Name: a novel bioreactor for high density cultivation of diverse microbial communities
Uploaded: 2015-12-25T13:00:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities at JoVE.com

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

1. Reactor Assembly
<ol>
	<li>Arrange the reactor components according to the schematic in Figure 1.
	<ol>
		<li>Place the reactor (R) on a mixing plate, add a stir bar to the reactor. Place the recycle tank (RT) beside the stir plate and reactor so that the effluent (top) port of the tank is directed towards the edge of the lab bench.</li>
		<li>Place the waste container (W) underneath the effluent (top) port of the recycle tank (RT). Place the feed tank (FT) next to the recycle tank (RT). 
		Note: The feed tank has a total capacity of 5 L.</li>
	</ol>
	</li>
	<li>Secure the reactor (R) against tipping with an appropriately sized stand and clamp. Likewise, secure the recycle tank (RT) to prevent movement.</li>
	<li>Insert neoprene peristaltic pump tubing in the recycle (Pump A) and feed (Pump B) pump heads. Refer to the Materials table for additional tubing specifications. Install the pump heads onto the pump drives with the screws provided with the pump drives.</li>
	<li>Connect Pump A&#39;s tubing to the ports on the reactor and the recycle tank. Insert the end of Pump B&#39;s tubing into the feed tank and the recycle tank. Connect the top reactor port to the recycle tank with tubing. Apply clamps to the tubing at the reactor ports. 
	Note: Photosynthetic communities may benefit from artificial illumination provided by lamps.</li>
</ol>
2. Preparation of Stock Solutions, Influent/Feed Solutions, and Algae Inoculant
<ol>
	<li>Prepare the mineral stock solution. Add the following to a 1 L volumetric flask with 500 ml of deionized water: 200 g sodium bicarbonate, 40 g monobasic potassium phosphate, 4 g magnesium sulfate, 4 g ferric chloride, 4 g calcium chloride, 1 g copper chloride, 1 g cobalt chloride hexahydrate, 1 g nickel chloride hexahydrate, 1 g zinc sulfate heptahydrate. Add an additional 400 ml of deionized water. Swirl forcefully to encourage dissolution of salts. Following dissolution of salts, add deionized water to bring the total volume of solution to 1 L.</li>
	<li>Prepare the ammonia stock solution. In a 1 L volumetric flask, dissolve 38.214 grams of ammonium chloride in approximately 900 ml of deionized water. After dissolution, add deionized water to bring the total volume up to 1,000 ml. 
	Note: 1 ml of stock solution diluted to 1 L yields a 10 mg L-1 NH4+-N solution.</li>
	<li>Prepare the nitrate stock solution. In a 1 L volumetric flask, dissolve 72.413 g of potassium nitrate in approximately 900 ml of deionized water. After dissolution, add deionized water to bring the total volume up to 1,000 ml. 
	Note: 1 ml of stock solution diluted to 1 L yields a 10 mg L-1 NO3--N solution.</li>
	<li>Prepare feed/influent solution. To make a feed solution containing 20 mg L-1 NH4+-N and 20 mg L-1 NO3--N, dilute 2 ml of ammonia stock solution and 2 ml of nitrate stock solution to 1 L total volume. Prior to dilution, add 0.5 ml mineral solution/L of solution being made. Prepare 5 L of influent in total to start up the reactor.</li>
	<li>Prepare the algae inoculant.
	<ol>
		<li>Collect a large volume (at least 10 L) of water from an algae-containing water body such as a stream or pond. Allow the algae to settle by leaving the water samples undisturbed for 24 hr.</li>
		<li>Decant and discard the clear (non-algae containing) water at the top of the samples, leaving a concentrated algae suspension within the sample bottles. Combine the algae suspension from all of the samples into one container and repeat the settling and decanting steps.</li>
		<li>Measure the biomass within the concentrated sample.
		<ol>
			<li>Dry a paper vacuum filter (0.45 &#181;m MCE vacuum filter) and aluminum weigh boat O/N in an oven which has been set to 103 &#176;C After cool-down in a desiccator for 30 min at RT measure the combined mass of the filter and weigh boat.</li>
			<li>Vacuum filter 20 ml of concentrated algae suspension and return the filter and weigh boat to oven to dry O/N.</li>
			<li>Measure the combined mass of the filter and weigh boat. Calculate the biomass density within the concentrated sample. 
			Note: The total volume of water sample that investigators will need to collect will depend upon the source water body.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Seeding and Starting the Reactor
<ol>
	<li>Add 750 ml of feed solution to the reactor. Fill the recycle tank with 500 ml of feed solution.</li>
	<li>Use a long pipette to gently add an inoculate suspension containing 1.5 g of algae near the bottom of the reactor. Allow the inoculum to settle to the bottom of the reactor, ensure this by visual observation, before proceeding to the next step.</li>
	<li>Once the cells have settled, remove the tube clamps and turn on Pump A to a slow flow rate (10 revolutions min-1/38 ml min-1). Air trapped in the tubing will be expelled into the reactor. 
	Note: The addition of 750 ml to the reactor will prevent any biomass disturbed by the pump from leaving the reactor. Squeeze the tubing to ensure that all air has been expelled.</li>
	<li>Gradually add feed solution to the recycle tank as the solution is pumped into the reactor. Continue the addition until both the reactor and the recycle tank are at capacity and effluent starts to exit the recycle tank via the top port. 
	Note: The volume of feed solution to be added to the recycle tank will vary with the volume of the inoculant added to the reactor.</li>
	<li>Pour the remaining feed solution into the feed tank.</li>
	<li>Set the recycle pump (Pump A) to 19 revolutions min-1, establishing a recycle flow rate of 72.5 ml min-1. Observe the algae begin to loft from the bottom of the reactor. Using the gradations on the reactor, determine the algae biomass zone height. Ensure that the height is constant before proceeding to the next step.</li>
	<li>Turn on the mixing plate at very low speed; a setting of 1 or 2 is appropriate to start. The mixing bar will assist in lofting biomass further, but aggressive mixing will cause algae to leave the reactor, enter the recycle tank, and leave in the effluent. Set mixing speed at a setting needed to establish a clear algae boundary within the reactor (Figure 2A); the algal biomass zone should be approximately 10-15 cm in height.</li>
	<li>Start the feed pump after observing a clear boundary between the algal plug and the reactor fluid. Set the pump to 25 revolutions min-1, establishing a flow rate of 1.5 ml min-1. Observe the reactor fluid exit the effluent port due to gravity and displacement caused by the incoming influent stream.</li>
</ol>
4. Sample Collection and Analysis
<ol>
	<li>Carry out sample collection activities prior to performing maintenance on the reactor system. Collect 20 ml of effluent and influent samples daily. Collect effluent samples from within the recycle tank. Collect influent samples directly from the feed tank.</li>
	<li>Vacuum filter samples to remove suspended solids prior to storage and analysis.</li>
	<li>Store the influent and effluent samples at -20 &#176;C until further analysis. Limit the number of freeze thaw cycles samples are subjected to. If needed, samples can be split into aliquots to maintain sample integrity.</li>
	<li>Carry out sample analysis for nitrate, nitrite, and ammonia using standard techniques17. 
	Note: The authors utilized Ion Chromatography (IC) to produce the results presented herein. Refer to the Materials table for specification.</li>
</ol>

