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Microreactor and probe chip for propane-eaters

Microreactor and probe chip for propane-eatersGlenSummer 2009Aerobes that eat propaneRhodococcus rhodochrous PNKb1ATCC 21197, 21198, $240, identified by vendor as propane-eaterNocardia paraffinicum3.0 mL cell suspension 11 mol n-propanol in 40 minutes Arthrobacter CRL-60Pseudomonas fluorescensBrevibacterium butanicumMycobacterium austroafricanumATCC 29678 $195, identified by vendor as propane-eaterMixed cultureATCC 21032 $240, identified by vendor as a mix of propane-eaters

Ashraf, W., Mihdhir, A., Murrell, C. FEMS Microbiol. Lett. 1994, 122, 1-6.Babu, J.P., Brown, L.R. Appl. Environ. Microbiol. 1984, 48, 2, 260-264.www.atcc.org

Mechanism of enzyme action (propane oxygenase) is not agreed upon on literature: some say it is a specialization of cytochrome P450, other say P450 is not involved because CO experiments do not show CO-inhibition of activity.

Enzyme is elusive because activity has half-life of 150 minutes and cells do not break easily? - from review, 1994, fems microbiol lett Ashraf et al.2Anaerobes that eat propane

Gram-positive (i.e. high peptidoglycan content in cell wall, unlike most bacteria of human disease), spore-forming bacteria

Thermophilic, marine species of genus Desulfotomaculum; this genus also rots sealed, canned food

2 C3H8 + 5 SO42- + 4 H+ 5 H2S + 2 HCO3- 12 mM H2S formed in 3 months from bacteria floating in one bottle of 100 mL medium and 56 mL gas

Kniemeyer , O. et al. Nature, 2007, 449, 898-902. www.atcc.org

ATCC sells 13 species of Desulfotomaculum, and one of them (D. reducens, ATCC BAA-1160, $195) has a completely known genome.

DSMZ (Germany) sells 29 species of Desulfotomaculum

Neither vendor identifies any strain as a propane-oxidizing strain.Can any Desulfotomaculum species oxidize propane? Authors did not identify the strain.3Anaerobic mechanism proposed

Hiro, F. J. Phys. Chem. B, 2002, 106, 7688-7692.Widdel, F., Boetius, A., Rabus, R. Prokaryotes, 2006, 2, 10281049.Becker, A., Fritz-Wolf, K., Kabsch, J., Knappe, S., Schulz, S., Wager, A.F.V. Nature Struct. Biol., 1999, 6, 969-975.

Can we see this step?isopropylsuccinateEnzyme mech based on a well-used motif Enzymes that use the glycyl radical mechamism are difficult to isolate:

They need another enzyme to get activatedExposure to O2 will break the peptide bond between Gly and CysAttack on primary carbon proposed

During the 50 days of propane consumption, some formation of n-propylsuccinate occurs.

n-propylsuccinateKniemeyer , O. et al. Nature, 2007, 449, 898-902. www.atcc.org

This step was newly proposed in 2007. It happens for only about 1/3 of the propane oxidation reactions

Primary C-H bond cleavage is more difficult than secondary.Evidence of attack primary C

Metabolites were extracted in CH2Cl2 and methylated.

GC-MS was compared to standard sample of n-propylsuccinate

We have GC-MS in the shared instrument rooms.Kniemeyer , O. et al. Nature, 2007, 449, 898-902

Planktonic anaerobes, anchored aerobesAnaerobes: Kniemeyer et al. used planktonic DesulfotomaculumBut microbes live in sediments ( =>native state is anchored)Anchored state might be easier to maintain over several days inside a hollow-fiber membrane microbioreactor ; Chung et al did this for 50 daysSlow rxn rate for anaerobes => cell maintenance is necessary

Aerobes: Babu and Brown used cell suspensions of Nocardia paraffinicum (planktonic)But microbes live in soil, sediments => native state is anchoredPropane oxidation occurs in minutes, so microbioreactor does not need to maintain cells for long.Anchoring to membrane increases cell density in reactor higher concentration of metabolites for better NMR detectionAnchoring to membrane with ~0.1 m pores allows controlled flow of nutrients, waste

Wang, S.-J., Zhong, J.-J., Bioreactor Engineering, Bioprocessing for Value-Added Products From Renewable Resources: New Technologies and Applications, edited by Yang, S.-T., San Diego: Elsevier, 2007, pp. 131-162.

