Aerobic
and Anaerobic
Estexified
Fav
Acids
Environments
Biodegradation
of Soy Diesel
John F. Stok’*, Paige Follisl, Gianni noro’,
BuzzelE’, and W. Michael Griffin’+
of the Methyl
in Freshwater
and
Robert
DOnofiiO',
lDepamnent of Biological Sciences, Duquesne University,
PA 15282-1502 U.S.A.
Joy
Pittsburgh
%ational E~~vironmental
Technologies Assessment Center, 615
William Pitt Way, Pittsburgh PA, 15238 U.S.A.
*corresponding author
*current address: Griffin Technical Services, 439 Monmouth
Cranberry, PA 16066
email: StolzFduq3 .cc.duq.edu
Phone
(412) 396-6333
Faiv
(412) 396-5907
Soil
Dr.,
Aerobic
and Anaerobic
Biodegradation
Freshwater
and Soil Environments
of Soy
Dies&
in
John F. Stoiz’, Paige Folk’, Joy Buzzellil, Gianni Flora’, ROM
Donotio’, and W. Michael Griffin’
‘Deparment
of Biological Sciences, Duquesne University,
PA 15282-1502
U.S.A.
Pimburgh
‘National Environmental Technologies Assessmem Cemer, 6 15
William Pitt Way, Pittsburgh PA, 15238 U.S..4.
email: Stolz@duq3 .cc.duq.edu
Abstract
Soy diesel
is composed
long
fatty-acids
chain
demonstrated
under
test
in
otic
and
analysis.
of a mixture
(e-g,,
both
freshwater
and
anoxic
conditions
as determined
Bacterial
populations
of magnitude
carbon.
Soy diesel
was completely
aerobic
flasks
by
seain
using
and
by
GSOY,
using
soy diesel
oxygen
(GSOY)
or nitrate
revealed
Phylogenetic
that
Proteobacteria
Burkholden~a
as the
the
and
analysis
two
smains
related
by flask
by
4 to 5
sole
source
by
in anaerobic
7 days
flasks,
An aerobic
bacterium,
as the
were
to tie
members
common
cepacia and Burholderia
as
were
of carbon
with
elecmon
gene
of the
soil
sequence
p
bacteria
solanacearum
2
in
Hl,
terminal
of 16s rIWA
of
bacterium,
strain
as the sole source
(Hl)
was
environments
degraded
a denitn’cfying
isolated
acceptor.
soy diesel
14 days
soil
increased
gas chromatography.
and
esterified
C 18-C 20). Its biodegradation
orders
determined
of methyl
respectively.
In subsequent
experiments,
GSOY, B. cepacia
25416) and B, sokmaceamm (seain AWlQA) were
shown to express an inducible esterase and produce
(ATCC
methanol when
grown on soy diesel- Preliminary
evidence
suggests that the fatty acid is then either degraded via poxidation
results
under
or incorporated
demonseate
both
of soil and
that
directly
soy diesel
oxic and anoxic
freshwater.
into phospholipids.
conditions
is readily
These
biodegraded
by the natural
flora
INTRODUCTION
Biodiesel, derived from crop oils such as soybean, rapeseed, and
sunfiower, is composed of small number of methyiated fatty acids.
Current interest centers on its use as an alternative or additive to
petroleum diesel and as a replacement for other petroleum based
products (e.g., Mxicants,
cutting fluids). In trials where biodiesel
was used by itself or in blends with petroleum diesel, CO, particulate,
NOx, sulfur and aromatic emissions were reduced while not
sacrificing engine performance{ 9,19,27,30).
Biodegradation
microorganisms
of hydrocarbons
by natural populations
of
represents one of the primary mechanisms by which
perroleum and diesel products are removed from the environment
(1,2,14,23,34).
however,
Many of the components of petroleum diesel,
are recalcitrant to microbial degradation or are simply
nonbiodegradable(
of hydrocarbons
1,2). Petroleum diesel consists of a complex array
including akanes, branched akanes, cycloalkanes,
and aromatic compounds(3). While many species of microorganisms
can degrade the simple alkane components, other classes of diesel
hydrocarbons
such as branched and aromatic hydrocarbons
susceptible to microbial atta&( 32). This is particularly
are less
true when
oxygen is not available as the terminal electron acceptor. Soy diesel,
or methyl soyate, is a less complex mixture having a limited variety
of stNctures
as compared to petroleum diesel, and is comprised
predominantly of 8 different C&18 fatty acid methyl esters
(FAME). These include methyl esters of oleate, palmitate, stearate,
linoleate, myristate, iso-stearate, laurate, and linolenate( 26).
