Name: continuously-stirred anaerobic digester to convert organic wastes into biogas: system setup and basic operation
Uploaded: 2012-07-13T12:00:00
Description: Watch this Scientific Journal Video about Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation at JoVE.com

This research is supported is supported by the USDA through the National Institutes of Food and Agriculture (NIFA), grant number 2007-35504-05381; by grant no. 58872 from NYSERDA and NYC-123444 through the Cornell University Agricultural Experiment Station's federal formula funds from the USDA NIFA.

1. Digester Construction
<ol>
 <li>Select a digester vessel that contains all the features shown in Fig. 1 (a cone is not necessary), and your desired working volume (typically between 1-10 L). If your digester vessel is not equipped with a heated-water jacket, place the digester in some other temperature-controlled environment, such as a heated water bath or incubation chamber.</li>
 <li>Secure the vessel in a vertical position in an area with sufficient horizontal bench space for placement of remaini...

<ol>
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B., Al Seadi, T., Oleskowicz-Popiel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+anaerobic+digestion+and+biogas+utilization.">The future of anaerobic digestion and biogas utilization.</a> Bioresour. Technol. 100, 5478-5484 (2009).</li><li>Hoffmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+shear+on+performance+and+microbial+ecology+of+completely-stirred+anaerobic+digesters+treating+animal+manure.">Effect of shear on performance and microbial ecology of completely-stirred anaerobic digesters treating animal manure.</a> Biotechnol. Bioeng. 100, 38-48 (2008).</li><li>Clesceri, L. S., Greenberg, A. E., Eaton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard Methods for the Examination of Water and Wastewater. , (1998).</li><li>Amani, T., Nosrati, M., Sreekrishnan, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+digestion+from+the+viewpoint+of+microbiological,+chemical,+and+operational+aspects:+a+review.">Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects: a review.</a> Environmental Reviews. 18, 255-278 (2010).</li></ol>

The anaerobic digestion system presented in this article provides a general introduction and some basic guidelines for the treatment of most substrates in an experimental context. The wide variety of substrate types, digester configurations, operating parameters, and also the unique ecology of the mixed-microbial community underlying these systems precludes outlining hard quantitative metrics, which can be applied universally. Despite all this variability, all anaerobic digestion systems follow a well-characterized ser...

