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Glen Summer 2009. Aerobes that eat propane Rhodococcus rhodochrous PNKb1 ATCC 21197, 21198, $240, identified by vendor as propane-eater Nocardia paraffinicum.

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Presentation on theme: "Glen Summer 2009. Aerobes that eat propane Rhodococcus rhodochrous PNKb1 ATCC 21197, 21198, $240, identified by vendor as propane-eater Nocardia paraffinicum."— Presentation transcript:

1 Glen Summer 2009

2 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 μmol n-propanol in 40 minutes Arthrobacter CRL-60 Pseudomonas fluorescens Brevibacterium butanicum Mycobacterium austroafricanum ATCC $195, identified by vendor as propane-eater Mixed culture ATCC $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,

3 Anaerobes 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 C 3 H SO H +  5 H 2 S + 2 HCO mM H 2 S 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, 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.

4 Anaerobic mechanism proposed Hiro, F. J. Phys. Chem. B, 2002, 106, Widdel, F., Boetius, A., Rabus, R. Prokaryotes, 2006, 2, 1028–1049. Becker, A., Fritz-Wolf, K., Kabsch, J., Knappe, S., Schulz, S., Wager, A.F.V. Nature Struct. Biol., 1999, 6, Can we see this step? isopropylsuccinate Enzyme mech based on a well-used motif Enzymes that use the glycyl radical mechamism are difficult to isolate: They need another enzyme to get activated Exposure to O2 will break the peptide bond between Gly and Cys

5 Attack on primary carbon proposed During the 50 days of propane consumption, some formation of n-propylsuccinate occurs. n-propylsuccinate Kniemeyer, O. et al. Nature, 2007, 449, This step was newly proposed in It happens for only about 1/3 of the propane oxidation reactions Primary C-H bond cleavage is more difficult than secondary.

6 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,

7 Planktonic anaerobes, anchored aerobes  Anaerobes: Kniemeyer et al. used planktonic Desulfotomaculum  But 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 days  Slow 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 anchored  Propane 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 detection  Anchoring to membrane with ~0.1 μm pores allows controlled flow of nutrients, waste

8 Planktonic microbioreactor Six microbioreactors on one PDMS-on-glass chip Bacteria in a constantly flow in a loop to prevent biofilm formation Each flow loop has nine segments Each segment is individually isolated for cleaning during the experiment Lysis buffer is pumped from on-chip well to kill and to detach biofilm cells Extra growth medium is pumped from on-chip well to re-dilute cells for control of population size. Balagaddé, F.K.,You, L., Hansen, C.L., Arnold, F.H., Quake, S.R. Science, 2005, 309,

9 Make the Soft Lithography Master 1. Design and print mask. 2. Spin SU-9 photoresist to Si wafer 3. Expose wafer through mask, develop and bake photoresist 4. The wafer is now the master template. Resolution ~ 20 μm McDonald, J.C., Duffy, D.C., Anderson, D.T., Chiu, D.T., Wu, H., Schueller, O.J.A., Whitesides, G.M. Electrophoresis, 2000, 21,

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

11 Make two layers Top PDMS layer for air channels Air pressure ≈ +5 psi PDMS 4mm thick for channel stability Bottom PDMS layer for fluid channels PDMS 30 μm thick via spin-coat onto master, 2000 rpm, 30 s. Channels 100 μm wide x 9 μm high Valve = intersection of fluid and air channels Unger, M.A., Chou, H-P., Thorsen, T., Scherer, A., Quake, S.R. Science, 2000, 288, Thorsen, T., Maerkl, S.J., Quake, S.R. Science, 2005, 298, McDonald, J.C., Duffy, D.C., Anderson, D.T., Chiu, D.T., Wu, H., Schueller, O.J.A., Whitesides, G.M. Electrophoresis, 2000, 21, 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 $ from local vendor RS Hughes (Pacoima, CA) Peristaltic pump made of PDMS channel intersections

12 Add a drainage capillary for NMR detection In lieu of six separate reactors, use only two. Use extra space on chip for PDMS peristaltic pump Pump cell suspension segment at a time to NMR detection device Connect chip to NMR detector via 100-μm capillary NMR detection device Fused silica capillary

13 Immobilization: denser cell growth Membrane Flat membrane Hollow-fiber membrane Free microcarriers Commercial designer particles Packed bed Foam Polymer Gel Beads Raschig ring Coke Activated carbon 17 mL

14 Hollow-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

15 Separation of (g), (l) nutrients possible 0.33 mm ID 0.66 mm OD μm pores 1.47 mm ID 1.96 mm OD 8 mm ID Chung, B.H., Chang, H.N., Kim, I.H. Enzyme Microb. Technol., 1987, 9,

16 1.3 cm OD microbioreactor 15 cm 1/16 “ 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 ¼” tubing. 9 mm OD

