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

1. Asamblea Reactor <ol><li> Colocar los componentes del reactor de acuerdo con el esquema de la Figura 1. <ol><li> Coloque el reactor (R) en una placa de mezcla, añadir una barra de agitación en el reactor. Coloque el depósito de reciclado (RT) al lado de la placa de agitación y el reactor de manera que el puerto efluente (parte superior) del depósito se dirige hacia el borde de la mesa de laboratorio. </li><li> Colocar el contenedor de residuos (W) por debajo del puerto efluente (parte superior) del depósito de reciclado (RT). Coloque el tanque de alimentación (FT) al lado del depósito de reciclado (RT). Nota: El tanque de alimentación tiene una capacidad total de 5 L. </li></ol></li><li> Asegure el reactor (R) en contra de inflexión con un stand de tamaño adecuado y la abrazadera. Del mismo modo, asegurar el depósito de reciclado (RT) para evitar el movimiento. </li><li> Inserte neopreno tubo de la bomba peristáltica en el reciclaje (bomba A) y alimentar cabezas (bomba B) de la bomba. Consulte la tabla de Materiales para las especificaciones de tubos adicionales. Instale los cabezales de bomba en las unidades de bombeo con la provi tornillosded con las unidades de bombeo. </li><li> Conectar la tubería de la bomba de A a los puertos en el reactor y el depósito de reciclado. Inserte el extremo de la tubería de la bomba B en el tanque de alimentación y el depósito de reciclado. Conecte el puerto del reactor superior al depósito de reciclaje con el tubo. Aplicar las abrazaderas a la tubería en los puertos del reactor. Nota: Las comunidades fotosintéticas pueden beneficiarse de la iluminación artificial proporcionada por lámparas. </li></ol> 2. Preparación de las soluciones de archivo, Soluciones influente / piensos y algas inoculante <ol><li> Preparar la solución mineral de valores. Añadir lo siguiente a un matraz aforado de 1 L con 500 ml de agua desionizada: 200 g de bicarbonato de sodio, 40 g de fosfato monobásico de potasio, 4 g de sulfato de magnesio, 4 g de cloruro férrico, 4 g de cloruro de calcio, 1 g de cloruro de cobre, 1 g de cobalto hexahidrato de cloruro, 1 g de hexahidrato de cloruro de níquel, 1 g de sulfato de zinc heptahidratado. Añadir un adicional de 400 ml de agua desionizada. Agitar enérgicamente para fomentar la disolución de sales. Siguiendo dissolución de sales, añadir agua desionizada para llevar el volumen total de solución a 1 L. </li><li> Prepare la solución de amoníaco de valores. En un matraz aforado de 1 L, se disuelven 38.214 gramos de cloruro de amonio en aproximadamente 900 ml de agua desionizada. Después de la disolución, agregue agua desionizada para llevar el volumen total de hasta 1.000 ml. NOTA: 1 ml de solución madre diluidos a 1 L se obtiene un 10 mg L -1 NH 4 + -N solución. </li><li> Preparar la solución de nitrato de valores. En un matraz aforado de 1 L, se disuelven 72,413 g de nitrato de potasio en aproximadamente 900 ml de agua desionizada. Después de la disolución, agregue agua desionizada para llevar el volumen total de hasta 1.000 ml. Nota: 1 ml de solución madre diluidos a 1 L se obtiene un 10 mg L -1 NO 3 - -N solución. </li><li> Preparar la solución de alimentación / influente. Para hacer una solución de alimentación que contiene 20 mg L -1 NH 4 + N y 20 mg L -1 NO 3 - -N, diluir 2 ml de unammonia solución madre y 2 ml de solución de nitrato a volumen total de 1 litro. Antes de la dilución, añadir 0,5 ml de solución mineral / L de solución que está siendo hecho. Preparar 5 L de influente en total para poner en marcha el reactor. </li><li> Preparar el inoculante algas. <ol><li> Reunir un gran volumen (al menos 10 L) de agua de un cuerpo de agua que contiene algas, tales como una corriente o estanque. Permita que las algas se asiente dejando las muestras de agua en reposo durante 24 horas. </li><li> Decantar y desechar el claro (no-algas que contiene) de agua en la parte superior de las muestras, dejando una suspensión de algas concentrado dentro de las botellas de muestra. Combinar la suspensión de algas de todas las muestras en un recipiente y repetir el asentamiento y los pasos de decantación. </li><li> Mida la biomasa dentro de la muestra concentrada. <ol><li> Secar un filtro de vacío de papel (filtro de vacío 0,45 micras MCE) y aluminio pesan barco