Anaerobic digestion (AD) is a mature technology involving the biologically mediated conversion of complex organic waste substrates into useful biogas with methane as the energy carrier. There are many benefits of anaerobic treatment, including minimal energy and nutrient inputs and reduced biosolids production compared to aerobic treatment 10. In addition, the versatility of the mixed microbial community inherent to these systems renders a wide variety of organic substrates suitable as feedstocks 11,12. Indeed, it is due to these benefits that a growing number of applications for AD are being adopted outside of conventional municipal wastewater treatment, particularly in the industrial, municipal (e.g., food waste), and agricultural sectors 4,7,13. AD experienced its first major proliferation beginning in the 1980s in response to the national energy crisis of the previous decade. As the world faces a growing global energy crisis, coupled with environmental degradation, greater focus is now being placed on biofuel technologies and the waste-to-energy concept in particular. For example, in the U.S., anaerobic digestion can generate 5.5% of the total electrical power needs 8.
This has increased the demand for well-controlled experimental research at the pilot- and laboratory-scale to assess the suitability of new organic waste materials and waste mixtures for anaerobic digestion 14. We intend to provide a generic model for the construction, inoculation, operation, and monitoring of a laboratory-scale anaerobic digester that will be suitable for robust assessments. Anaerobic digesters exist in many different configurations. A few common configurations include the: continuously-stirred tank reactor (CSTR) with continuous influent feeding; continuously stirred anaerobic digester (CSAD) with periodic influent feeding; plug flow (PF), upflow anaerobic sludge blanket (UASB); anaerobic migrating blanket reactor (AMBR); anaerobic baffled reactor (ABR), and anaerobic sequencing batch reactor (ASBR) configurations 9,15. The CSTR and CSAD configuration have been widely adopted for laboratory-scale experiments due to its ease of setup and favorable operating conditions. Because of continuous mixing, the hydraulic retention time (HRT) is equal to the sludge retention time (SRT). The SRT is the important design parameter for ADs. The configuration is also conducive to controlled experiments because of a greater spatial uniformity of parameters, such as chemical species concentrations, temperature, and diffusion rates. It should be noted, however, that the optimal full-scale configuration for an anaerobic digester depends on the particular physical and chemical qualities of the organic substrate among other nontechnical aspects, such as target effluent quality. For example, dilute waste streams with relatively high soluble organic content and little particulates, such as brewery wastewater, typically experience greater energy conversion in an high-rate upflow bioreactor configuration (e.g., UASB) rather than a CSAD configuration. Regardless, there are fundamental operating parameters that are essential to successful digestion and relevant to all configurations, which justify a generic explication of using this configuration.
Indeed, every AD system containing a diverse, open community of anaerobic microbes will serially metabolize the substrate to methane (the final end-product with the lowest available free energy per electron). The metabolic pathways involved in this process constitute an intricate food web loosely categorized into four trophic stages: hydrolysis; acidogenesis; acetogenesis; and methanogenesis. In hydrolysis, complex organic polymers (e.g., carbohydrates, lipids, and proteins) are broken down to their respective monomers (e.g., sugars, long-chain fatty acids, and amino acids) by hydrolyzing, fermentative bacteria. In acidogenesis, these monomers are fermented by acidogenic bacteria to volatile fatty acids (VFAs) and alcohols, which in acetogenesis, are further oxidized to acetate and hydrogen by homoacetogenic and obligatory hydrogen-producing bacteria, respectfully 5. In the final step of methanogenesis, acetate and hydrogen are metabolized to methane by acetoclastic and hydrogenotrophic methanogens. It is important to recognize that the overall AD process, by relying on an interconnected series of metabolisms by different groups of microbes, will depend on the successful function of each member before the system as a whole will perform optimally. The design and construction of an AD bioreactor system should always take into consideration the requirement to completely seal the bioreactor. Small leaks in the top of the bioreactor (separating the headspace) or in the gas-handling system may be difficult to detect, and therefore the system should be pressure tested before use. After ensuring a leak-free setup, failures with anaerobic digester studies often stem from errors during inoculation, culturing, and day-to-day operation. As a result, digesters have a reputation as being intrinsically unstable and prone to unexpected failure. Why is it then that full-scale digesters have been operated under stable conditions for decades 13? Failure is likely to stem from improper handling by the operator, especially during the startup period during which the microbial community must slowly acclimate to the organic waste composition and strength. Therefore, our goal is not only to provide a methodology for constructing an AD system, but to also elucidate the processes of inoculation, operation, and monitoring of these systems.
The first section of the article will explain how to construct the CSTR or CSAD system, while the second section will provide a procedure for digester inoculation with active methanogenic biomass. It is more practical and less time-consuming to inoculate digesters with active methanogenic biomass from the mixed-liquor or effluent of an operating digester that is treating a similar substrate than to attempt to develop a sufficient biomass from an incipient culture. The third section of the article will cover operating considerations, such as feeding substrate, decanting effluent, and troubleshooting various reactor problems. Feeding substrate and decanting effluent for this system will be conducted on a semi-continuous basis (i.e., periodic feeding and decanting while most of the biomass and mixed liquor stays in the bioreactor). The frequency in which the digester is fed/decanted is the prerogative of the operator. In general, feeding/decanting more frequently and at regular intervals will promote greater digester stability and consistency in performance between feeding cycles. The fourth section will introduce a basic monitoring protocol to be used during the experimental period. Several standard analyses, which are outlined in Standard Methods for the Examination of Water and Wastewater 16 (Table 1, 2), will be required for characterization of the substrate and proper system monitoring. In addition to the measured variables, an important aspect of monitoring is to check that the digester system components are functioning properly. Regular maintenance to the digester system will preempt major system problems that could otherwise jeopardize the long-term performance and stability of the digester. For example, a failure of the heating element, leading to a drop in temperature, could cause the accumulation of volatile fatty acids by reducing the metabolic rate of methanogens. This problem would be compounded if the system lacked sufficient alkalinity to maintain the pH above inhibitory levels for methanogens. It is also important to detect and close possible leaks after unexpected drops in biogas production rates. Therefore, duplication within the experimental design by, for example, running two bioreactors side-by-side under the exact operating conditions, is important to detect unexpected performance losses caused by system malfunctions, such as small leaks.

