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

1. Reaktoranordnung <ol><li> Ordnen die Reaktorkomponenten nach dem Schema in Figur 1. <ol><li> Setzen Sie den Reaktor (R) auf einer Mischplatte, fügen Sie einen Rührstab in den Reaktor. Platzieren der Rückführungstank (RT) neben der Rührplatte und eines Reaktors, so daß das Abwasser (oben) Anschluss der Flasche in Richtung auf den Rand der Laborbank gerichtet sind. </li><li> Setzen Sie den Abfallbehälter (W) unterhalb des Abwassers (oben) Anschluss des Rückführungstank (RT). Transporteur-Tank (FT) neben dem Rückführungstank (RT). Hinweis: Der Beschickungsbehälter hat eine Gesamtkapazität von 5 L. </li></ol></li><li> Sichern Sie den Reaktor (R) gegen Kippen mit einem passenden Stativ und Klemme. Ebenso sichern die Rückführungstank (RT), um eine Bewegung zu verhindern. </li><li> Legen Neopren-Schlauchpumpenschlauch in den Papier (Pumpe A) und Futtermittel (Pumpe B) Pumpenköpfe. Beziehen sich auf die Materialtabelle für zusätzliche Schlauchspezifikationen. Installieren Sie die Pumpenköpfe auf die Pumpenantriebe mit den Schrauben provimit den Pumpenantriebe ded. </li><li> Schließen Pumpe A der Schlauch an den Häfen an der Reaktor und der Rückführungstank. Führen Sie das Ende Pump Bs Schlauch in den Vorlagebehälter und die Rückführungstank. Schließen Sie die obere Reaktor Port in den Papierbehälter mit Schlauch. Bewerben Schellen auf den Schlauch an den Reaktor Ports. Hinweis: Photosynthetic Gemeinden können von künstlicher Beleuchtung durch Lampen profitieren. </li></ol> 2. Herstellung von Stammlösungen, Zulauf / Beschickungslösungen und Algen Impflegierung <ol><li> Bereiten Sie die Mineralgrundlösung. Fügen Sie folgende in einem 1-L-Kolben mit 500 ml VE-Wasser: 200 g Natriumhydrogencarbonat, 40 g Kaliumdihydrogenphosphat, 4 g Magnesiumsulfat, 4 g Eisenchlorid, 4 g Calciumchlorid, 1 g Kupferchlorid, 1 g Kobalt Chlorid-Hexahydrat, 1 g Nickelchlorid-Hexahydrat, 1 g Zinksulfatheptahydrat. Fügen Sie eine zusätzliche 400 ml VE-Wasser. Wirbeln mit Nachdruck um die Auflösung von Salzen zu fördern. Nach dissolution von Salzen, fügen VE-Wasser, um das Gesamtvolumen der Lösung zum 1 L. </li><li> Bereiten Sie die Ammoniak-Stammlösung. In einem 1 l-Messkolben 38,214 g Ammoniumchlorid in etwa 900 ml entionisiertem Wasser. Nach dem Auflösen hinzufügen VE-Wasser, um das Gesamtvolumen auf 1000 ml zu bringen. Hinweis: 1 ml der Stammlösung auf 1 l verdünnt ergibt eine 10 mg L -1 NH 4 + -N-Lösung. </li><li> Bereiten Sie die Nitratstammlösung. In einem 1 l-Messkolben 72,413 g Kaliumnitrat in etwa 900 ml entionisiertem Wasser. Nach dem Auflösen hinzufügen VE-Wasser, um das Gesamtvolumen auf 1000 ml zu bringen. Hinweis: 1 ml der Stammlösung auf 1 l verdünnt ergibt eine 10 mg L -1 NO 3 - -N-Lösung. </li><li> Bereiten feed / Zulauflösung. Einen Rohstofflösung mit 20 mg L -1 NH 4 + -N und 20 mg L -1 NO 3 - -N, 2 ml der einmmonia Stammlösung und 2 ml Nitratstammlösung auf 1 L Gesamtvolumen. Vor der Verdünnung, 0,5 ml Minerallösung / l-Lösung gemacht. Bereiten 5 l Zulauf insgesamt zur Inbetriebnahme des Reaktors. </li><li> Bereiten Sie die Algen Impfmittel. <ol><li> Sammeln ein großes Volumen (mindestens 10 l) Wasser aus einer Algenhaltigen Wasserkörpers, wie beispielsweise einem Strom oder einem Teich. Lassen Sie die Algen, indem Sie die 24 Stunden lang ungestört Wasserproben zu begleichen. </li><li> Dekantieren und entsorgen Sie die clear (nicht-Algen enthalten) Wasser an der Spitze der Proben, so dass eine konzentrierte Algensuspension innerhalb der Probenflaschen. Kombinieren Sie die Algensuspension von allen Proben in einem Container, und wiederholen Sie das Absetzen und Dekantierstufen. </li><li> Messen Sie die Biomasse im konzentrierten Probe. <ol><li> Trocknen Sie ein Papier Vakuumfilter (0,45 um MCE Vakuumfilter) und Aluminium wiegen Boot O / N in einem Ofen, der auf 103 ° C eingestellt wurde nach dem Abkühlen in einem Exsikkator für 30 Minuten bei RT messen die Zusammenarbeitmbined Masse des Filters und wiegen