O / N en un horno que ha sido ajustado a 103 ° C Después de enfriamiento en un desecador durante 30 min a TA medir la combined masa del filtro y pesar barco. </li><li> Filtro de vacío de 20 ml de suspensión concentrada de algas y regresar el filtro y pesan barco al horno para secar O / N. </li><li> Medir la masa combinada del filtro y pesar barco. Calcular la densidad de la biomasa dentro de la muestra concentrada. Nota: El volumen total de la muestra de agua que necesitan los investigadores para recolectar dependerá de la masa de agua de origen. </li></ol></li></ol></li></ol> 3. Siembra e inicio del Reactor <ol><li> Añadir 750 ml de solución de alimentación al reactor. Llenar el depósito de reciclaje con 500 ml de solución de alimentación. </li><li> Utilice una pipeta larga para agregar suavemente una suspensión inóculo que contiene 1,5 g de algas cerca de la parte inferior del reactor. Permitir que el inóculo a asentarse en el fondo del reactor, a asegurar esto por observación visual, antes de proceder al siguiente paso. </li><li> Una vez que las células se han asentado, retire las abrazaderas del tubo y encienda la bomba A a una velocidad de flujo lento (10 revolutions min -1 / 38 ml min -1). El aire atrapado en el tubo será expulsado en el reactor. Nota: La adición de 750 ml al reactor evitará que cualquier biomasa perturbado por la bomba de salir del reactor. Apriete el tubo para asegurar que todo el aire ha sido expulsado. </li><li> Gradualmente añadir solución de alimentación al tanque de reciclaje como la solución se bombea en el reactor. Continuar la adición hasta que tanto el reactor y el depósito de reciclado están en capacidad y se inicia de efluentes para salir del depósito de reciclado a través del puerto superior. Nota: El volumen de la solución de alimentación que se añaden al tanque de reciclaje variará con el volumen de la inoculante añadido al reactor. </li><li> Verter la solución de alimentación restante en el tanque de alimentación. </li><li> Ajuste la bomba de reciclaje (bomba A) a 19 revoluciones min -1, estableciendo un caudal de reciclaje de 72,5 ml min -1. Tenga en cuenta las algas comienzan a desván de la parte inferior del reactor. Utilizando las gradaciones en el reactor, determine la bi algasaltura de la zona omass. Asegúrese de que la altura es constante antes de proceder al siguiente paso. </li><li> Encienda la placa mezcladora a velocidad muy baja; un valor de 1 o 2 es apropiado para comenzar. La barra de mezcla ayudará a lofting biomasa más lejos, pero mezclado agresivo hará que las algas que abandonar el reactor, introduzca el depósito de reciclado, y dejar en el efluente. Ajuste de mezcla de velocidad en un entorno necesario para establecer una frontera clara algas dentro del reactor (Figura 2); la zona de la biomasa de algas debe ser de aproximadamente 10-15 cm de altura. </li><li> Inicie la bomba de alimentación después de observar un límite claro entre el enchufe de algas y el líquido del reactor. Ajuste la bomba a 25 revoluciones min -1, estableciendo un caudal de 1,5 ml min -1. Observe la salida de fluido del reactor el puerto de efluentes debido a la gravedad y el desplazamiento causado por la corriente influente entrante. </li></ol> 4. Recolección y Análisis <ol><li> Llevar a cabo actividades de recogida de muestras prio para realizar tareas de mantenimiento en el sistema de reactor. Recoger 20 ml de muestras de efluentes y afluentes diaria. Recoger muestras de efluentes desde dentro del depósito de reciclado. Recoger muestras del influente directamente desde el tanque de alimentación. </li><li> Muestras de filtros de vacío para eliminar los sólidos antes de su almacenamiento y análisis suspendidos. </li><li> Guarde el afluente y efluente muestras a -20 ° C hasta su posterior análisis. Limite el número de ciclos de congelación y descongelación muestras son sometidos. Si es necesario, las muestras se pueden dividir en alícuotas para mantener la integridad de la muestra. </li><li> Llevar a cabo análisis de muestras para el nitrato, nitrito y amoníaco utilizando técnicas estándar 17. Nota: Los autores utilizaron la cromatografía iónica (IC) para producir los resultados que se presentan en este documento. Consulte la tabla de Materiales para la especificación. </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.

