bioaugmentation experiments with pseudomonas stutzeri kc...
TRANSCRIPT
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National Academies Committee
“Future Biotechnology Products and Opportunities to Enhance Capabilities of the Biotechnology Regulatory System”
San Francisco, June 27, 2016
Bioaugmentation experiments with
Pseudomonas stutzeri KC:
lessons learned and new insights
Craig Criddle
Holly Sewell
Stanford University
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Pseudomonas stutzeri KC
Aquifer isolate discovered by serendipity (Criddle et al., 1990).
pH 8. Injected into an aquifer in Schoolcraft, MI, in 1996 &
1998.
Denitrifying bacterium ( NO3- NO2
- N2O N2 )
Secretes a biomolecule that provides pathway control:
degrades carbon tetrachloride without chloroform production.
Motile rod chemotactic toward nitrate
Criddle et al, 1990. Applied Eviron Microbiol. 56 (11): 3240-3246.
Witt et al, 1999.. Environ. Science and Technol.33: 2958-2964.
Hyndman et al., 2000. Ground Water 38(3): 462-474.
Dybas et al., 1998. Environ. Science and Technol.32 (22): 3598-3611.
Dybas et al. 2002. Envir. Sci. Technol. 36: 3635-3644
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Pyridine-2,6-(bis)thiocarboxylate (PDTC)
Secreted biomolecule identified by Lee et al. (1999)
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P. stutzeri KC
CTN1 spontaneously lost this ~148 kbp fragment
Genes for PDTC production were independently identified
by Lewis et al. (2000) and Sepulveda-Torres et al. (1999)
The KC genome was recently sequenced; now under analysis
Lewis et al. reported that a KC
mutant (CTN1 ) spontaneously
lost the ability to degrade CT and
produce PDTC.
The lost ~148 kbp fragment
contained the pdt genes.
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Slightly
alkaline
conditions
(pH ~8)
Decreased
availability
of Fe
CO2 + nonvolatile products
e-
Cu
Activation of fur
Transcription of
pdt locus
Translation of
mRNA to produce
protein products
Synthesis of PDTC
Export of PDTC
Cu binding to PDTC
PDTC
fur
pdt
1
2
3
4
5
6
7
Cu
8
Cu*
PDTC-Cu
activation
(reduction)
9
Actively
respiring
cells
containing
electron
transport
chains
Pseudomonas
stutzeri KC
CCl4 10
Current understanding of mechanism (Criddle et al., 2013)
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Full-scale
demonstration
Schoolcraft, Michigan
Nitrate and carbon tetrachloride-contaminated aquifer
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At pH 8, KC was favored over native Schoolcraft
microflora under denitrifying conditions.
Criddle et al., 2013. Chapter 9 in: Bioaugmentation for Remediation.
Editors: H. F. Stroo, A. Leeson, and C. H. Ward. SERDP and ESTCP
Remediation Technology Monograph Series. Series Editor: C. Herb
Ward, Springer Science + Business Media, New York, p. 257-285.
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KC only wins in the first step of denitrification.
KC wins Indigenous microflora win
Criddle et al., 2013. Chapter 9 in: Bioaugmentation for Remediation.
Editors: H. F. Stroo, A. Leeson, and C. H. Ward. SERDP and ESTCP
Remediation Technology Monograph Series. Series Editor: C. Herb
Ward, Springer Science + Business Media, New York, p. 257-285.
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Equilibration tankRecirculat ion
pump
Nutrient sBase
Static mixer
Injection lines
50 ‘
Ferment ers for inoculum
Extraction wells
pumps
• Closely-spaced wells (1 m apart) installed
•Weekly base addition
•Inoculation
•Weekly addition of base plus acetate.
Schoolcraft Bioaugmentation Tests (1998-2002)
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Weekly alkalinity addition prepared the site for bioaugmentation with strain KC.
pH along Transect A
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Starter culture:
50 gallons
grown on
nutrient broth
Steam-sterilized
tanks aerated
with filter-
sterilized air
Growth in filter-
sterilized
groundwater,
supplemented
with acetate,
phosphate
Colony on
agar plate Shake flask
Pressure monitoring at well
heads during injection
Inoculation (Day 117)
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Downstream degradation of carbon tetrachloride
Appearance of the biocurtain
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Strain KC on sediments – Day 152
Distance from gallery
Depth (m)
Strain KC (cfu/gram)
Aquifer flora (cfu/gram)
0.3 meter 13.7 1.35 x 106 1.6 x 10 6
19.8 2.5 x 105 2.1 x 10 6
22.8 1.1 x 106 3.9 x 10 6
1 meter 13.7 0 2.5 x 10 4
19.8 0 1.8 x 10 5 22.8 0 8.5 x 10 4
2 meters 13.7 0 9.0 x 10 4
19.8 0 1.0 x 10 5 22.8 0 1.4 x 10 5
Strain KC was initially only detected in a 0.3 m
strip close to the injection well gallery.
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Strain KC on sediments – Days 336-342
…but as pH increased downstream of the injection
well gallery, KC was detected downstream.
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We now know that KC possesses an
integrative and Conjugative Element (ICE): Mobile chromosomal elements that have the ability to be
transferred between cells by conjugation like a plasmid
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Integrative and Conjugative Element (ICE):
n
attL attR Integration & Excision Mobility Accessory
pdt genes
Addiction Toxin/Antitoxin
Module
Direct Repeat
Target Gene: tRNA-pro
Holly Sewell
ICE found in P. stutzeri strains. Accessory modules identified:
• KC – pdt genes • SLG510A3-8 - Cu resistance • 19SMN4
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• Application of strong selection pressures (pH adjustment to 8) constrained the organism, locking it into a specific location.
• KC is an example of natural genetic engineering involving horizontal gene transfer of an ICE. Such elements could potentially escape constraints that limit spread of strain KC.
Lessons and questions