Chung, B.H., Chang, H.N., Kim, I.H., Enzyme and Microbial Technology, 1987, 9, 345-349.

7Planktonic microbioreactor

Six microbioreactors on one PDMS-on-glass chip

Bacteria in a constantly flow in a loop to prevent biofilm formationEach flow loop has nine segmentsEach segment is individually isolated for cleaning during the experimentLysis buffer is pumped from on-chip well to kill and to detach biofilm cellsExtra growth medium is pumped from on-chip well to re-dilute cells for control of population size.Balagadd, F.K.,You, L., Hansen, C.L., Arnold, F.H., Quake, S.R. Science, 2005, 309, 137-140.

For single-cell MRI, add hydrodynamic focusing to one or two loop systems, to isolate single cells. Flow the single cell to the NMR sample channel.8 Make the Soft Lithography MasterDesign and print mask.

Spin SU-9 photoresist to Si wafer

Expose wafer through mask, develop and bake photoresist

The wafer is now the master template. Resolution ~ 20 m

McDonald, J.C., Duffy, D.C., Anderson, D.T., Chiu, D.T., Wu, H., Schueller, O.J.A., Whitesides, G.M. Electrophoresis, 2000, 21, 27-40.

Make the PDMS layer on the master

McDonald, J.C., Duffy, D.C., Anderson, D.T., Chiu, D.T., Wu, H., Schueller, O.J.A., Whitesides, G.M. Electrophoresis, 2000, 21, 27-40.

Place newly cast PDMS layer onto glass (flat).Align PDMS and flat.Using air plasma,

Make two layersTop PDMS layer for air channelsAir pressure +5 psi PDMS 4mm thick for channel stability

Bottom PDMS layer for fluid channelsPDMS 30 m thick via spin-coat onto master, 2000 rpm, 30 s.Channels 100 m wide x 9 m high

Valve = intersection of fluid and air channels

Unger, M.A., Chou, H-P., Thorsen, T., Scherer, A., Quake, S.R. Science, 2000, 288, 113-116.

Thorsen, T., Maerkl, S.J., Quake, S.R. Science, 2005, 298, 580-584.

McDonald, J.C., Duffy, D.C., Anderson, D.T., Chiu, D.T., Wu, H., Schueller, O.J.A., Whitesides, G.M. Electrophoresis, 2000, 21, 27-40.

The two layers are different types of PDMS: one layer contains free Si-H groups, the other layer contains free vinyl (ethenyl) groups; the two bond covalently and become a monolith.General Electric RTV 615 silicone potting compound kit, 1 pint for $109.55 from local vendor RS Hughes (Pacoima, CA)

Peristaltic pump made of PDMS channel intersections E.V. ROBERTS sells 6 pints for 93.90 each.18027 BISHOP AVECarson, CACall: 310-204-6159Fax: 310-202-7247

R S HUGHES PACOIMA sells one pint for 109.5510639 GLENOAKS BLVDPACOIMA, CACall: 818 686-9111Fax: 818 686-197311Add a drainage capillary for NMR detectionIn lieu of six separate reactors, use only two.Use extra space on chip for PDMS peristaltic pumpPump cell suspension segment at a time to NMR detection deviceConnect chip to NMR detector via 100-m capillary

NMR detection device

Fused silica capillary13C has Larmor frequency 100 MHz at 9.4 T c=lambda nu lambda=c/nu = 3.0x108 m/s / 100 000 000 s-1