<table border="1">
 <tbody>
 <tr>
 <td>Reactor Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Heated Recirculator</td>
 <td>VWR Scientific</td>
 <td>13271-063 VWR</td>
 <td>For use with a heating jacket reactor system</td>
 </tr>
 <tr>
 <td>Variable Speed Electric Lab Stirrer</td>
 <td>Cleveland Mixer Co.</td>
 <td>(Model 5VB)</td>
 <td>This mixer model facilitates mounting with a ring stand</td>
 </tr>
 <tr>
 <td>Wet-Type Precision Gas Meter</td>
 <td>Ritter Gasmeters</td>
 <td>(Model TG-01)</td>
 <td>This model needs a minimum flow of (0.1 L/h) and can handle a maximum flow of 30 L/h</td>
 </tr>
 <tr>
 <td>Gas Bubbler</td>
 <td>Chemglass</td>
 <td>(Model AF-0513-20)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gas Sampling Tube</td>
 <td>Chemglass</td>
 <td>(Model CG-1808)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axial Impeller</td>
 <td>Lightnin'</td>
 <td>R04560-25 Cole-Parmer</td>
 <td>Impeller blades with 7.9375 mm bore diameter</td>
 </tr>
 <tr>
 <td>Impeller Shaft</td>
 <td>Grainger</td>
 <td>2EXC9 Grainger</td>
 <td>1.83 m stainless steel rod with 7.9375 mm O.D. (needs to be cut to appropriate size)</td>
 </tr>
 <tr>
 <td>Cast Iron Support Stands</td>
 <td>American Educational Products</td>
 <td>(Model 7-G16)</td>
 <td>For mixer mounting</td>
 </tr>
 <tr>
 <td>Three-Prong Extension Clamp</td>
 <td>Talon</td>
 <td>21572-803 VWR</td>
 <td>For mixer mounting</td>
 </tr>
 <tr>
 <td>Regular Clamp Holder</td>
 <td>Talon</td>
 <td>21572-501 VWR</td>
 <td>For mixer mounting</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Masterflex</td>
 <td>WU-07523-80 Cole-Parmer</td>
 <td>For effluent decanting</td>
 </tr>
 <tr>
 <td>L/S Standard Pump Head</td>
 <td>Masterflex</td>
 <td>EW-07018-21 Cole-Parmer</td>
 <td>For effluent decanting -accessory to peristaltic pump</td>
 </tr>
 <tr>
 <td>L/S Precision Pump Tubing</td>
 <td>Masterflex</td>
 <td>EW-06508-18 Cole-Parmer</td>
 <td>For effluent decanting - accessory to peristaltic pump</td>
 </tr>
 <tr>
 <td>Analysis Equipment/Reagents</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td> 
<td>pH Analysis</td>
 </tr>
 <tr>
 <td>pH Meter</td>
 <td>Thermo Fisher Scientific - Orion</td>
 <td>1212000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td> 
 <td>&nbsp;</td>
 <td>&nbsp;</td>
<td>Total and Volatile Solids Analysis (Standard Methods: 2540-B,E)</td>
 </tr>
 <tr>
 <td>Glass Vacuum Dessicator</td>
 <td>Kimax</td>
 <td>WU-06536-30 Cole-Parmer</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Porcelain Evaporating Dishes</td>
 <td>VWR</td>
 <td>89038-082 VWR</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lab Oven</td>
 <td>Thermo Fisher Scientific</td>
 <td>(Model 13-246-516GAQ)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Medium Chamber Muffle Furnace</td>
 <td>Barnstead/ Thermolyne</td>
 <td>F6010 Thermo Scientific</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td> 
 <td>&nbsp;</td>
 <td>&nbsp;</td>
<td>Total Volatile Fatty Acid Analysis (Standard Methods: 5560-C)</td>
 </tr>
 <tr>
 <td>Large Capacity Variable Speed Centrifuge</td>
 <td>Sigma</td>
 <td>WU-17451-00 Cole-Parmer</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laboratory Hot Plate</td>
 <td>Thermo Scientific</td>
 <td>(Model HP53013A)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Large Condenser</td>
 <td>Kemtech America</td>
 <td>(Model C150190)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Acetic Acid Reagent [CAS: 64-19-7]</td>
 <td>Alfa Aesar</td>
 <td>AA33252-AK</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Chemical Oxygen Demand (Standard Methods: 5520-C)</td>
 </tr>
 <tr>
 <td>COD Block Heater</td>
 <td>HACH</td>
 <td>(Model DRB-200)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Borosilicate Culture Tubes</td>
 <td>Pyrex</td>
 <td>(Model 9825-13)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium Dichromate Reagent [CAS: 7778-50-9]</td>
 <td>Avantor Performance Materials</td>
 <td>3090-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mercury II Sulfate Reagent [CAS: 7783-35-9]</td>
 <td>Avantor Performance Materials</td>
 <td>2640-04</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ferroin Indicator Solution [CAS: 14634-91-4]</td>
 <td>Ricca Chemical</td>
 <td>R3140000-120C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ammonium iron(II) sulfate hexahydrate [CAS: 7783-85-9]</td>
 <td>Alfa Aesar</td>
 <td>13448-36</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Gas Composition by Gas Chromatography Analysis</td>
 </tr>
 <tr>
 <td>Gas Chromatograph</td>
 <td>SRI Instruments</td>
 <td>Model 8610C</td>
 <td>Must be equipped with a thermal conductibility detector (TCD), using below mentioned column and carrier gas operated at an isothermal temperature of 105&deg;C</td>
 </tr>
 <tr>
 <td>Helium Gas</td>
 <td>Airgas</td>
 <td>He HP300</td>
 <td>To be used as the carrier gas</td>
 </tr>
 <tr>
 <td>Packed-Column</td>
 <td>Restek</td>
 <td>80484-800</td>
 <td>To be used for N2, CH4, and CO2 separation</td>
 </tr>
 </tbody>
</table>

