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11-2005
Isolation and characterization of hydrocarbon
degrading bacteria from environmental habitats in
Western New York State
Katarina Malatova
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Malatova, Katarina, "Isolation and characterization of hydrocarbon degrading bacteria from environmental habitats in Western New
York State" (2005). Thesis. Rochester Institute of Technology. Accessed from
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ISOLATION AND CHARACTERIZATION OF
HYDROCARBON DEGRADING BACTERIA
FROM ENVIRONMENTAL HABITATS IN
WESTERN NEW YORK STATE
Katarina Malatova
November, 2005
A thesis submitted in partial fulfillment of the requirement for the Degree of
Master of Science in Chemistry.
Approved:
G. A. Takacs
Chemistry Advisor
Name Illegible
Research Advisor
Terence Morrill
Department Head
Department of Chemistry
Rochester Institute of Technology
Rochester, NY 14623-5203
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Katarina Malatova
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Signature
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Copyright Release Form
ISOLA TION AND CHARACTERIZATION OF
HYDROCARBON DEGRADING BACTERIA
FROM ENVIRONMENTAL HABITATS IN
WESTERN NEW YORK STATE
I, Katarina Malatova, hereby grant perrmsslOn to the Wallace
Memorial Library of RIT, to reproduce my thesis in the whole or in the part.
Any use will not be for commercial use or profit.
Katarina
Malatova
Signature: _
____
__
Date:
/1/.2..3 j oS"
11
Abstract
Screening
of
hydrocarbon
in Western New York State
of
20 distinct
species.
All
by
degrading
microorganisms
technique,
selective enrichment
strains were cultivated
in liquid
carbon and
energy
the
Serratia marcescens, Acinetobacter baumannii
genera
dioxide
source.
Bacterial
effectively
measurements
the biodegradation rates
evaluate
The
period.
strains capable of
evolution experiments were used as the major
in biometric flasks. The
of oil
chemical
composition
of
of
by
the
isolated from
and
residual
hydrocarbons
Pseudomonas
indicator
of microbial
evolution
providing
collection
media with crude oil as a sole
degrading
CO2
in the
resulted
habitats
three
rates
significant
oil
degradation
shown
to
within a short
determined
was
to
Carbon
sp.
have
data
belong
by
gas-
chromatographic techniques.
The
results
indicate that the highest
highest degradation efficiency
crude
Smakover
oil was
of
significantly
bacterial
Serratia
consortiums
degradation
organic
and
of
industrial
The
crude oil and also
waste.
oil
a
The bacterial
high
with
a
and the
Leepershank
composition
of
higher
Additionally, biodegradation
of
content of aromatic and cyclic
agents was observed
results also suggest
by
GR1 (not
yet
that the application of
of
two isolated strains enhanced the
lead to
a successful utilization of complex
combinations
baumannii demonstrated the highest
all
due to
solubilizing
marcescens.
containing
Mexican
of
crude
was prolonged.
reduced
hydrocarbons. Noticeable formation
identified clone)
Mexican
hydrocarbons
saturated and substituted
dioxide
of carbon
of medium chain alkanes were observed on
degradation
oil, whereas
accumulation
mixture
growth and
tested bacterial blends.
hi
of
CO2
GR1
clone
evolution on
and
both
Acinetobacter
substrates
among
Acknowledgements
This thesis
Due
was an experience that was challenging,
be
credit must
given to several people
Takacs. I thank him for his
experience
to thank
in teaching
throughout
my
as well
research
I
would
like to
during measurements
and
in
work.
explore alternative methods
research
Dr. Paul
Rosenberg. I
It
work.
my
at
contribute with
our
to
work
like to
recognize
of
work and studies
on
me.
generous
would
my
for my
led
as a
help
to
education.
and guidance
Craig
and
for
assistant with
Dr.
committee
teaching
I
long
me
like to thank Dr. Paul
graduate
which
His
and support
research
greatly benefited my
closely
during
career as well
in
to me. I also would like
independence in
which
serve
conversations,
Rochester Institute
my
of
Dr. Gerald
major advisor
valuable
Tom Allston for his
pleasure
instructions for my future teaching
Department
ideology
for agreeing to
would
my
and enjoyable.