Preliminary
studies on its toxicity and biodegradability
4
have
suggested that it is less toxic than petroleum diesel and
biodegradabie
information
(26-28). These studies, however, have provided little
on the range of environmental
COnditions
under which
biodiesel can be degraded, the by-products formed, the rates and
pathway of degradation,
and the organisms involved in the process.
We report here the results of our study on the biodegradation
of soy diesel in freshwater and soil environments
anoxic conditions, and the identification
including
under oxic and
of soy degrading bacteria
species which can grow on soy diesel anaerobically
with
nitrate as the terminal electron acceptor. We also discuss the
proposed pathway of degradation based on the current results.
MATERIALS
Aerobic
AND
METHODS
flask tests. Aerobic flask tests were set up in 250
ml Erlenmeyer flasks. Each flask contained 100 ml of sterile minimal
salts media (MS&I, 0.2g MgSOeiH20,0.02g CaClz, 1.0 g KHzPOa I..Og
KzHPO4, Log NH&l,
and 10 ml base mineral mix (24) per liter of
distilled water) with a final pH of 7.0. Test flasks were amended
with 0.1 ml (88mg) of filter-sterilized
EMironmental,
Incorporated,
soy diesel (Interchem
Leawood, KS) and inoculated
with lml
of freshwater sample (Squaw Run Pond or Old Hickory Lake), lgm of
soil sample (top soil), or 2OOd of a 7 day pure culture grown on soy
diesel. Two different controls were used. The first set of controls
(soy diesel controls) included MS&fand soy diesel without inocuhm
in order to determine if there were any abiotic breakdown
of the
methyl esters. The second set of controls (bacterial controls)
included MS&l and the inoculum without soy diesel. This conmol was
used to determin e the amount of growth which occurred as a result
of nutient
Flasks were placed in a gyrotory water bath
carryover
(Model G76, New Brunswick Scientific) or in a gyrotory shaker
(Model GlO, New Brunswick Scientific) at 22-230 C for 14 days. Ah
tests were run in triplicate.
Anaerobic
flask tests. Anaerobic flask tests were set up in
125m.l Wheaton septum top bottles. Each botie contained lOOmI of
the MS&/Iwith KNO3 (Zig/L) and was rendered anoxic by sparging
with oxygen-free
CO2 and N2 (20:80) ( 18). After autoclaving,
the
medium was amended with O.lml of filter sterilized soy diesel and
inoculated with either lg of soil, Lm.l of degassed freshwater sample,
or >, or 2OOd of a 7 day pure culture grown on soy diesel using
anaerobic technique(l8).
Three controls were done. The first
contained MSM with soy diesel but no inoculum (again to test the
stability of the methyl esters), the second was inocuIated MSM
without soy diesel (the bacterial growth control), and the third was
inoculated MSM with soy diesel but without nitrate (NO3 -minus
medium). This last con~ol was done to determine if any growth
occurred without the terminal electron acceptor. In addition, the
production
of nitite
was monitored using the methods from Parsons
et al. (29). Flasks were shaken on a gyrotory shaker (Model GlO, New
Brunswick Scientific) at 200 rpm at 22-230 C for 14 days. All tests
were run in triplicate.
Bacterial
epifluorescence
enumeration.
with auidine
Direct cell counts were done by
orange using the method from Parsons
et aL(29) with a Nikon Microphot SA. Samples were taken in tandem
witi samples for the GC analysis at 0, 7, and 14 days and all counts
were done in tipkate.
GAS chromatography.
Samples were extracted by the
method of Douglas et al.{ 10) using methylene chloride. One microliter
of diluted sample ( MO) was analyzed on a Hewlett Packard 5890
Series II gas chromatograph
equipped with a high-resolution
fused
capillary column (J&W Scientific DB-5, 30 m, 0.25 ID mm, 0.25 mm
film) and a spLit/spIitless injection port. Helium was the carrier gas.