Boot. </li><li> Vakuumfilter 20 ml konzentrierte Algensuspension und kehren Sie den Filter und wiegen Boot zum Ofen zum Trocknen O / N. </li><li> Messung der Gesamtmasse des Filters und Wägeschiffchen. Berechnen Sie die Biomassendichte innerhalb der konzentrierten Probe. Hinweis: Das Gesamtvolumen der Wasserprobe, die Ermittler beenden, müssen Sie hängt von der Quellwasserkörper abhängen. </li></ol></li></ol></li></ol> 3. Säen und Starten des Reactor <ol><li> Add 750 ml Speiselösung in den Reaktor. Füllen Sie den Rückführungstank mit 500 ml Speiselösung. </li><li> Verwenden Sie eine lange Pipette vorsichtig fügen Sie ein inoculate Suspension, die 1,5 g Algen in der Nähe des Bodens des Reaktors. Ermöglichen das Inokulum auf den Boden des Reaktors absetzen, sicherzustellen, dass dies durch visuelle Beobachtung, bevor sie zu dem nächsten Schritt. </li><li> Sobald die Zellen angesiedelt haben, entfernen Sie die Schlauchklemmen und schalten Sie Pumpe A zu einem langsamen Fließgeschwindigkeit (10 revolutions min -1 / 38 ml min -1). Luft in der Röhre eingefangen werden in den Reaktor ausgestoßen wird. Anmerkung: Die Zugabe von 750 ml in den Reaktor werden beliebige Biomasse von der Pumpe gestört ab dem Verlassen des Reaktors zu verhindern. Drücken Sie den Schlauch, um sicherzustellen, dass alle Luft entwichen ist. </li><li> Speiselösung Fügen Sie stufenweise in den Papierbehälter, wenn die Lösung in den Reaktor gepumpt. Die Zugabe, bis sowohl dem Reaktor und dem Rückführungstank ausgelastet sind und Ablauf beginnt, den Rückführungstank über die obere Öffnung zu schließen. Hinweis: Das Volumen der Zulauflösung in den Papierbehälter zugesetzt werden, dass mit dem Volumen der Impfmittel in den Reaktor gegeben variieren. </li><li> Gießen Sie die restliche Speiselösung in den Vorlagebehälter. </li><li> Stellen Sie die Rückführungspumpe (Pumpe A) bis 19 Umdrehungen min -1, zur Schaffung einer Recycling-Fließgeschwindigkeit von 72,5 ml min -1. Beachten Sie die Algen anfangen, aus dem Boden des Reaktors Dachboden. Mit den Abstufungen am Reaktor, bestimmen die Algen biomass Zonenhöhe. Sicherzustellen, dass die Höhe, bevor zum nächsten Schritt konstant ist. </li><li> Schalten Sie die Mischplatte mit sehr geringer Geschwindigkeit; einer Einstellung von 1 oder 2 ist angebracht, um zu starten. Das Mischungs bar wird in lofting Biomasse weiter zu unterstützen, aber aggressive Misch verursacht Algen, um den Reaktor zu verlassen, geben Sie das Rückführungstank, und lassen Sie im Abwasser. Stellen Sie die Mischgeschwindigkeit bei einer Einstellung erforderlich, um eine klare Grenze Algen innerhalb des Reaktors (2A) zu schaffen; die Algenbiomasse Zone sollte ca. 10-15 cm hoch sein. </li><li> Starten Sie die Förderpumpe nach Beobachtung eine klare Grenze zwischen der Algen Stecker und der Reaktorflüssigkeit. Die Pumpe auf 25 Umdrehungen min -1, zur Schaffung einer Strömungsrate von 1,5 ml min -1. Beachten Sie die Reaktorfluidaustritts das Abwasser-Anschluss durch die Schwerkraft und die Verschiebung von der ankommenden Zulaufstrom verursacht. </li></ol> 4. Probengewinnung und Analyse <ol><li> Durchführung Probenerhebung prioder eine Wartung des Reaktorsystems. Sammeln Sie 20 ml des Abwassers und Zulauf Proben täglich. Sammeln Abwasserproben aus dem Rückführungstank. Sammeln Zulaufproben direkt aus dem Vorlagebehälter. </li><li> Vakuumfilter Proben Schwebstoffe vor der Speicherung und Analyse entfernen. </li><li> Lagern Sie den Zu- und Ablauf-Proben bei -20 ° C bis zur weiteren Analyse. Beschränken Sie die Anzahl der Frost-Tau-Zyklen Proben werden unterzogen. Bei Bedarf können Proben in Aliquots aufgeteilt, um Probenintegrität aufrechtzuerhalten. </li><li> Führen Sie die Probenanalyse für Nitrat, Nitrit und Ammoniak unter Verwendung von Standardverfahren 17. Hinweis: Die verwendeten Ionenchromatographie (IC) Autoren, die Ergebnisse hier vorgestellt zu produzieren. Beziehen sich auf den Werkstofftabelle für die Spezifikation. </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.