Lodge for his instructions
Jeffrey
for solving problems,
was
will cherish
In addition, I
has been very
the GC/MS instrument. I also
Rosenberg
evaluating my
His
Dr.
acknowledge
on
with
genuine guidance, advice and confidence
second research advisor
my
starting
illuminating
received
many
valuable
professional goals.
Dr. Terence Morrill, Head
Technology, for granting
me
the
of
Chemistry
opportunity to
to the success and high reputation of the
Chemistry
Department.
Finally, I
emotional support
am
in
thankful
to my
the process of
family
members
accomplishing my
IV
for their
studies at
RIT.
encouragement
and
Table
of
Contents
Copyright Release Form
ii
Abstract
iii
Acknowledgements
iv
Table
of
Contents
List
of
Figures
List
of
Tables
List
of
Pictures
1.
v
vii
x
xi
INTRODUCTION
1
1 1 Hydrocarbons
2
1
5
.
.2
Microorganisms
8
1.3 Metabolic machinery
1
1
1
2.
.4
8
Aerobic degradation
.3.1
Anaerobic degradation
.3.2
Experimental
13
19
goals
MATERIALS AND METHODS
2. 1 Liquid
2.2 Solid
20
20
media
20
media
2.3 Hydrocarbons
20
2.4 Chemicals
21
2.5 Microorganisms
2.6
Screening
of
and their
isolated
2.7 Biodegradation
isolation
microorganisms
of crude oil
in
v
21
by microliter plate
abiometric system
technique
22
23
2.8 Measurement
2.9 Hydrocarbon
24
analysis
2.10 Identification
2. 1 1
24
of cell growth and cell concentration
26
of microorganisms
26
Laboratory equipment
3. RESULTS AND DISCUSSION
3.1 Cell
3.2
counts and characterization of
Screening
flasks
3.3
27
of
27
bacterial isolates
bacterial isolates for
utilization
of
hydrocarbons in
shake
31
experiments
Screening
of
bacterial
isolates
for
utilization
of
hydrocarbons
41
microtiter plate experiments
3.4 Biodegradation
of
crude
oil
in biometric flasks,
evolution
of
dioxide
carbon
54
3.5 Biodegradation
of crude
oil
and
organic
compounds
by
the
bacterial
71
consortium
3.6 Identification
in
of
hydrocarbon-degrading isolated
strains
85
4. CONCLUSIONS
90
5. FUTURE PROSPECTS
93
6. REFERENCES
95
vi
List
Figure 1.1 Structure
Figures
of
4
of crude oil constituents
Figure 1.2 Degradation
of alkanes
by Acinetobacter sp
10
Figure 1.3 Degradation
of alkanes
by Rhodococcus sp
11
Figure 1.4 Anaerobic degradation
Figure 3.2.1 Growth
on mixture of
and cell yield of
bacteria isolated from Genesee
river sediment
33
bacteria isolated from Canandaigua Lake
and cell yield of
hydrocarbons
Figure 3.2.3 Growth
mixture of
16
hydrocarbons
hydrocarbons
Figure 3.2.2 Growth
mixture of
of saturated and aromatic
34
and cell yield of
bacteria isolated from Toomey's Corner
hydrocarbons
Figure 3.2.4 Growth
on
35
isolated bacteria
of
on
hexadecane
as sole carbon and
energy
37
source
Figure 3.2.5 Growth
isolated bacteria
of
on
heptadecane
as sole carbon and
energy
38
source
Figure 3.2.6 Growth
carbon and
on
energy
of
isolated bacteria
on
2,6,10,14-tetramethylpentadecane
39
source
Figure 3.3.1 Growth
of
isolated
clones
from Genesee River
sediment on various
hydrocarbons
Figure 3.3.2 Growth
43
of
isolated
clones
from Canandaigua Lake
on
hydrocarbons
Figure 3.3.3 Growth
as sole
various
44
of
isolated
clones
hydrocarbons
from Toomey's Corner
soil
on
various
45
vu
Figure 3.3.4 Growth
of