The injection port and detector temperatures where 300°C and
32O”C, respectively.
The oven temperature was maintained
for 4 min followed by a programmed
to 195’C. A three-point
calibration
at 150°C
temperature rise of 2.O“C&in
curve was used to establish the
linear range for analysis and for quantification
of sample analytes.
Sample analyte recovery was corrected for through the use of an
internal
standard, methyl myristate. Although methyl myristate can
be a component of soy diesel, it was only present in trace amounts in
the soy diesel used in these experiments.
Isolation
and culture
of the soy degrading
bacteria
The aerobic soy degrading bacterium, strain GSOY,was isolated by
streak plating using MSM with 1.5% agar and soy diesel as the sole
source of carbon. The anaerobic soy degrading bacterium, HI, was
isolated by serial dilution using MS&l with n&ate and soy diesel as
the sole carbon source. Pure cultures were grown in septum top
tubes(20ml)
containin g 1Oml of sparged MSM with nitrate amended
with 10 ml of soy diesel.
Pure cultures of Burkholdeia
solanaceanun
cepacia (ATCC 25416), B.
(strain AWIQA, kindly provided by Dr. iMark ScheLI,
of Georgia) and Psendomonas okovoms
University
(ATCC 8062)
were maintained on nutrient broth (DISCO.).
Phylogeneric
The 16s rRNA gene sequences for
analysis.
GSOY,HI, and P. oleovorans were obtained using PCR amplification
with forward and reverse sequencing ptiers
( 15,21) and have been
deposited in Genbank (assention numbers U16142, U16144, and
uXXXX.K respectively). Additional
sequences were obtained directly
from Genbank and the Ribosomal Database Project (22). Sequences
were aligned using the Clustal W program (17) and phylogenetic
analysis done using a version
of PHYLIP (maxium parsimony,lZ)
and
MEGA (neighbor joining, 20). Evolutionary distances were estimated
with the correction of Jukes and Cantor from 1200 bases. Bootstrap
numbers are based on 1,000 trees.
Esterase
activity.
Esterase activity was detected using the
methods in Shum and Markovitz
(33). C&s from flask tests or pure
cultures grown on soy diesel were harvested by centigation
(5,000 x g, 20 min), lysed by sonication (three 30 set bursts with a
Braunsonic Ultrasonicator),
(supernatant)
and fractionated into cytoplasmic
and membrane (pellet) fractions by centigation
at
200,000 x g for 1 hr. Membrane fkacuons were run on native
polyaaylamide
gels (8% resolving gel, 4% stacking gel, 1% CHAPS
detergent) and then stained with a naphthyi acetate and fast blue RR.
Control cultures were grown on nuuient broth or MSivI with oleic
acid (lg/L)
and harvested
as described
above.
Methanol detection. The presumptive analysis of
methanol was determined with Draeger tubes (Fisher Scienufic) .
Flasks were capped with a polypropylene
stopper and evacuated
8
through the Draeger tube at 2Opsi for 2min. Crimp top Wheaton
bottles were measured in the same manner through the septum . The
results were determined
using the methanol Draeger scale using the
relative values: -=0 (none detected), + -300, ++ =500-2000, and
*>2m.
RESULTS
Environmental
Samples.
of bacterial populations
Soy diesel stimulated
the growth
in freshwater and soil flask tests boti under
oxic and anoxic conditions. Initial studies on population
growth done
under otic conditions over 21 days using a freshwater inocuhun
showed that after an initial lag phase of about 2 to 4 days, the
populations
grew exponentially
up until 7 days and then maintained
stationary phase (Figure 1). Subsequently, samples for bacterial
counts and GC analysis were taken at 0, 7, and 14 days.
Flask test analyses using freshwater inoculurn showed the
concomitant growth of microbial populations with the disappearance
of the fatty acid methyl esters (FAMES) of soy diesel (Figures 2A and
3A). Bacterial populations
increased from 104 to 1010 cells/ml for
Old Hickory Lake samples (Figure 2A) and from 105 to 109 cells/ml
for Squaw Run Pond samples in the aerobic flask tests (Figure 3A).