En esta sección se iniciará con una discusión de las variaciones de protocolo necesarios para hacer frente a posibles problemas operativos, así como el uso de diferentes comunidades microbianas. Se discutirán los puntos fuertes de este diseño del reactor, incluyendo la capacidad para gobernar el control de flujo de oxígeno y la formación de flóculos de alta densidad dentro del reactor. También se mencionan los desafíos actuales y las posibles vías de investigación. Protocolo...

Las aguas residuales municipales se trata comúnmente con los procesos de lodos activados con el fin de reducir los sólidos en suspensión (SS), demanda biológica de oxígeno (DBO), de nitrógeno orgánico e inorgánico, y contenido de fósforo 5,6. El proceso de lodos activados, un medio de tratamiento secundario de aguas residuales, implica la oxidación del carbono orgánico en un tanque de aireación lleno de un licor mixto de las aguas residuales de entrada y de microorganismos heterótrofos reciclado (comúnmente conocida como lodo activado) 5-7. El licor mixto entra entonces en un relativamente grande clarificador (tanque de sedimentación), donde el lodo se asienta para la recolección más fácil, ya sea ser eliminados o reciclados de nuevo al tanque de aireación, mientras que el, aguas residuales tratadas aclarado puede seguir un tratamiento terciario o desinfección antes de ser liberado en aguas receptoras 5-7. La separación eficiente de las aguas residuales tratadas y los sólidos (lodos) en el clarificador secundario es esencial para la función apropiada de una erasistema de tratamiento de tewater, como cualquier lodos activados continuar más allá de los clarificadores aumentará la DBO y SS en el efluente 5-8. Un número de procesos biológicos alternativos existe para el tratamiento secundario de las aguas residuales, que reducen o eliminan la necesidad de grandes tanques de clarificación, incluyendo el crecimiento adjunto (biofilm) reactores, biorreactores de membrana (MBR), y los reactores de lodo granular. En los reactores de biopelículas, la formación de biopelículas, en el que los microorganismos de forma natural agregada y adjuntar como una capa sobre una superficie sólida, permite la retención de biomasa y acumulación sin la necesidad de un tanque de clarificación. Reactores de biopelícula se pueden clasificar en tres tipos: reactores de lecho empacado, reactores de lecho fluidizado, y contactores biológicos rotatorios. Los reactores de lecho Almuerzos, tales como un filtro de escurrimiento y torres biológicas, utilizan una superficie de un sólido crecimiento estacionario 5,6. Los reactores de lecho fluidizado (FBR) dependen de la fijación de los microorganismos a las partículas,tales como arena, carbón activado granular (GAC), o cuentas de vidrio, que se mantienen en suspensión por una alta tasa de flujo ascendente 9,10. Rotación de reactores biológicos dependen de biopelículas formadas en los medios de comunicación adjuntos a un eje de rotación que permiten la biopelícula para ser alternativamente expuestas al aire y el líquido se está tratando 5,6. MBR utilizan unidades de filtración de membrana, ya sea dentro del biorreactor (configuración sumergido) o externamente a través de recirculación (configuración corriente lateral) 5,11. Las membranas sirven para lograr una buena separación de la biomasa y las partículas sólidas del líquido tratado 11,12. Reactores de lodo granular son reactores de flujo ascendente en el que la formación de gránulos extremadamente densas y bien de sedimentación de los microorganismos se produce cuando están expuestos a alta flujo ascendente de aire superficial velocidades 13. Como otra alternativa al proceso de lodos activados, un sistema de reactor de flujo ascendente novela, que ahora se llama un biorreactor de alta densidad (HDBR), fue designed y construido por Ventas y Shieh (2006) para estudiar la eliminación de DQO por lodos activados de las corrientes de residuos sintéticos en bajas condiciones de F / M que se sabe que causan la formación de lodos de decantación pobres (es decir, aumento de volumen de lodos) 1,7,14. El sistema utilizado HDBR modificado reactores de lecho fluidizado que normalmente consisten en un reactor de flujo ascendente y un depósito de reciclaje externo. Reactores de lecho fluidizado se suelen funcionar con velocidades de flujo corriente de reciclo suficientemente alta para mantener el sustrato de crecimiento de biopelículas en suspensión pero bajas lo suficiente para que se retiene el sustrato biofilm-cubierto. A diferencia de reactores de lecho fluidizado, el HDBR describe en Sales y Shieh (2006) utilizó relativamente bajas tasas de flujo corriente de reciclo que, junto con aireación externo, impedían la interrupción de la zona de biomasa formada dentro del reactor 1. Estudios posteriores han demostrado la capacidad de este diseño del reactor para tratar con éxito una serie de flujos de nitrógeno mediante nitrificación / bacterias desnitrificantes 3,4. En todo espárragoIES formación de una zona de biomasa densa estable dentro del HDBR eliminó la necesidad de un proceso / sedimentación floculación externa a 1-4. Como se presenta aquí, el uso de la HDBR para crecer cultivos densos también se ha probado en un fotobiorreactor (PBR) de configuración para el cultivo de algas. Se discuten las ventajas e inconvenientes de este novedoso sistema de reactor para el cultivo de algas y su potencial para superar un gran obstáculo en la comercialización de biocombustibles de algas asociadas con la biomasa de cosecha (es decir, una buena separación sólido-líquido 15,16). El siguiente protocolo describe los pasos necesarios para montar, puesta en marcha, muestra a partir, y mantener un HDBR con algas como la comunidad microbiana de interés. También se mencionan las variaciones en el protocolo de puesta en marcha y funcionamiento de las culturas heterótrofos y nitrificantes / desnitrificantes. Por último, las ventajas generales, desventajas, y las incógnitas de esta novela diseño del reactor se resaltarán.