isolated
from Genesee River
clones
sediment on various
48
organic compounds
Figure 3.3.5 Growth
of
isolated
clones
from Canandaigua Lake
on various organic
49
compounds
Figure 3.3.6 Growth
of
isolated
clones
from Toomey's Corner
on various organic
50
compounds
Figure 3.4.1 Production
of carbon
dioxide
during
Leepershank
crude oil
degradation
by isolated bacteria
Figure 3.4.2 Gas
55
chromatographic analysis of
Leepershank
crude oil
before
and after
degradation
58
Figure 3.4.3 Production
of carbon
dioxide
Mexican
during
crude oil
degradation
isolated bacteria
61
Figure 3.4.4 Gas
chromatographic analysis of
Mexican
crude oil
before
and after
degradation
64
Figure 3.4.5 Production
of carbon
dioxide
during
Smakover
crude oil
degradation
isolated bacteria
Figure 3.4.6 Gas
by
67
chromatographic analysis of
Figure 3.5.1 Production
of
by
of carbon
dioxide
Smakover
during
69
crude oil
Mexican degradation
by
isolated bacteria
72
Figure 3.5.2 Growth
patterns
in CFU/ml
of
bacterial
mixture cultivated on
Mexican
75
crude oil
Figure 3.5.3 GC
mixtures
chromatograms
of
Mexican
crude
oil
inoculated
with
bacterial
78
mixtures
vni
Figure 3.5.4 Production
of carbon
dioxide
during
C1M I degradation
by
mixtures of
isolated bacteria
Figure 3.5.5 Growth
82
patterns
in CFU/ml
of
bacterial
mixture cultivated on
CEVI I
84
organic waste
IX
List
Table 3.1.1 Bacterial
population
of
in the
Tables
original natural
habitats
and
in
an enrichment
28
medium
Table 3.1.2 Microscopic
Table 3.3.1 Growth
and
characterization of
ability
of
isolated
isolated bacterial
strains
to
utilize
30
strains
different hydrocarbons
as
46
sole carbon source
Table 3.3.2 Growth
and
ability
of
isolated
strains
to
use
different
organic compounds
51
as carbon source
Table 3.4.1 Degradation
of
Leepershank
Table 3.4.2 Degradation
of
Mexican
Table 3.4.3 Composition
Table 3.5.1 Cell
of
crude oil
Smakover
counts of mixture of
Table 3.5.2 Degradation
of
crude oil
Mexican
by isolated bacteria
hydrocarbons
by
isolated bacteria.
cultivated on
crude oil
Mexican
hydrocarbons
by
.
.65
74
isolated
77
counts of mixture of
Table 3.6.1 Biochemical
crude oil
mixture of
bacteria
Table 3.5.3 Cell
.
69
crude oil
bacteria
57
bacteria
characterization of
cultivated on
CBVI II
isolated bacteria
83
87
List
Picture 3.4.1 View
on
Leepershank
Picture 3.4.2 View
on
Mexican
Picture 3.4.3 View
on
Smakover
Pictures 3.5.1 Visual
Picture 3.6.1 View
Pictures
crude oil
crude oil
biodegradation in biometric flasks
biodegradation in biometric flasks
crude oil
observations of
on
of
biodegradation in biometric flasks
biodegradation
final hydrocarbon degraders
XI
59
65
70
80
89
Introduction
1. INTRODUCTION
In
influence
last years,
the
of
human
a
large
activity.
As
a
previous
Most
of
years, the
frequency
increased
interest
the
of scientists
environment, especially the
damage
caused
and risk of oil pollution
the petroleum goes in the ecosystem
via
leak
to investigate the
marine
changed
have become
people
result, many
protect ecosystems as well as to evaluate the
the
have been
number of ecosystems
by
the need to
aware of
contamination.
has lead to
oil
distribution
During
extensive research.