Control flask tests which contained only MS&l and inoculum
increased only two orders of magnitude. Bacterial populations
increased from 105 to 109 cells/ml for Old Hickory&&e
samples and
from 105 to 1010 cells/ml for Squaw Run Pond samples in the
anaerobic flasks that had been amended with nitrate (Figures 2B and
3B respectively).
Again, the population
in control flasks not amended
with soy diesel increased less than two orders of magnitude.
9
Anaerobic flask tests using MSM prepared without the addition of
KNO3 had bacterial counts no greater than controls (2.8 x 10’
cdls/mI vs. 2.1 x 10’ ceIIs/ml for Old Hickory Lake, 2.7 x 10” cells/ml
vs. 2.1 x lo5 ceils/ml for Squaw Run Pond).
FAMES were reduced to undetectable levels by seven days in
the aerobic freshwater (Figures 2 and 3) and soil flask tests (Figure
4). Appreciable
degradation occu.rred by day 7 with complete
disappearance
by day 14 in anaerobic freshwater and soil flasks.
There also was a noticeable loss of FAME after 14 days in the sterile
controls (Figures 2,3 and 4) but no increase in microbial population
(Figures 2 and 3).
Soy degrading
isolates. Nine isolates of aerobic soy
degrading bacteria were obtained from Old Hickory Lake
enrichments. All nine strains were identified as strains of
Bu.rMzuMe&
cepada based on physiological characteristics (Rapid
ID). One strain, GSOY,was selected for 16s rRNA sequence analysis.
Four strains of soy degrading bacteria were isolated from Old
Hickory Lake samples using anaerobic conditions with nitrate as the
terminal elecmon acceptor. They were shown to also grow aembidly
on soy diesel, but were not further characterized biochemically.
One
smin, HI, was chosen for 16s rRNA sequence tiysis.
Phylogenic
analysis. Phylogenetic
analysis with maximum
parsimony and neighbor joining methods using the 16s rRNA
sequence revealed that both GSOYand Hl were members of the p
sub&s of the Proteobacteria (Figure 5). GSOYwas most closdy
related to B. cepada and B. gladioli, while HI was most closely
related to the plant pathogen B. sdanaceamm
and B. pickettii (Table
10
1). Our analyses have also shown that the original peW0leu.m
degrading bacterium, Pseudomonas okovorans, is a -{
Proteobacterium
most closely related to P. mendocina (Figure 5).
Soy diesel degradation
in pure cultures. Strains
of
Burkholden’a cepacia (GSOY and ATCC 25416) and E. solanaceanun
(strain AWMA) were tested for their ability to grow on soy diesel.
Populations of GSOYincreased from lo4 cells/ml to 10’ cells/ml in 14
days with the concomitant
Populations of B. so&aceincreased from 10’ cells/ml
complete utilization
disappearance of FAMES (Figure 6).
grown under anoxic conditions
to lo6 cells/ml in 14 days also with the
of FAMB and production of nitrite (Figure 7).
Membrane fractions of all srrains tested showed that when the
organisms were grown on soy diesel, they expressed an inducible
esterase (Figure 8). It was not expressed when the cells were grown
on either nutrient broth (Figure 8) or MS&i with oleate (data not
shown). Furthermore, methanol was detected in only cultures fed soy
diesel (Table 2).
DISCUSSION
The data presented here show that soy dies4 can support the
growth of bacteria with concomitant disappearance of FA8Es. It can
be degraded under both oxic and anoxic conditions by the microbial
populations
found in freshwater and soil environments.
An extended
lag phase was observed in all experiments using natural samples or
pure cultures
(Figure
which
had not previously
1). This lag phase was eliminated
been exposed
when samples
to soy diesel
were
preadapted with soy diesel (e.g., 7 day enrichments or pure cultures)
suggesting that the organisms don’t grow
on
the soy diesel until the
esterase is expressed..