= 3 x 10-2 m = 3 cm Stripline is lambda/4, so stripline is 7.5 mm long for 13C.12Immobilization: denser cell growthMembraneFlat membrane Hollow-fiber membrane

Free microcarriersCommercial designer particles

Packed bedFoamPolymer GelBeadsRaschig ringCokeActivated carbon

17 mLJ. R. Lloyd, C. L. Harding, L. E. Macaskie BIOTECHNOLOGY AND BIOENGINEERING, VOL. 55, NO. 3, AUGUST 5, 1997

S. Raihan, N. Ahmed, L. E. Macaskie, J. R. Lloyd, Appl Microbiol Biotechnol (1997) 47: 352-357

Akay, G., Erhan, E., Keskinler, B. Biotechnology and Bioengineering, 2005, 90, 2, 180-190.

Wang, S.-J., Zhong, J.-J., Bioreactor Engineering, Bioprocessing for Value-Added Products From Renewable Resources: New Technologies and Applications, edited by Yang, S.-T., San Diego: Elsevier, 2007, pp. 131-162.

13Hollow-fiber membrane Even distribution of cells

High surface area / volume

Control of nutrient and waste flows

I would use one 1.1-mm fiber in a glass tube microbioreactor

http://www.dic.co.jp/en/products/membrane/images/hollow_fiber.gif

Wang, S.-J., Zhong, J.-J., Bioreactor Engineering, Bioprocessing for Value-Added Products From Renewable Resources: New Technologies and Applications, edited by Yang, S.-T., San Diego: Elsevier, 2007, pp. 131-162.

14Separation of (g), (l) nutrients possible

0.33 mm ID0.66 mm OD0.4-0.6 m pores1.47 mm ID1.96 mm OD8 mm IDChung, B.H., Chang, H.N., Kim, I.H. Enzyme Microb. Technol., 1987, 9, 344-349.By inserting a single PP fiber into a single silicone tube, I can use a 2mm channel in PDMS on glass, instead of an 8mm glass tube.

Other materials:

Romicon hollow tubes, acrylic PM100, XM50Koch Membrane Systems, Inc. 850 Main Street, Wilmington, Massachusetts, USA01887-3388Telephone: 1-888-677-5624 Telephone:+1-978-694-7000 Fax: +1-978-657-5208

Capillary: fused silica, Polymicro, TSP100375, ID 100 um, OD 363 um, $10/m for 50m, $15/m for 10m. http://www.polymicro.com/productpdfs/TSP_TSG_3_08.pdf

Upchurch (now called Idex Corp.) connectors capillary to glass.N-124S NANOPORT ASSEMBLY, STANDARD, 6-32 CONED, 360m OD

If holes are 1mm diam. X 1mm thickness, then N-121SNANOPORT ASSEMBLY, STANDARD, 6-32 FLAT, 360/510m OD, 1mm x 1mm

For connecting two tubes (1/16 ID x peristaltic pump tube to 100 um ID x 360 um OD fused silica capillary) use Upchurch / IDEX part # 1356KIT, PEEK MICROTIGHT FITTINGS

John (800) 426-0191 x [email protected]

Loctite UV-activated 3105 glue 800-LOCTITEhttp://65.213.72.112/tds5/docs/3105-EN.PDF

151.3 cm OD microbioreactor15 cm1/16 ID x 0.025 OD peristaltic pump tubing feeds hollow-fiber membrane directly.This is for seeding the fiber with live cells, and for collecting metabolites.1/16 ID x 0.025 OD peristaltic pump tubing takes output to optical microscope and to NMR. Tubing fits through rubber septum with sealant.Glass reactor contains a single Romicon XM50 hollow-fiber tube, 1.1 mm in diameter.Nutrients and dissolved propane gas flow around outside of hollow-fiber membrane, via peristaltic pump and tubing.9 mm ODOr connect using IDEX polypropylene Luer part #p-857x or p-854x