<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.

El HDBR se utilizó para cultivar algas lo largo de varias proporciones de las concentraciones de amoníaco y nitrato influente, mientras se mantiene un contenido de nitrógeno total en la alimentación a 40 mg -NL -1. Afluente y efluente muestras fueron tomadas a diario; muestras de densidad de biomasa fueron tomadas al principio y al final de cada condición. El reactor tuvo un promedio de 3-5 días para alcanzar el equilibrio de estado estacionario después se cambiaron las condiciones. Más de una amplia ...

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 biorreactor Novela de alta densidad de cultivo de diversas comunidades microbianas

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 vistas.  Drexel University. A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Video: A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

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

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

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

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

Gradiente de densidad multicapa polimerización (DGMP): una técnica novedosa para la creación de multi-compartimiento, Andamios personalizables para Ingeniería de Tejidos

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

Investigación

Bioingeniería

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.

Diseño de un Carga mecánica Biaxial biorreactor para Ingeniería de Tejidos

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.

La operación de un biorreactor de sobremesa

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.

Construcción y caracterización de un Fold biorreactor Novela 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.

El cultivo de células de mamífero utilizando un solo uso Sistema Neumático biorreactor

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.

Construcción y configuración de un biorreactor fotosintético de algas a escala de banco con control de temperatura, luz y pH para pruebas de crecimiento cinético

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.

Optimización de procesos mediante microbiorreactores automatizados de alto rendimiento en el cultivo de células de ovario de hámster chino

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.

Identificación de alto rendimiento de la resistencia a Pseudomonas syringae pv. Tomate en tomate usando el ensayo de inundación de plántulas

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.

Generación de condiciones ambientales dinámicas utilizando un dispositivo microfluídico de alto rendimiento

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