Phylogenetic analysis based on 16s rRNA sequence has
revealed interesting relationships
in the soy degrading bacteria. The
anaerobic soy degrading bacterium main HI is closely related to the
plant pathogen B. solanaceannn, while GSOYis closely related to B.
cepacia and B. gladioli also plant pathogens. These remits initially
suggested that the soy degrading bacteria might be a closely related
phyiogenetic group (e.g., confined to the p Proteobactexia). However,
examination
of other species including Acinetobacter Iwoffti,
Pseudomonas oleovorans, and a marine isolate (Pseudomonas sp.
srrain Marsoy) show this is not the case. Rather, the presence of an
inducible esterase is the defining characteristic of a FAME degrader.
Our results also have begun to elucidate the pathway of soy
diesel degradation.
by an inducible
The first step is the cleavage of the methyl group
esterase. An inducible esterase has been.detected in
all speties we have examined which can grow on soy diesel (GSOY, B.
cepacia ATCC 25416, B. solanacearum SIX&I AWl0A, Acinetobacter
lwoffi strain RAG-l). This was not suprising given that Pseudomonas
sp. and B. cepacea had previously been shown to degrade short
chain FAMES to the fatty acid and methanol (3 1,33) and esterase
activity had been detected in B. solanaceanun (7).
Once the free fatty acids are formed, they may enter
intermediary
oxidation(
metaboI.ism
to be oxidized
to acetate via p-
3 2) or be directly incorporated into phospholipids.
experiments
Labeling
using 14C-labeled oleic acid revealed the presence of
labeled phospholipid
in both GSOYand B. cepacia (M. Davis, personal
12
communication). Furthermore, the cells accumulate conspicuous
InEacellular inclusions suggestive of poly-&hydroxyalkanoates
or
unmetabolized
provided
FAME ( 16,3 2). These inclusions could apparently
substrate for growth as growth occurred in control flasks
without soy diesel when washed cell suspensions of preadapted
cultures were used (Figure 7).
The biodegradation
of FAME3 under anoxic conditions also
has precedent. Eubacterium limosum and ,4cerobacterizm woodii
have been shown to anaerobically metabolize methyl esters of
acetate and proprionate with CO2 and H2 (25). Our results show that
B. solanaceanzm is able to grow anaerobically with soy diesel as the
sole source of carbon with nitrate as the terminal electron acceptor.
It expresses the inducible esterase under these conditions producing
both methanol and nitrite. This result also suggests that other
electron acceptors such as Fe@) and sulfate may be suitable so long
as the species has an inducible esterase.
It is es-ted
that 3.3 million barrels of petroleum
are consumed daily for transportation,
industrial
diesel
residential, commercial, and
uses (24). Petroleum diesel is involved on average in 21%
of all spill incidents ( 11). Although petroleum diesel is degradable
under oxic conditions, it is recalcitrant to deosdation
under
anaerobic conditions (45). Thus, the environmental impact of a
biodiesel spill, as a result of transportation, use, and storage, should
be far less than petroleum diesel especially
under anoxic
conditions.
However, the ability of microbes to uBlize soy diesel under anoxic
conditions with alternative electron acceptors such as N03, may
impact its long term stability in pipelines and storage tanks.
ACKNOWLEDGEMENTS
We acknowledge
the Ribosomal Data Base Project and James Garey
for assistance in sequence comparisons. The work was supported
under a cooperative
agreement with the USDA.
REFERENCES
1. Atlas, R.M. 1981. Microbial degradation
hydrocarbons:
an environmental
of petroleum
perspective. Microbid.
Revs.
45:180-209.
2. Atlas, RM. 1990. Microbial hydrocarbon
bioremediation
degradation-
of oil spills. J. CTltem.Tech.Biotechol. 52149-156.
3. FmycIopedia of Chemical Technology, 3rd ed., vol. 17, 119130 (John Wiley & Sons, 1982).
4. Bartha, R and L Boss-
1984. The treament
and disposal of
pemoleum wastes. In, RM. Atlas (ed), Peuoleum Microbiology,
553-578, Maanillan
5. Boss-
p.
Publishing, New York.
L and R Bartha
1984. The fate of petroleum
ecosystems. In, R.M. Atlas (ed.), Petroleum Microbiology,
in soil
p. 434-476,
Macmillan Publishing, New York.
6. Belch, W.& G.E. Fox, L.J. Magrum, C-R Woese, and RS.
Wolfe. 1979 Methanogens:
group. Microbial.