16Optical microscopic observation to check for escaping cells (cellular attachment)Glass plate, 1 x 5Glass plate, 1 x 5 5 length allows glass to overhang microscope stage on both sidesPDMS sandwiched in the middle here, in the shape of one microfluidic channel, 100 m wide, 10 m deep.360 m holes for metabolite throughput: connections to 1/16 ID peristaltic pump tubing made by Nanoport PEEK connectors (IDEX Corp)This hole on top plateThis hole on bottom plateOutput goes to NMRThis replaces NMR until the bioreactor is surely filled.17Detachment of bacteria from bioreactor

Johansen, C., Falholt, P., Gram, L.,. Applied Environmental Microbiology, 1997, 63, 3724 3728.Bockelmann, U., Szewzyk, U., Grohmann, E. Journal of Microbiological Methods, 2003, 55, 201 211.For use of live cells in the stripline probe, cells detach from hollow-fiber membrane walls with buffered mixture of -glucosidase, -galactosidase.

Commercial enzyme cocktail Pectinex Ultra SP-L works, too, but may be more expensive.

NMR detection deviceMixture of Alpha-glucosidase, Beta-galactosidase works for bacterial detachment. If you use lipase, you will kill because lipase acts like surfactant.

Pectinex: I have called Novozyme for pricing.

18Strip of Cu instead of coilvan Bentum, P.J.M., Janssen, J.W.G., Kentgens, A.P.M., Bart, J., Gardeniers, J.G.E., Journal of Magnetic Resonance, 2007, 189, 104-113.

A Dutch group uses a strip of copper instead of a coil to receive and to send rf signals. Their 1H frequency is 600 MHz.

Thin strip part of Cu is 35 m thick, 1 mm long, 500 m wide, etched out of the Cu foil coating the Rogers 5870 PTFE material.

B1 homogeneity for pulses perpendicular to B0 field is shown in yellow in this Maxwell equations simulation:Microfluidic channels may run here(axial view)Homogeneity might be better with stripline than with microcoils?

Axial cross-section of half of a four-turn 3D coil. Sample would go into the rectangle.Axial cross-section of half of a three-turn 2D coil (spiral). Sample would go into the rectangle.van Bentum, P.J.M., Janssen, J.W.G., Kentgens, A.P.M., Bart, J., Gardeniers, J.G.E., Journal of Magnetic Resonance, 2007, 189, 104-113.http://www.ndt-ed.org/EducationResources/CommunityCollege/MagParticle/Graphics/coil1.gif

Double-resonance stripline probe

van Bentum, P.J.M., et al. Journal of Magnetic Resonance, 2007, 189, 104-113.

Bart, J., Kolkman, A.J., Oosthoek-de Vries, A.J., Koch, K., Nieuwland, P.J., Janssen, J.W.G., van Bentum, P.J.M., Ampt, K.A.M., Rutjes, F.P.J.T., Wijmenga, S.S., Gardeniers, J.G.E., Kentgens, A.P.M. J. Am. Chem. Soc. 2009, 132, 14, 5014-5015.microreactorStripline is easier to build than coil (sputter Cu or anodically bond Cu strip)

Tune, match via dielectric plunger5-mm microcoil versus stripline

Human cerebrospinal fluid using 256 scans, 6-s 90 pulsewidth on 5mm commercial microcoilHuman cerebrospinal fluid using 4608 scans, 7-s 90 pulsewidth on stripline Bart, J., Kolkman, et al. J. Am. Chem. Soc. 2009, 132, 14, 5014-5015.Stripline gets all the peaks, and there is some sensitivity enhancement, but might take too long to measure short-lived intermediate metabolites?

Authors say sensitivity can be further enhanced by using a less lossy substrate. Maybe Forturan glass?

Authors say line broadening can be fixed by adding a lock channel to the rf circuit.

22Ways to improve sensitivity

Bart, J., Kolkman, et al. J. Am. Chem. Soc. 2009, 132, 14, 5014-5015 online supplementKnapkiewicz, P., Walczak, R., Dziuban, J.A. Optica Applicata, 2007, 37, 65-72.Olson, D.L, Peck, T.L., Webb, A.G., Magin, R.L., Sweedler, J.V. Science, 1995, 270, 1967-1970.