17 Optical microscopic observation to check for escaping cells (cellular attachment) Glass plate, 1” x 5” 5” length allows glass to overhang microscope stage on both sides PDMS sandwiched in the middle here, in the shape of one microfluidic channel, 100 μm wide, 10 μm deep. 360 μm holes for metabolite throughput: connections to 1/16” ID peristaltic pump tubing made by Nanoport PEEK connectors (IDEX Corp) This hole on top plate This hole on bottom plate Output goes to NMR

18 Detachment of bacteria from bioreactor Johansen, C., Falholt, P., Gram, L.,. Applied Environmental Microbiology, 1997, 63, 3724– 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 α- glucosidase, β-galactosidase. Commercial enzyme cocktail Pectinex Ultra SP-L works, too, but may be more expensive. NMR detection device

19 Strip of Cu instead of coil 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, A Dutch group uses a strip of copper instead of a coil to receive and to send rf signals. Their 1 H frequency is 600 MHz. Thin strip part of Cu is 35 μm thick, 1 mm long, 500 μm wide, etched out of the Cu foil coating the Rogers 5870 PTFE material. B1 homogeneity for pulses perpendicular to B 0 field is shown in yellow in this Maxwell equations simulation: Microfluidic channels may run here (axial view)

20 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, http ://

21 Double-resonance stripline probe van Bentum, P.J.M., et al. Journal of Magnetic Resonance, 2007, 189, 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, microreactor Stripline is easier to build than coil (sputter Cu or anodically bond Cu strip) Tune, match via dielectric plunger

22 5-mm microcoil versus stripline Human cerebrospinal fluid using 256 scans, 6-μs 90 ⁰ pulsewidth on 5mm commercial microcoil Human cerebrospinal fluid using 4608 scans, 7-μs 90 ⁰ pulsewidth on stripline Bart, J., Kolkman, et al. J. Am. Chem. Soc. 2009, 132, 14,

23 Ways to improve sensitivity Bart, J., Kolkman, et al. J. Am. Chem. Soc. 2009, 132, 14, online supplement Knapkiewicz, P., Walczak, R., Dziuban, J.A. Optica Applicata, 2007, 37, Olson, D.L, Peck, T.L., Webb, A.G., Magin, R.L., Sweedler, J.V. Science, 1995, 270, Use nonconductive substrate instead of Si. Nanolab has anodic bonding machine Forturan glass: bond at 250⁰C, under 2kV Flow two microfluidic channels along length of stripline (one on each face of strip) vs. Forturan glass Si

24 Rogers 5870 can be ordered as a PTFE sandwiched between two copper foil layers (35 μm) A piece of Rogers 5870 with copper foil cladding

25 Mill channel into dielectric surface (fluoropolymer): 100 μm wide, 10 μm deep Thinnest capillary wall for 100μm ID = 64 μm; Height of homogeneous B 1 field of stripline ≈ 50 μm Therefore, sample must flow into shallow channel in dielectric. Gershenfeld et al used capillary, but did not show homogeneity of B 1 Bentum et al used channel, and showed limited homogeneity height

26 Copper ground planes Copper to become the planar NMR rf strip PTFE to become the microfluidic channel bed Etch away copper on portions of one piece, for microfluidic channel

27 Mill a channel: 100 μm wide, 10 μm 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 μm 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 nanoliters

28 Copper ground planes Copper to become the planar NMR rf strip PTFE to become the microfluidic channel bed Shorter piece of Rogers 5870 Longer piece of Rogers 5870

29 Mill channel into dielectric surface (fluoropolymer): 100 μm wide, 10 μm deep Thinnest capillary wall for 100μm ID = 64 μm; Height of homogeneous B 1 field of stripline ≈ 50 μm Therefore, sample must flow into shallow channel in dielectric. Channel volume on order of tens of nanoliters Affix planktonic microbioreactor here. Connect to NMR sample channel via capillary tubing

30 Affix Quaker-design planktonic microbioreactor onto dielectric, and cap NMR sample channel Affix planktonic microbioreactor here. Connect to NMR sample channel via capillary tubing Stripline-bearing Rogers 5780 piece will form lid to this channel. Use PTFE adhesive on PTFE parts only, and press hard.

31 Single-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.

32 Can we see this step? Widdel, F., Boetius, A., Rabus, R. Prokaryotes, 2006, 2, 1028–1049

33 Another use for hollow-fiber Butterfield DA, Bhattacharya D, Daunert S, Bachas L. J Membr Sci 2001, 181, 29–37 Charcosset, C. Biotechnology Advances, 2006, 24, 482–492. Bouchard, L., Burt, S.R., Anwar, M.S., Kovtunov, K.V., Koptyug, I.V., Pines, A. Science 2008, 319, 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.

34 Monitor growth inside tube Linton, E. A., Higton, G.. Knowles, C. J., and Bunch, A. W. Enzyme and Microbial Technology, 1989, 11, Hydrostatic pressure increases as growth blocks pores in tube walls; Pressure transducer at inlet can measure this.

35 Growth 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 > 1psi

36 Immobilized cells grow more densely Pore size = 100 μm Cuture time = 30 days Polymer disk dimensions: 2.3 cm diameter, 50 mm height I do not have an easy way of observing these cells via NMR; 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 paddles


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