This fact
of coastal oil refineries.
Approximately
environment.
by the growing
and
five
its fate in the
million
tons of
crude oil and refined oil enter the environment each year as a result of anthropogenic
sources such as oil spills
indicated that
land
pipeline
spill
(1999)
and
most of the oil comes
spills.
resulting from
(Hinchee
the
and
Extensive
grounding
Kitte, 1995). Past
from tankers, barges
in marine,
changes
of the
Exxon Valdez
the Prestige spill
et
al.,
2004; Tazaki
Shipping
consequences
terrestrial
spills as
accidents
include serious,
already known
of oil and estimate
a serious
1970s. Conventional
and
impact
widespread and
sources.
method.
as
terrestrial ecosystems
the Nahodka oil spill, the Erica
engineers
the attention of
(Braddock
This
can
help
on
et
al.,
1995;
the surrounding environment. The
marine
It is very important to
of oil
include
was
ecosystems,
characterize oil
to predict the
behavior
is necessary to
select an
environmentalists
the environment. It
The recovery
remediation methods
on
long-term damage to
and natural resources.
the long-term impact
clean-up
well
from
al., 2004).
have
life, human health
appropriate
et
(1989),
and other vessels as well
(2002), have recently increased
environmentalists, chemists, biotechnologists
Khodijah
as
analysis of reported oil spills
studied
extensively
physical removal
since the
of contaminated
Introduction
material.
These
use
methods also
chemicals, especially shoreline cleaners,
(Riser-Roberts 1992). The
often organic solvents with or without surfactants
cleaners with surfactants
emulsify the
adsorbed
transported deeper into the shoreline soil. The
conventional
sorbents.
skimming
Sorbents
However,
help
They
organisms.
There is
of
cleaning
additional
an
of
of oil
includes the
well as
up in the landfills. Most
of
the
their emulsion with oil cause
losses due to
evaporation
(involves only
aromatic
increased interest in promoting
chemicals
sites.
to
offers a
These
the
use of
using
the oil
of
low
physicochemical
toxicity, to
increase the
compounds) play
oil
aquatic
recovery
hydrocarbons,
molecular
a major role
less
expensive and
Compared
alternative
technology for
the molecules in the
environmental methods
methods are
environment.
very feasible
considered an effective
majority
oil solvent mixtures are collected
is
in
the oil spill environments (Mills et al., 2003).
oil-polluted
bioremediation
is
agents, as
abiotic
of
shoreline
to transform oil to a transportable form for short-term storage.
and photooxidation
decontamination
are
which entrain adjacent waters or
produce another source of pollution and also
Additionally,
dispersion
oil,
Mechanical recovery
most of the used sorbents end
methods use chemical
cost.
methods.
which
for
to
do
in the
not
physiochemical
an oil spill response.
treatment of oil pollution.
crude oil and refined products are
One
process
introduce
methods,
This technique
reason
is
that the
biodegradable.
1.1 Hydrocarbons
Petroleum
products
pharmaceutical and plastic
and
other organic
are
used
as
fuels,
solvents
industries. Petroleum is
compounds,
including
some
and
feedstocks in the textile,
a complex mixture of
hydrocarbons
organometallo-constituents.
Petroleum
Introduction
constituents represent: saturates, aromatics, resins and asphaltenes
are
defined
as
hydrocarbons containing
to their chemical structures into alkanes
the
highest
the
rings
saturated
hydrocarbon
are
and
usually
Resins
structure with
(Harayama, 2004). Petroleum
from the
very low
of
The
molecular
molecule
may
enzymes.
where microbial
degradation does
affects
hydrocarbon,
the
sulfonate groups, ether
Adding
hydrocarbons to
not
have very
with one
In
complex and
oxygen
and
sulfur
reservoirs
or
comparison
non-
contain
asphaltenes
and
varies
the
its biodegradation in two
easily
mostly
atoms
widely in
that cannot react
occur.