Use nonconductive substrate instead of Si.Nanolab has anodic bonding machineForturan glass: bond at 250C, under 2kV

Flow two microfluidic channels along length of stripline (one on each face of strip)

vs.Forturan glassSiRogers 5870 can be ordered as a PTFE sandwiched between two copper foil layers (35 m)

A piece of Rogers 5870 with copper foil cladding

Capillary: fused silica, Polymicro, TSP100375, ID 100 um, OD 363 um, $10/m for 50m, $15/m for 10m. http://www.polymicro.com/productpdfs/TSP_TSG_3_08.pdf

Upchurch (now called Idex Corp.) connectors capillary to glass.N-124S NANOPORT ASSEMBLY, STANDARD, 6-32 CONED, 360m OD

If holes are 1mm diam. X 1mm thickness, then N-121SNANOPORT ASSEMBLY, STANDARD, 6-32 FLAT, 360/510m OD, 1mm x 1mm

John (800) 426-0191 x [email protected]

Loctite UV-activated 3105 glue 800-LOCTITEhttp://65.213.72.112/tds5/docs/3105-EN.PDF24Mill channel into dielectric surface (fluoropolymer): 100 m wide, 10 m deepThinnest capillary wall for 100m ID = 64 m;

Height of homogeneous B1 field of stripline 50 mTherefore, sample must flow into shallow channel in dielectric.

Gershenfeld et al used capillary, but did not show homogeneity of B1 Bentum et al used channel, and showed limited homogeneity heightGershenfeld et al used capillary, but I cannot because thinnest capillary walls for 100 m ID = 64 mheight of most homogeneous part of microstrip B1 fieldPTFE is millable via computer numerically controlled milling (CNC); mill is manufactured by Roland, U.K.

Vendor of Roland: http://www.sourcegraphics.com/ 3D printers Roland ASD

eBay for new Roland mill, but no returns:http://cgi.ebay.com/Roland-MDX-15-CNC-Mill-3D-Scanner-NEW-in-Box_W0QQitemZ370229635969QQcmdZViewItemQQssPageNameZRSS:B:SRCH:US:102#ht_3842wt_732

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Copper ground planesCopper to become the planar NMR rf stripPTFE to become the microfluidic channel bed

Etch away copper on portions of one piece, for microfluidic channel Mill a channel:100 m wide, 10 m deep

Place input and output where convenient.

For example, if ends are convenient, then glue glass strip or PTFE strip to ends, and etch 360 m hole in each for IDEX Nanoport capillary connections

Stripline-bearing Rogers 5780 piece will form lid to this channel. Use PTFE adhesive on PTFE parts only, and press hard.

Channel volume on order of tens of nanolitershttp://www.reltekllc.com/teflon-adhesives.htmSells PTFE adhesive kit including PTFE etchant.

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Copper ground planesCopper to become the planar NMR rf stripPTFE to become the microfluidic channel bedShorter piece of Rogers 5870

Longer piece of Rogers 5870Mill channel into dielectric surface (fluoropolymer): 100 m wide, 10 m deep

Thinnest capillary wall for 100m ID = 64 m;

Height of homogeneous B1 field of stripline 50 mTherefore, sample must flow into shallow channel in dielectric.

Channel volume on order of tens of nanolitersAffix planktonic microbioreactor here.Connect to NMR sample channel via capillary tubingGershenfeld et al used capillary, but I cannot because thinnest capillary walls for 100 m ID = 64 mheight of most homogeneous part of microstrip B1 fieldPTFE is millable via computer numerically controlled milling (CNC); mill is manufactured by Roland, U.K.