Dieser Abschnitt wird mit einer Diskussion der Protokollvariationen benötigt, um mögliche Betriebsprobleme sowie unter Verwendung verschiedener mikrobieller Gemeinschaften Adresse starten. Die Stärken dieser Reaktorkonstruktion diskutiert werden, einschließlich der Fähigkeit zur Steuerung der Sauerstofffluss und die Bildung von Flocken hoher Dichte in dem Reaktor zu regeln. Aktuelle Herausforderungen und mögliche Wege der Untersuchung wird ebenfalls erwähnt werden. Protokoll Nuanc...

Kommunales Abwasser ist häufig mit Belebtschlammverfahren, um die suspendierten Feststoffe (SS), biologischen Sauerstoffbedarf (BSB), organische und anorganische Stickstoff und Phosphorgehalt 5,6 zu verringern behandelt. Das Belebtschlammverfahren, ein Mittel zur Abwasserbehandlung vor, beinhaltet die Oxidation von organischem Kohlenstoff in einem Belüftungstank mit einer Mischflüssigkeit von ankommenden Abwasser- und Recycling heterotrophen Mikroorganismen gefüllt 5-7 (üblicherweise als Belebtschlamm genannt). Der gemischten Flüssigkeit tritt dann in eine relativ große Absetzbecken (Absetzbecken), wo der Schlamm absetzt zur leichteren Erfassung, um entweder entsorgt oder recycelt zurück in den Belüftungstank, während das geklärte, behandeltes Abwasser zu tertiären Behandlung oder Desinfektion weiter, bevor es in freigesetzt werden Vorfluter 5-7. Effiziente Trennung des gereinigten Abwassers und Feststoffe (Schlamm) im Nachklärbecken ist für die ordnungsgemäße Funktion der a wartewater Behandlungssystem, wie jeder Belebungs weiterhin über die Klärbecken wird das BOD und SS im Abwasser 5-8 erhöhen. Eine Anzahl von alternativen biologischen Verfahren zur Zweitbehandlung von Abwasser, die Verringerung oder Beseitigung der Notwendigkeit für große Klärung Tanks, einschließlich befestigt-Wachstum (Biofilm) Reaktoren, Membranbioreaktoren (MBR) und granulare Schlammreaktoren existieren. Biofilmreaktoren, die Bildung von Biofilmen, in dem Mikroorganismen natürlich aggregierten und befestigen sich als Schicht auf einer festen Oberfläche, ermöglicht eine Biomasserückhaltung und Akkumulation, ohne die Notwendigkeit für ein Klärbecken. Biofilmreaktoren können in drei Typen eingeteilt werden: Festbett-Reaktoren, Fließbettreaktoren und Scheibentauchkörper. Festbett-Reaktoren, wie ein Tropfkörper und biologische Türme, nutzen eine stationäre feste Wachstumsoberfläche 5,6. Wirbelschichtreaktoren (FBRS) hängen von der Befestigung von Mikroorganismen an Teilchen,wie Sand, Aktivkohlegranulat (GAC) oder Glasperlen, die in der Suspension durch einen hohen Aufwärtsströmungsgeschwindigkeit 9,10 gehalten werden. Rotierenden biologischen Reaktoren hängen von Biofilmen auf Medien zu einem rotierenden Welle befestigt ermöglicht den Biofilm gebildet, um abwechselnd Luft ausgesetzt werden, und der zu behandelnden Flüssigkeit 5,6. MBRs verwenden Membranfiltrationseinheiten, entweder innerhalb des Bioreaktors (untergetaucht Konfiguration) oder extern über Rückführung (Nebenstromkonfiguration) 5,11. Die Membranen dienen dazu, eine gute Trennung von Biomasse und Feststoffpartikeln aus der behandelten Flüssigkeit 11,12 zu erzielen. Granuläre Schlammreaktoren mit Aufwärtsströmung Reaktoren, in denen die Bildung von extrem dichte und gut Absetzen Granulat von Mikroorganismen auftritt, wenn sie hohen Oberflächenluftaufwärtsströmungsgeschwindigkeiten ausgesetzt sind 13. Als weitere Alternative zu dem Belebtschlammverfahren, einem neuartigen Aufstromreaktor System, jetzt eine hohe Dichte Bioreaktor (HDBR) war designed und Vertrieb und Shieh (2006) gebaut, um CSB-Entfernung von Belebtschlamm aus synthetischen Abfallströmen in niedrigen F / M Bedingungen, die bekannt sind, um die Bildung von schlechten Vorklärschlamm verursachen (dh Blähschlamm) 1,7,14 studieren. Das verwendete HDBR System modifizierten Fließbett-Reaktoren, die typischerweise aus einem Aufstromreaktor und einer externen Rückführungstank. Wirbelschichtreaktoren werden typischerweise mit Rückführstrom Fließgeschwindigkeiten hoch genug ist, um die Biofilmwachstum Substrat in Suspension zu halten, jedoch niedrig genug betrieben, daß die Biofilm-bedeckten Substrats beibehalten wird. Anders als Wirbelbettreaktoren, in Verkaufs beschriebene HDBR und Shieh (2006) verwendet, relativ niedrigen Rückführstrom Flussraten, die zusammen mit externe Belüftung verhindert Störungen des Biomasse-Zone innerhalb des Reaktors 1 ausgebildet. Nachfolgende Studien haben dieses Reaktordesign Fähigkeit, erfolgreich zu behandeln eine Reihe von Stickstoffflüssen mit nitrifizierenden / denitrifizierenden Bakterien 3,4 demonstriert. In allen Studies die Bildung einer stabilen, dichten Biomasse Zone innerhalb des HDBR eliminiert die Notwendigkeit für eine externe Flockung / Sedimentationsverfahren 1-4. Wie wir hier berichten, hat sich der Einsatz der HDBR dichte Kulturen wachsen auch in einem Photobioreaktor (PBR) Konfiguration für die Kultivierung von Algen getestet. Wir diskutieren die Vor-und Nachteile dieser neuen Reaktorsystem zur Algenanbau und sein Potenzial zur Überwindung eine große Hürde bei der Kommerzialisierung von Biokraftstoffen mit Algenbiomasseernte (dh gute Fest-Flüssigtrennung 15,16) verbunden sind. Das folgende Protokoll beschreibt die Schritte notwendig, um zusammenzubauen, Inbetriebnahme, Probe aus, und unterhält ein HDBR mit Algen als der mikrobiellen Gemeinschaft von Interesse. Variationen in der Start und Betrieb Protokoll heterotrophe Nitrifikanten und / denitrifizierenden Kulturen werden ebenfalls erwähnt werden. Schließlich werden allgemeine Vorteile, Nachteile und Unbekannten dieser neuartigen Reaktordesign hervorgehoben.

