain nombre de processus biologiques alternatives existent pour le traitement secondaire des eaux usées, qui réduisent ou éliminent la nécessité pour les grands réservoirs de clarification, y compris la croissance jointe (biofilm) réacteurs, bioréacteurs à membranes (MBR) de, et les réacteurs de boues granulaires. Dans les réacteurs à film biologique, la formation de biofilms, dans lequel les micro-organismes naturellement ensemble et fixer comme une couche sur une surface solide, permet la rétention et l&#39;accumulation de la biomasse sans la nécessité d&#39;une cuve de clarification. Biofilm réacteurs peuvent être classés en trois types: les réacteurs à lit garni, réacteurs à lit fluidisé, et contacteurs biologiques rotatifs. Des réacteurs à lit paniers, comme un filtres bactériens et des tours biologiques, utilisent une surface de croissance solide fixe 5,6. Les réacteurs à lit fluidisé (FBR) dépendent de la fixation de micro-organismes à des particules,tel que du sable, du charbon actif granulaire (GAC), ou des billes de verre, qui sont maintenus en suspension par un courant ascendant de vitesse élevé 9,10. Rotation des réacteurs biologiques dépendent de biofilms formés sur des supports fixés à un arbre rotatif permettant le biofilm pour être alternativement exposées à l&#39;air et le liquide à traiter de 5,6. MBR membrane utilisent des unités de filtration, soit dans le bioréacteur (configuration immergée) ou en externe via recirculation (configuration courant latéral) 5,11. Les membranes servent à obtenir une bonne séparation de la biomasse et de particules solides à partir du liquide traité 11,12. Réacteurs de boue granulaire sont des réacteurs à flux ascendant dans lequel la formation de granules extrêmement dense et bien-décantation de micro-organismes se produit quand ils sont exposés à courant ascendant d&#39;air superficielle des vitesses élevées 13. Dans une autre variante au procédé à boues activées, un système de réacteur à courant ascendant roman, maintenant appelé un bioréacteur à haute densité (HDBR), était designed et construit par des ventes et Shieh (2006) pour étudier la DCO par boues activées à partir de flux de déchets synthétiques dans des conditions de faible F / M qui sont connus pour provoquer la formation de mauvaise boues de décantation (c.-à-gonflement des boues) 1,7,14. Le système de HDBR utilisée à jour réacteurs à lit fluidisé qui se composent typiquement d&#39;un réacteur ascendant et un réservoir de recyclage externe. Réacteurs à lit fluidisé sont généralement utilisés avec des débits de flux de recyclage suffisamment élevées pour maintenir le substrat de croissance du biofilm en suspension mais suffisamment basse de sorte que le substrat recouvert d&#39;un biofilm est conservée. Contrairement réacteurs à lit fluidisé, l&#39;HDBR décrit dans les ventes et Shieh (2006) a utilisé des débits des cours d&#39;eau de recyclage relativement faibles qui, avec l&#39;aération externe, ont empêché la rupture de la zone de biomasse formée dans le réacteur 1. Des études ultérieures ont démontré la capacité de cette conception du réacteur pour traiter avec succès une gamme de flux d&#39;azote à l&#39;aide de nitrification / dénitrification bactéries 3,4. Dans tous les studies la formation d&#39;une zone dense de la biomasse stable dans la HDBR a éliminé la nécessité d&#39;un processus / d&#39;une sédimentation de floculation externe 1-4. Comme nous rapportons ici, l&#39;utilisation de la HDBR de cultiver des cultures denses a également été testé dans un photobioréacteur (PBR) pour la configuration de la culture d&#39;algues. Nous discutons les avantages et les inconvénients de ce nouveau système de réacteur pour la culture d&#39;algues et de son potentiel pour surmonter un grand obstacle à la commercialisation de biocarburants d&#39;algues associés à la récolte de biomasse (à savoir, une bonne séparation solide-liquide 15,16). Le protocole suivant décrit les étapes nécessaires à l&#39;assemblage, au démarrage, à partir de l&#39;échantillon, et de maintenir un HDBR avec des algues que la communauté microbienne d&#39;intérêt. Variations dans le démarrage et le fonctionnement protocole de cultures hétérotrophes et nitrification / dénitrification seront également mentionnés. Enfin, les avantages, les inconvénients, générales et inconnues de cette nouvelle conception de réacteur seront mis en évidence.

<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.

Le HDBR a été utilisé pour cultiver des algues sur plusieurs rapports de concentrations d&#39;ammoniaque et influent nitrate, tout en maintenant une teneur totale en azote dans la charge à 40 mg -NL -1. Influent et des échantillons d&#39;effluents ont été prélevés quotidiennement; Des échantillons de densité de la biomasse ont été prélevés au début et à la fin de chaque état. Le réacteur a fallu en moyenne 3-5 jours pour atteindre l&#39;état d&#39;équilibre après conditions ont été mod...

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 bioréacteur pour Novel Haute Culture de la densité des communautés microbiennes diverses

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 vues.  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.

Vidéo: 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

Gradient de densité multicouche de polymérisation (DGMP): une nouvelle technique pour la création multi-compartiment, échafaudages personnalisables pour l&#39;ingénierie tissulaire

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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Bio-ingénierie

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.

Conception d&#39;un bioréacteur de chargement mécanique biaxial pour l&#39;ingénierie tissulaire

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.

Microfluidique picolitres bioréacteur pour Analyse microbienne unicellulaire: Fabrication, configuration du système, et l&#39;opération

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.

Fonctionnement d&#39;un bioréacteur de laboratoire

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.

Construction et caractérisation d&#39;un pli bioréacteur roman 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 culture de cellules de mammifères en utilisant un système pneumatique bioréacteur à usage unique

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.

Construction et mise en place d&#39;un bioréacteur photosynthétique Algal à échelle de banc avec surveillance de la température, de la lumière et du pH pour les tests de croissance cinétique

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.

Optimisation des processus à l’aide de micro-bioréacteurs automatisés à haut débit dans la culture chinoise de cellules d’ovaires de Hamster

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.

Identification à haut débit de la résistance aux seringues Pseudomonas pv. Tomate dans la tomate à l’aide de semis 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.

Génération de conditions environnementales dynamiques à l’aide d’un dispositif microfluidique à haut débit

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