Vendor of Roland: http://www.sourcegraphics.com/ 3D printers Roland ASD

eBay for new Roland mill, but no returns:http://cgi.ebay.com/Roland-MDX-15-CNC-Mill-3D-Scanner-NEW-in-Box_W0QQitemZ370229635969QQcmdZViewItemQQssPageNameZRSS:B:SRCH:US:102#ht_3842wt_732

29Affix Quaker-design planktonic microbioreactor onto dielectric, and cap NMR sample channel

Affix planktonic microbioreactor here.Connect to NMR sample channel via capillary tubingStripline-bearing Rogers 5780 piece will form lid to this channel. Use PTFE adhesive on PTFE parts only, and press hard.

Gershenfeld et al used capillary, but I cannot because thinnest capillary walls for 100 m ID = 64 mheight of most homogeneous part of microstrip B1 fieldPTFE is millable via computer numerically controlled milling (CNC); mill is manufactured by Roland, U.K.

Vendor of Roland: http://www.sourcegraphics.com/ 3D printers Roland ASD

eBay for new Roland mill, but no returns:http://cgi.ebay.com/Roland-MDX-15-CNC-Mill-3D-Scanner-NEW-in-Box_W0QQitemZ370229635969QQcmdZViewItemQQssPageNameZRSS:B:SRCH:US:102#ht_3842wt_732

30Single-cell imaging: no?

These are images of very large cell, the Xenopus oocyte.

Even for a macroscopic cell, the resolution is low , and the individual pixels are distinguishable.

This is the T1 weighted image. Purea, A., Neuberger, T., Webb, A.G., Concepts in Magnetic Resonance Part B: Magnetic Resonance Engineering,, 2004, 1, 7-14.

Can we see this step?Can we see this step?Widdel, F., Boetius, A., Rabus, R. Prokaryotes, 2006, 2, 10281049Another use for hollow-fiber

Butterfield DA, Bhattacharya D, Daunert S, Bachas L. J Membr Sci 2001, 181, 2937Charcosset, C. Biotechnology Advances, 2006, 24, 482492.Bouchard, L., Burt, S.R., Anwar, M.S., Kovtunov, K.V., Koptyug, I.V., Pines, A. Science 2008, 319, 442-445. Binding enzymes to membranes for high-throughput catalysis can be random (A) B is far more desirable

Characterization of true bound orientation is laborious, via enzyme kinetics assays

Microfluidic MRI can show B / A ratio and other orientation details, just as done for hydrogenation, in a single experiment.

This is also a good way to characterize site-specifically membrane-immobilized catalytic enzyme bioreactors (cell-free):

33Monitor growth inside tube

Linton, E. A., Higton, G.. Knowles, C. J., and Bunch, A. W. Enzyme and Microbial Technology, 1989, 11, 238-288.Hydrostatic pressure increases as growth blockspores in tube walls;

Pressure transducer at inlet can measure this.

Flow rate of growth medium is 10 mL/h in shell space (outside of hollow-fibers).34Growth inside tube affects productivity

Total protein, including fiber walls, lumen, and outside of fibers.Protein on inside surfaces of fiber walls only.Pressure indicates when to switch to maintenance medium.Fiber walls will not retain original pore size when pressure > 1psiBuy transducer and build this feature only if overgrowth becomes a problem. Is there a transducer sensitive enough for one fiber? This study used a nine ROMICON PM100 fibers, length 15 cm.35Immobilized cells grow more densely

Pore size = 100 mCuture time = 30 days

Polymer disk dimensions: 2.3 cm diameter, 50 mm height

I do not have an easy wayof observing these cells viaNMR; they are stuck.

Polymer is a polystyrene developed by the authors, prepared from monomers and Span 80 surfactant in 220 mL water at RT with three orthogonal mixing paddlesThis is one example of many immobilization methods. The hollow-fiber membrane method has seen lowest residence time for E. coli enzymatic activity

Wang, S.-J., Zhong, J.-J., Bioreactor Engineering, Bioprocessing for Value-Added Products From Renewable Resources: New Technologies and Applications, edited by Yang, S.-T., San Diego: Elsevier, 2007, pp. 131-162.

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