First,
ways.
with available or
compound
Usually,
very high.
to be in a
A
the
inducible
physical state
the larger and more complex
the more slowly it is oxidized. Also the degree of
linkages, halogens
microbial attack
represents
hydrocarbons to
degradation. Compounds
aliphatic
Saturates
alkyl groups.
many nitrogen,
the structure may determine the
the structure of a
persistent.
affects
according
composition of particular petroleum product
contain groups or substituents
Second,
substitution
structure
resin
weight
hydrocarbons'
chemical
different
from different
recovered
are categorized
Aromatic hydrocarbons
and asphaltenes
addition
compositional and physical properties.
the
They
and cycloalkanes.
substituted with
fractions,
aromatic
polar compounds.
unknown carbon
ranges
(paraffins)
percentage of crude oil constituents.
several aromatic
to
double bonds.
no
(Figure 1.1). Saturates
side -chains
and
that
contain
branched
increases
(Riser-Roberts, 1992).
carbon
the
amine,
chains
methoxy
are
susceptibility
and
generally
of
cyclic
Introduction
H2
H,
'
H
H2C
^C
H2
\
^2
^CH2
111
H,C
3
H,
HC^
%CH,2
;
c
I
CH,
H,
(A)
CH,
H,C
CH,
(B)
s
s
I
I
s
s
s
s
(C)
Oh
(D)
OH
(E)
(F)
Figure 1.1
Structures
species,
elemental
(A) substituted cyclopentane, cyclohexane, bicyclic
(C) thiophene,
(xylene, naphthalene, perylene),
substituted
pyrrole,
carbazole,
pyridine,
(E) phenol,
(D)
of crude oil constituents:
(B)
substituted
aromatics
sulfur, nonyl mercaptan,
long chain alcohol, (F) asphaltene
model molecule
(Machin
et al.,
2005).
Introduction
Hydrocarbon
composition affects their physicochemical properties.
in their solubility, from
polar
compounds,
such as
The solubilization is
Many
polar
not
microorganisms,
high
determining
the degradation of
microorganisms
substrates
increase
the
surface
modify their
cell
relative nonvolatile.
spreading
for
and
dispersion
microbial attack.
causes changes
The
of
Viscosity
of
can
of
area
to
absorption
polluting
oils
is
The variability in the
of
concentration
water were related to
(Cybulski
or
an
very
et
al.,
viscous
important
individual hydrocarbons
all
fractions
high concentrations, only those fractions
shown
other
hand,
these
2003; Carvalho
and
property.
very
and
volatile or
It determines the
physicochemical character of
concentration of organic compounds
It has been
On the
excrete
the hydrocarbon mixture and also the surface area available
in the behavior
down. Also the
substrate.
and
putida
increase its affinity for hydrophobic
be very fluid
tolerance. At low concentrations,
present.
of the
surface
and, thus facilitate their
Fonseca, 2004). Hydrocarbons
hydrocarbons.
Bacillus laterospor
and
non-
hydrocarbons.
Pseudomonas aeruginosa, Pseudomonas
as
Bacillus subtilis, Bacillus cereus, Bacillus licheniformis
emulsifiers that
low solubility
molecular weight polynuclear aromatic
the only factor
such
to very
such as methanol
compounds,
Hydrocarbons differ
in
as well as mixtures.
the environment also affects the
are
likely
most susceptible
of contaminants
hydrocarbons
will
to be attacked.
level
However,
at
to degradation will be broken
affect
the number of organisms
that the higher concentrations of gasoline in contaminated
higher counts
of microorganisms
(Doong
and
Wu, 1995).
1.2 Microorganisms
In
species
recent years,
that are effective
many
microbial
degraders
of
ecologists
hydrocarbons in
have identified
natural
various
microbial
Introduction
environments.