7. Baptist, JX,
reevaluation
of a unique biological
Revs. 43:260-296.
CJt
zone electrophoresis
Shaw, and M. Mandel. 1971. Comparative
of enzymesof Pseudomonassohaceanzm and
Pseudomonas cepacia. J. Bat. 108:799-803.
9.
J. and K Fouhy. 1993. Vegetable oils: from table
Chowdhury,
to gas tank. them. fig.
10. Douglas,
February,3 S-39.
G.S., K.J. McCarthy,
Stehhauer,
D-T. Dahkn,
RC. Prince, and D.L Ehendorf.
hydrocarbon
analyses for environmental
JA
Seavy, W.G.
1992. The use of
assessment and
remediation, p.l-20. In, Kostecki, P.T. and E.J. Calabrese (ed.),
Contaminated
soikdiesel
fuel contamination
p.l-20, Lewis
Publishers, Chelsea, Michigan.
11. Emergency
Response Notification
12. Fekenstein,
J. 1988. Phylogenies from molecular
inference and reliability.
Service.
1991.
EPA.
sequences:
AMU. Rev. Genet. 22521-526.
14. Floodgate, G. 1984. Ln R.M. Atlas (ed.), Petroleum Microbiology,
p. 355398,
Macmillan
15. Giovannoni,
Publishing, New York.
S. 1991. Polymerase Chain Reaction Ln E.
Stackebrandt and M. Goodfellow (ed.) Nucleic Acid Techniques
. in
Bacterial Systexnatics, p. 177-204, J. Wiley and Sons, New York.
16. Griffin, W.M. and RW. Traxler.
of an n-akane
1983. Intracellular
grown A&x=obacrer. Dev. Indust. Microbial.
inclusions
24:389-
400.
17. Higgins, D. G. and P.M. Sharp. 1989. Fast and sensitive
multiple sequence alignments on a miuocomputer.
CABIOS 515 l-
1.53.
18. Hungate, R.E. 1969. A roll tube method for cultivation
anaerobes.
Methods Mcrobiol.
3B:117-132.
15
of stict
19.
Isigigiir,
k,
HamduIlahpw,
Turkish
F. Karaosmanoglu,
and 6L
origin
Giilder.
H.A. Moy,
1993.
F.
Safflower
seed oil of
Appl. Biockm. BiotechoL
as a diesel fuel alternative.
40/41:89-105.
20. Kumar,
S., K. Tamura,
and M. Nei. 1993. MEGA:
Evolutionary
Genetics Analysis,
Evolutions
Genetics,
version
LO. Institute
The Pennsylvania
Molecular
of Molecular
State University,
University
Park, PA 16802.
21. Lane, DJ. 1991. 16s/23s
and M. GoodfeUow
p. 115-176,
22. Larson,
N. G-J. Olsen,
T. Macke,
data base project.
23.
Leahy,
low-sulfur
B.L. Maidak,
T.L. Marsh
and C.R Woese. The
T. 1993. Demand,
1990. Microbial
R.
ribosomal
outlook
annual
supply,
supplement
25. Lui, S. and JXSulfital994.
degradation
of
Mcrobiol. Revs. 54:305-315.
and price outlook
diesel fuel. In Energy Information
term energy
methyl
M J. McCaughey,
Nucleic Acid Res. 21:3021-3023.
in the environment.
24. Lidderdale,
in Bacterial
J. Wiley and Sons, New York.
J.G. and RJL Colwell.
hydrocarbons
In E. Stackebrandt
(ed.) Nucleic Acid Techniques
Systematics,
Overbeek,
rRXA sequencing.
Adminismtion.
DOE-E&0202(
Anaerobic
for
Short-
93).
biodegradation
of
esters by Acetobacterium woodii and Eubacterium hnosum.
J. Iud. Microbial. 13:321-327.
26.
Midwest
and toxicity
received
Biofuels
technical
from National
Inc.
1993.
information.
Soy Diesel in biodegradability
In, Biodiesel
Soy Diesel Development
information
kit
Board, Overland
KS.
1G
Park.,
27. Newman, A. 1993. Growing
TechoL
your own died
fuel. &wiron.