<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.

Die HDBR wurde verwendet, um Algen über mehrere Verhältnisse von Zulauf Ammoniak und Nitrat-Konzentrationen zu kultivieren, während eine Gesamtstickstoffgehalt in der Beschickung bei 40 mg -NL -1. Zu- und Ablauf-Proben wurden täglich genommen; Biomassedichte Proben wurden zu Beginn und am Ende jeder Bedingung aufgenommen. Der Reaktor hat im Durchschnitt 3-5 Tage, um stationäre Gleichgewicht zu erreichen, nachdem Bedingungen geändert wurden. Über einen weiten Bereich der Zulaufbedingungen eine deutliche...

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.

A Novel Bioreaktor für High Density Anbau von Diverse mikrobiellen Gemeinschaften

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

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

Research

JoVE Journal

Bioengineering

16.8K Aufrufe.  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.

Serie: 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

Density Gradient Multilayered Polymerisation (DGMP): A Novel Technique für das Erstellen von Multi-Fach, Anpassbare Scaffolds für das Tissue Engineering

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

Forschung

Biotechnik

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.

Entwurf eines Biaxiale Mechanische Belastung Bioreaktor für Tissue Engineering

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.

Betrieb einer Tischbioreaktor

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.

Konstruktion und Charakterisierung eines neuartigen Bioreaktor Stimmlippen

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.

Anbau von Säugetierzellen mit einem Einweg-Bioreaktorsystem Pneumatische

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.

Aufbau und Aufbau eines handelsüblichen Algen-Photosynthese-Bioreaktors mit Temperatur-, Licht- und pH-Monitoring für kinetische Wachstumstests

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.

Prozessoptimierung mit automatisierten Mikro-Bioreaktoren mit hohem Durchsatz in der chinesischen Hamster-Ovarialzell-Kultivierung

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.

High-Throughput Identifikation der Resistenz gegen Pseudomonas syringae pv. Tomate in Tomaten mit 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.

Erzeugung dynamischer Umgebungsbedingungen mit einem hochdurchsatzstarken mikrofluidischen Gerät

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