Many
these
of
contaminated coastal areas.
carbon
sources,
derivates.
such
The
as
microbial
They
were
aliphatic
microorganisms
have been isolated from
consortia
isolated
and
their ability to metabolize various
on
compounds
aromatic
were
obtained
where maximum specific growth rate or maximum
was used as
the selection criterion.
fungi,
play
yeast and microalgae
the central
cell growth and
alkanes
are
Sun
at
el.,
capable
most
of petroleum
at
oil
microorganisms
el.,
for
is already
biodegradation,
metabolic
2004).
rather
natural populations
adapted
2004; Trindade
There
survival and proliferation
natural ecosystems and either
a
several
However, bacteria
force for
that low
petroleum
molecular weight
out
carry
more
2004; Oteyza
indigenous
at
el,
to
extensive
at
el.,
2005;
community
relying
These
Secondly,
microbial population.
in
on
and
are
Vogel,
indigenous
degrade hydrocarbons. First,
years.
environment.
or
microbial
2001; Richard
advantages
microorganisms
independently
bacteria,
that environmental conditions
provided
diverse
cell concentration
al., 2004).
an adequate
in that
culture
hydrocarbons to satisfy their
cultures
have developed through many
hydrocarbons is distributed among
in
utilize
activity (Capelli
are
than adding
et
final
driving
than pure cultures (Ghazali at el.,
el.,
favorable for oil-degrading
at
Mixed
enrichment
el., 2004).
at
number of studies report
rapidly.
ecosystems there
of extensive
1999; Kim
A large
chlorinated
microorganisms such as
(Riser-Roberts, 1992; Bundy
needs.
energy
2004; Gerdes
In many
by
the ability of microorganisms to
degraded
biodegradation
be degraded
in hydrocarbon degradation. The
role
biodegradation is
can
their
and
by
originally
procedures,
Petroleum hydrocarbons
heavily
microorganisms
are
the ability to utilize
This
population occurs
combination metabolizes various
Introduction
hydrocarbons. Many times,
when
contaminated
microbial
environment,
of nitrogen
especially
and
the
amount of microorganisms
seeding is
be
seems to
phosphorus,
sufficient
factor. It
limiting
the most
confirmed that these nutrients enhance growth of microorganisms, which
rapid
decomposition
Accepted
values
100:1. Nitrogen
for
of
contaminants
extract
or
be
and phosphorus can
kg dry
domestic
formaldehyde
was
et
a mixed microbial population
the N:P ratio at 16:1 when the optimum
than 100 mg N
(Chaineau
soilimates
sewage
found to be
nitrogen
source
the
in the
at
of
most
2003; Kim
nitrogen
did
satisfactory
at
a
sandy
and
matrix
el, 2004).
not
more
C: P,
with
is lower
Adding
yeast
beneficial. Urea
prove
nitrogen
was
al., 2004).
et
inorganic fertilizers
fertilization for
el,
leads to
C: N, 10:1;
soil are
supplied with common
(Ferguson
as
2005; Coulon
al.,
in the
Nutrient availability,
required.
not
is
source
(Riser-Roberts,
1992).
Microorganisms
are
products as a carbon and
degrading
electron
heterotrophs
acceptor)
nitrate or sulfate).
energy
use can
or anaerobic
be
with
source.
metabolic
The
(i.e. they
anaerobic
less free energy for initiation
machinery
to
use
petroleum
methabolic pathways that hydrocarbon-
either aerobic
(i.e. they
utilize oxygen as
the primary
utilize an alternative electron acceptor such as
Aerobic degradation usually
to be more effective than
require
equipped
proceeds more
degradation. One
and yield more
reason
energy
rapidly
is that
and
is
considered
aerobic reactions
per reaction.
Introduction
1.3 Metabolic machinery
1-3.1 Aerobic degradation
Aerobic biodegradation
studied
hydrocarbons
and
compounds
into
carbon
inorganic
reaction
because
utilize
the
The
as
recently.
dioxide,
aerobic
These
do
not
a
long
known
substrate
organic
and
to
microorganisms
microorganisms
under
decompose
well-
oxidize
aerobic
most organic
matter, such as sulfate, nitrate and
produce
proceeds
pathway
aerobic reactions require
complex
a
water and mineral
They
compounds.
products.
oil
is
and crude oil
anaerobic
of
ability
crude
discovered just
conditions was
other
However,
process.
hydrocarbons
of
hydrogen
most
sulfide
rapidly
less free energy for initiation
and
or
methane
most
as
efficiently,
and yield more
energy
per
reaction.
