Transcript of Glen Summer 2009. Aerobes that eat propane Rhodococcus rhodochrous PNKb1 ATCC 21197, 21198, $240,...
- Slide 1
- Glen Summer 2009
- Slide 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 mol n-propanol in 40 minutes
Arthrobacter CRL-60 Pseudomonas fluorescens Brevibacterium
butanicum Mycobacterium austroafricanum ATCC 29678 $195, identified
by vendor as propane-eater Mixed culture ATCC 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
- Slide 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 8 + 5 SO 4 2 - + 4 H + 5 H 2 S + 2 HCO 3 - 12 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, 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.
- Slide 4
- Anaerobic 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? 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
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- 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, 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.
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- 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
- Slide 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 m pores allows
controlled flow of nutrients, waste
- Slide 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. Balagadd, F.K.,You,
L., Hansen, C.L., Arnold, F.H., Quake, S.R. Science, 2005, 309,
137-140.
- Slide 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 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.
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- 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,
- Slide 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 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
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- 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-m capillary
NMR detection device Fused silica capillary
- Slide 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
- Slide 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
- Slide 15
- Separation of (g), (l) nutrients possible 0.33 mm ID 0.66 mm OD
0.4-0.6 m 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, 344-349.
- Slide 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
tubing. 9 mm OD
- Slide 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 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 plate This hole on bottom plate Output
goes to NMR
- Slide 18
- Detachment 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 device
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- 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, 104-113. 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 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 B 0 field is shown in yellow in this Maxwell equations
simulation: Microfluidic channels may run here (axial view)
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- 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
- Slide 21
- 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. microreactor Stripline is
easier to build than coil (sputter Cu or anodically bond Cu strip)
Tune, match via dielectric plunger
- Slide 22
- 5-mm microcoil versus stripline Human cerebrospinal fluid using
256 scans, 6-s 90 pulsewidth on 5mm commercial microcoil Human
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.
- Slide 23
- Ways to improve sensitivity Bart, J., Kolkman, et al. J. Am.
Chem. Soc. 2009, 132, 14, 5014-5015 online supplement Knapkiewicz,
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 machine Forturan glass: bond at
250C, under 2kV Flow two microfluidic channels along length of
stripline (one on each face of strip) vs. Forturan glass Si
- Slide 24
- Rogers 5870 can be ordered as a PTFE sandwiched between two
copper foil layers (35 m) A piece of Rogers 5870 with copper foil
cladding
- Slide 25
- Mill channel into dielectric surface (fluoropolymer): 100 m
wide, 10 m deep Thinnest capillary wall for 100m ID = 64 m; Height
of homogeneous B 1 field of stripline 50 m 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
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- 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
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- 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
nanoliters
- Slide 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
- Slide 29
- Mill channel into dielectric surface (fluoropolymer): 100 m
wide, 10 m deep Thinnest capillary wall for 100m ID = 64 m; Height
of homogeneous B 1 field of stripline 50 m 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
- Slide 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.
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- 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.
- Slide 32
- Can we see this step? Widdel, F., Boetius, A., Rabus, R.
Prokaryotes, 2006, 2, 10281049
- Slide 33
- Another use for hollow-fiber Butterfield DA, Bhattacharya D,
Daunert S, Bachas L. J Membr Sci 2001, 181, 2937 Charcosset, 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.
- Slide 34
- Monitor 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 blocks pores in
tube walls; Pressure transducer at inlet can measure this.
- Slide 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
- Slide 36
- Immobilized cells grow more densely Pore size = 100 m 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