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The authors have nothing to disclose and declare that they have no competing financial interests.

This section will start with a discussion of protocol variations needed to address possible operational issues as well as using different microbial communities. The strengths of this reactor design will be discussed, including the ability to govern control of oxygen flux and the formation of high density flocs within the reactor. Current challenges and possible avenues of investigation will also be mentioned.
Protocol nuances and variations 
The operation of HDBRs for cultivation...

Municipal wastewater is commonly treated with activated sludge processes in order to reduce the suspended solids (SS), biological oxygen demand (BOD), organic and inorganic nitrogen, and phosphorous content5,6. The activated sludge process, a means of secondary wastewater treatment, entails the oxidation of organic carbon in an aeration tank filled with a mixed liquor of incoming wastewater and recycled heterotrophic microorganism (commonly referred to as activated sludge)5-7. The mixed liquor then enters a relatively large clarifier (settling tank) where the sludge settles for easier collection, to either be disposed or recycled back to the aeration tank, while the clarified, treated wastewater can continue to tertiary treatment or disinfection before being released into receiving waters5-7. Efficient separation of the treated wastewater and solids (sludge) in the secondary clarifier is essential for the proper function of a wastewater treatment system, as any activated sludge continuing beyond the clarifiers will increase the BOD and SS in the effluent 5-8.
A number of alternative biological processes exist for secondary treatment of wastewater, which reduce or eliminate the need for large clarifying tanks, including attached-growth (biofilm) reactors, membrane bioreactors (MBRs), and granular sludge reactors. In biofilm reactors, the formation of biofilms, in which microorganisms naturally aggregate and attach as a layer on a solid surface, allows for biomass retention and accumulation without the need for a clarifying tank. Biofilm reactors can be classified into three types: packed bed reactors, fluidized bed reactors, and rotating biological contactors. Packed bed reactors, such as a trickling filters and biological towers, utilize a stationary solid growth surface5,6. Fluidized bed reactors (FBRs) depend on the attachment of microorganisms to particles, such as sand, granular activated carbon (GAC), or glass beads, which are kept in suspension by a high upward flow rate9,10. Rotating biological reactors depend on biofilms formed on media attached to a rotating shaft allowing the biofilm to be alternately exposed to air and the liquid being treated5,6. MBRs use membrane filtration units, either within the bioreactor (submerged configuration) or externally via recirculation (side-stream configuration)5,11. The membranes serve to achieve good separation of biomass and solid particles from the treated liquid11,12. Granular sludge reactors are upflow reactors in which the formation of extremely dense and well-settling granules of microorganisms occurs when they are exposed to high superficial air upflow velocities13.
As another alternative to the activated sludge process, a novel upflow reactor system, now called a high density bioreactor (HDBR), was designed and built by Sales and Shieh (2006) to study COD removal by activated sludge from synthetic waste streams in low F/M conditions that are known to cause the formation of poor settling sludge (i.e., bulking sludge)1,7,14. The HDBR system utilized modified fluidized bed reactors that typically consist of an upflow reactor and an external recycle tank. Fluidized bed reactors are typically operated with recycle stream flow rates high enough to keep the biofilm growth substratum suspended but low enough so that the biofilm-covered substrate is retained. Unlike fluidized bed reactors, the HDBR described in Sales and Shieh (2006) used relatively low recycle stream flow rates which, along with external aeration, prevented disruption of the biomass zone formed within the reactor1. Subsequent studies have demonstrated this reactor design&#39;s ability to successfully treat a range of nitrogen fluxes using nitrifying/denitrifying bacteria3,4. In all studies the formation of a stable, dense biomass zone within the HDBR eliminated the need for an external flocculation/sedimentation process1-4.
As we report here, the use of the HDBR to grow dense cultures has also been tested in a photobioreactor (PBR) configuration for the cultivation of algae. We discuss the benefits and drawbacks of this novel reactor system for algal cultivation and its potential for overcoming a large hurdle in the commercialization of algal biofuels associated with biomass harvesting (i.e., good solid-liquid separation15,16). The following protocol outlines the steps needed to assemble, startup, sample from, and maintain an HDBR with algae as the microbial community of interest. Variations in the startup and operation protocol of heterotrophic and nitrifying/denitrifying cultures will also be mentioned. Lastly, general advantages, disadvantages, and unknowns of this novel reactor design will be highlighted.

