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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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
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
Pyridine-2,6-(bis)thiocarboxylate (PDTC)
Secreted biomolecule identified by Lee et al. (1999)
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.
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)
Full-scale
demonstration
Schoolcraft, Michigan
Nitrate and carbon tetrachloride-contaminated aquifer
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.
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.
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)
Weekly alkalinity addition prepared the site for bioaugmentation with strain KC.
pH along Transect A
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)
Downstream degradation of carbon tetrachloride
Appearance of the biocurtain
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.
Strain KC on sediments – Days 336-342
…but as pH increased downstream of the injection
well gallery, KC was detected downstream.
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
• 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.