Sci.
27~427.
28. Osenga, M, 1993. What exactly is biodiesel?
Diesel progress
engines and drives. Ln, National Soy Diesel Development
Alternative
Board
Fuels Special Report.
29. Parsons, TX.,
Y. Maita,
and C.M. Talli (ed.).
1984.
A
Manual of Chemical and Biological Methods for Seawater Analysis
(Pergamon Press, Oxford, England1984).
3 0.
Peters,
J.F., C.H. Adams,
MC- Rand, MC. Ziemke. 198 1.
Test of plant oil (soybeans) as a diesel fuel. University of Alabama,
Huntsville,
3 1. Rakov,
report ff295.16.
D.Y.,
N.V.
Dormonina,
Y-k
Alieva. 1991. Growth of methylotrophic
Trotsenko
and
R.M.
bacteria on methyiacetate.
Prikl. Biokhim. IMikrobiol. 275 84-S 88.
32. Singer, ME. and W.R. Finn-.
of straight-chain
Microbiology,
33. shum,
AX.
Microbial
pollutants.
metabolism
p. l-60, Macmillan Publishing, New York
and AJ. Markovetz.
of undecylacetate
grown on 2-tidecanone.
34. Ward,
Microbial
and branched alkanes. Ia, RM. Atlas (ed.),
Petroleum
induction
1984.
D., RM.
degradation
1974.
Specificity
and
esterase from Psendonzonas cepacia
J. Bat. 118:890-897.
Atlas,
P.D. Boehm,
and J,A. Calder.
and the chemical evolution
Ambio. 9:3 3 3-340.
17
1980.
of Amoco Cadiz
Figure
legends
Figure I.. Bacterial
growth
under oxic conditions using freshwater
inocula. Old Hickory Lake ( W) with soy diesel, (a) without soy diesd;
Squaw Run Pond (0) with soy diesel, (0) without soy dies&; ( LL)soy
diesel
no
inocd~m,
Figure 2. Bacterial growth with the concurrent disappearance
of
fatty acid methyl esters with Old Hickory Lake samples. A) oxic
conditions, B) an&c conditions with nitrate as the t erminal electron
acceptor. (U) inoculum without soy diesel, (0) inoculum with soy
diesel,
(a)
FAMES
without inoculum, (0) FAMESwith inoculum.
Bacterial counts are in cells/ml, FAMES in mg.
Figure 3. Bacterial growth with the concurrent disappearance
fatty acid metiyi
of
esters with Squaw Run Pond samples. A) oxic
conditions, B) anoxic conditions with nitrate as the terminal electron
acceptor. (a) inoculum without soy dies& (0) inoculum with soy
diesel, (B) FAMES without inoculum, (a) FAMES with inocuhm~
Bacterial counts are in c&s/m.L, FAMES in mg.
Figure 4. Bacterial degradation
of soy diesel in soil flask tests based
on G-Canalysis (FAMES in mg). (0) oxic conditions without it~oculum,
(I) oxic condilions with inoculum, (I) anoxic conditions without
inoculum, ( q ) anotic conditions with inoculum.
18
Figure 5. Phylogenetic analysis of the soy degrading isolates based
on 16s rRNA sequence. Although both maximum parsimony and
neighbor joining methods were used only the PHILIP generated rree
is shown, Only bootstrap numbers greater than 50 are shown.
Figure 6. Soy diesel degradation
by strain GSOY,(0) inoculum
without soy diesel, (0) inoculum with soy diesel, (H) FAMES without
inoculum,
(a) FAMES with inoculum.
Figure 7. Soy diesel degradation
by Burkhoideria solanacearum
grown under anoxic conditions with soy diesel and nitrate. A) cell
growth and FAME disappearance, (0) inocuium without soy diesel,
(0) inoculum
with soy diesel, (w) FAMES without inoculum,
(a)
FAMES with inoculum. Bacterial counts are in cells/ml, FAMES in
mg/ml. B) nitrite production (0) cells/ml, (H) mm01 of nitite.