continuously-stirred anaerobic digester to convert organic wastes into biogas: system setup and basic operation

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier 1-3. Increasingly, AD is being used in industrial, agricultural, and municipal waste(water) treatment applications 4,5. The use of AD technology allows plant operators to reduce waste disposal costs and offset energy utility expenses. In addition to treating organic wastes, energy crops are being converted into the energy carrier methane 6,7. As the application of AD technology broadens for the treatment of new substrates and co-substrate mixtures 8, so does the demand for a reliable testing methodology at the pilot- and laboratory-scale.
Anaerobic digestion systems have a variety of configurations, including the continuously stirred tank reactor (CSTR), plug flow (PF), and anaerobic sequencing batch reactor (ASBR) configurations 9. The CSTR is frequently used in research due to its simplicity in design and operation, but also for its advantages in experimentation. Compared to other configurations, the CSTR provides greater uniformity of system parameters, such as temperature, mixing, chemical concentration, and substrate concentration. Ultimately, when designing a full-scale reactor, the optimum reactor configuration will depend on the character of a given substrate among many other nontechnical considerations. However, all configurations share fundamental design features and operating parameters that render the CSTR appropriate for most preliminary assessments. If researchers and engineers use an influent stream with relatively high concentrations of solids, then lab-scale bioreactor configurations cannot be fed continuously due to plugging problems of lab-scale pumps with solids or settling of solids in tubing. For that scenario with continuous mixing requirements, lab-scale bioreactors are fed periodically and we refer to such configurations as continuously stirred anaerobic digesters (CSADs).
This article presents a general methodology for constructing, inoculating, operating, and monitoring a CSAD system for the purpose of testing the suitability of a given organic substrate for long-term anaerobic digestion. The construction section of this article will cover building the lab-scale reactor system. The inoculation section will explain how to create an anaerobic environment suitable for seeding with an active methanogenic inoculum. The operating section will cover operation, maintenance, and troubleshooting. The monitoring section will introduce testing protocols using standard analyses. The use of these measures is necessary for reliable experimental assessments of substrate suitability for AD. This protocol should provide greater protection against a common mistake made in AD studies, which is to conclude that reactor failure was caused by the substrate in use, when really it was improper user operation 10.

Watch this Scientific Journal Video about Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation at JoVE.com

Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation

Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier ...

continuously-stirred-anaerobic-digester-to-convert-organic-wastes

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Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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 ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...