<table><tbody><tr><td>Aeration stone</td><td>Alita</td><td>AS-3015C</td><td></td></tr><tr><td>Aerator</td><td>Top Fin</td><td>Air-1000</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Anion analysis column</td><td>Shodex</td><td>IC SI-52 4E</td><td></td></tr><tr><td>Beaker (600 ml)</td><td>Corning Pyrex</td><td>1000-600</td><td>Used as mixing vessel (MV). Addition of hose barbs at the bottom and 500 ml levels. Outside diameter of hose barbs 3/8&quot;.</td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>C5670</td><td></td></tr><tr><td>Cation analysis column</td><td>Shodex</td><td>IC YS-50</td><td></td></tr><tr><td>Cobalt chloride hexahydrate</td><td>Sigma Aldrich</td><td>C8661</td><td></td></tr><tr><td>Copper chloride</td><td>Sigma Aldrich</td><td>222011</td><td></td></tr><tr><td>Ferric chloride</td><td>Sigma Aldrich</td><td>157740</td><td></td></tr><tr><td>Filter (vacuum)</td><td>Fisherbrand</td><td>09-719-2E</td><td>0.45 &amp;#956;m membrane filter, MCE, 47&amp;nbsp;mm diameter</td></tr><tr><td>Graduated cylinder (1,000 ml)</td><td>Corning Pyrex</td><td>3025-1L</td><td>Used as reactor vessel (R). Addition of hose barbs at bottom, 500 ml, and 1 L levels. Outside diameter of hose barbs 3/8&quot;.</td></tr><tr><td>HPLC/IC</td><td>Shimadzu</td><td>Prominence</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Masterflex L/S variable speed drive</td><td>Masterflex</td><td>07553-50</td><td>Drive for recycle and feed pumps (2 needed)</td></tr><tr><td>Nickel chloride hexahydrate</td><td>Sigma Aldrich</td><td>N6136</td><td></td></tr><tr><td>Potassium nitrate</td><td>Sigma Aldrich</td><td>P8291</td><td></td></tr><tr><td>(Monobasic) Potassium phosphate</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Pump head</td><td>Masterflex</td><td>07018-20</td><td>Recycle pump head</td></tr><tr><td>Pump head</td><td>Masterflex</td><td>07013-20</td><td>Feed pump head</td></tr><tr><td>Pump tubing</td><td>Masterflex</td><td>6404-18</td><td>Recycle pump tubing</td></tr><tr><td>Pump tubing</td><td>Masterflex</td><td>6404-13</td><td>Feed pump tubing</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Sigma Aldrich</td><td>Z0251</td><td></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.

The HDBR was used to cultivate algae over several ratios of influent ammonia and nitrate concentrations, while maintaining a total nitrogen content in the feed at 40 mg &#8211;N L-1. Influent and effluent samples were taken daily; biomass density samples were taken at the beginning and end of each condition. The reactor took on average 3-5 days to reach steady state equilibrium after conditions were changed. Over a wide range of influent conditions a distinct biomass zone was established, as observed previous ...

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 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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Research

JoVE Journal

Bioengineering

17.0K Views.  Drexel University. A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

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

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.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...

Environment

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.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

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.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.