Figure 8. Esterase activity in pure cultures. Lane 1, GSOY on soy
diesel, lane 2, GSOY on nutrient broth, lane 3, Burkizolderia cepacia on
soy diesel, lane 4, B. cepacia on nutrient broth, lane 5, B.
solanaceamm aerobic on soy diesel, lane 6, B. solauacearum aerobic
on nutrient broth, lane 7, B. solanacearzm anaerobic with ni=ate
and soy diesel, Each lane was loaded with 75 pg of total protein.
Arrow indicates esterase.
TABLE 1
Percent
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Simihrity
B. subdlus
E. coli
P. oleovorans
P. mendocina
123456789
80.0 83.2 84.5 833
86.0 86.9 86.6
97.3 96.2
97.5
P. aeruginosa
P. flavescens
83.9
87.0
97.5
98.7
96.7
f. lurescens
0. multigfobufiferum
M.Mga
GSOY
84.5
87.6
97.5
98.7
97.8
97.7
82.5
87.6
92.3’
93.5
913
93.1
92.3
Bur. gladioli
Bur. solanacearum
Hl
Bur. cepada
15 Bur. pickertii
81.5
88.8
903
913
90.7
92.1
90.8
92.5
10
81.5
84.7
86.5
87.6
87.0
87.6
87.6
86.1
85.0
11
82.3
85.8
87.3
883
87.5
88.3
88.3
87.0
86.0
98.8
12
80.2
84.1
84.5
85.8
86.7
85.0
86.0
85.0
84.0
93.9
94.5
13
14
15
79.5 813 80.6
83.1 853 84.2
83.5 86.9 84.6
84.7 87.9 85.9
853 87.5 86.0
84.5 87.9 85.4
85.0 87.9 8G.l
84.0 86.8 85.1
833 85.5 84.1
92.5 98.7 93.5
93.4 99.4 94.4
97.7.94-S 99.1
.93.497.G
94.4
16
81.6
85.3
86.3
87.9
87.4
8T.3
87.3
87.0
8G.I
97.7
38.3
34.7
93.3
98.3
34.3
16 5. andropogotia
(RDB ,Y): Bacillus subdlus (449), Gcherichia
coli (2X0), Pseudomonas
oleovonns
(this study),
Pseudomonas mendodna
(2007), Pseudomonas
aemginosa (2000), Pseudomonas
flavescens
(2005),
Oceanosphillum
mufrigiobulif&rxm
(1985 ), Marinomonas
~ga (1995), GSOY (this study), Burhofdek
gladioli
(1811), Burholdexia
soianoceanrm
(17861, Hl (this study), Burkhoidexia
cepacia (1809),
Organism
f?urkhofden’a
picketii
(1797),
Burkholderia
andropogonis
(1806).
TABLE 2
Methanol
Detection
Strain
Bwkhoideria cepacia
GSOY
B. sohaceanm
(aerobic)
B. solanaceamn (anaero bit j
Day 0
Day 7
22
Day 14
+
i-l-
+
-+t
+
+
i
t-it
1E-c07
lE+O4
3
7
DV
11
15
lEti
p---.
*..’
,
.:.
..’
.:’
,:-
lE+09
7
Days
lE+l
lE+10
lE+@J
G
d
7
Days
.-
-
14
lE+i I
lE+IO
..t
\.
....
\'*.
.:'
', *
\ -*.
.:'
'\, -. .*
:"
*.
:
',
-. :'
'\
“
,:' ' .
',
-
I E+O9
I E+O8
-30
IEt07
I E+O6
IE+O5
IE+O4
IEtO3
0
7
I4
Days
lE+IO
IEt09
-60
IE+O7
I E+06
I Et04
7
Days
.
Escherichia
-
coli
r
57
64
ri
Pseudomonas
63
oleovorans
LPseudomonas
mendocinrt
Havobacterium
Pseudomonas
Iutesccos
aeruginosa
1 O(
Burkholderia
84
95
100
Hl
Bnrkh olderia
Alcaligenes
eutroph
solanacearum
us
100
GSOY 1
51
Burkholderia
cepacia
99
Burkbolderia
gladioli
Burkholderia
caryophylli
Burkholderia
andropogonis
51
Bacillus
subtilus
pickcttii
OEiOO
7
Days
lE+o6
lE+O5
0
B
lE+O9
IE+o8
lE+o6