Microreactor and probe chip for propane-eaters

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Microreactor and probe chip for propane-eaters. Glen Summer 2009. Aerobes that eat propane. Rhodococcus rhodochrous PNKb1 ATCC 21197, 21198, $240, identified by vendor as propane-eater Nocardia paraffinicum 3.0 mL cell suspension  11 μ mol n- propanol in 40 minutes - PowerPoint PPT Presentation

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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 122JBATTS@IDEXCORP.COM

Loctite UV-activated 3105 glue 800-LOCTITEhttp://65.213.72.112/tds5/docs/3105-E