The hydrocarbons
Oxygen
serves as an
a new
fatty
known
as
fatty
ring
a series of enzyme-mediated reactions.
acceptor, while
an
donor
or
the
electron
involves
fatty acid is
sequential
initial
The
organic
energy
component of the
The
source.
formation
of
an
general
alcohol,
cleaved, releasing carbon dioxide and
carbon units shorter
pathway for
forming
a
aromatic
an
forming
than the parent molecule in a process
enzymatic
attack
involves
aromatics
hydrocarbons involves
a
group
of
cis-hydroxylation of the
diol (e.g. catechol) using dioxygenase. The
by dioxygenases, forming
substituted
by
.
general
structure
cleaved
The
is two
as
alkane
an
beta-oxidation.
monooxygenases
The
acid.
acid that
electron
functions
degradation pathway for
aldehyde and a
broken down
external
substance
contaminating
are
generally
a
dicarboxylic
proceeds
by
acid
(e.g.
ring
muconic acid).
initial beta-oxidation
is oxidatively
Oxidation
of the
of
sidechain,
Introduction
followed
by
branched
compound, such as pristane or phytane, may proceed
cleavage
forming a dicarboxylic
of
ring
the
bacterial
has
on
Acinetobacter
hydrocarbon
sp.
is
soil
actinomycetes and
associated
the
study
utilizes
to a
an
primary
of
alkane
was
as
a
alkane
monooxygenase
alcohol to allow
investigated
growth
dodecylcyclohexane
ring
by
for the
Koma (Koma
substrate.
GC/MS
suggests that strain
(Koma
et al.,
crude oil and the
showed
a novel
pathway
of
variety
and
Rhodococcus
are two
habitats. Intense interest
subsequent
breakdown
of alkylcyclohexane
et
microorganisms,
convert
the
and utilization
by Acinetobacter
al., 2003). Strain ODDK71 degraded
by
co-metabolism when
hexadecane
products
from
ODDK71 degraded dodecylcyclohexane
via a
analysis
of
co-metabolized
of microbial
The
ring
degradation
oxidation
of
pathway
of
dodecylcyclohexane
2003).
Significant
several
highly
omega oxidation
(terminal oxidation) to
oxidation and an alkyl sidechain oxidation pathways.
dodecylcyclohexane is
by
a
these bacteria in the last decade.
by
oxidation
alkylcyclohexanes (alkyl sidechain length of >12)
used
a
fungi. Acinetobacter
hydrocarbon (Figure 1.2). The degradation
ODDK71
was
with
strains often associated with petroleum contaminated
arisen
of the
The degradative pathway for
acid, instead of only monocarboxylic acid (Hamme et al., 2003).
Aerobic degradation in
including bacteria,
structure.
studies.
conelation
between aliphatic,
Rhodococcus
After
genera able to utilize such
oil pollution
of
second carbon atom
spectrum of
posses an alkane monooxygenase as
is
oxidized
hydrocarbons
fractions
(subterminal oxidation)
of
the
was observed
soil, representative strains of the
the ability to metabolize a broad
2O03). Rhodococcus
aromatic and asphaltic
in
Rhodococcus
hydrocarbons (Peressutti
Acinetobacter, but in
et
al.,
which the
leading to the production of
Introduction
ci5H3iCH3
rubredoxin
2
alkane
monooxygenase
(inducible)
>/
(ox)
y(
A
rubredoxin
H20
NADH
reductase
-^
(red)
'
(oxid)
^-*-
reductase
NAD+
.A
(red)
C15H31CH2OH
NAD+
alcohol
dehydrogenase
(constitutive)
NADH
C15H31CHO
NAD+
+
H20
aldehyde
dehydrogenase
(constitutive)
NADH
C15H31COOH
S-CoA
C15H31C-0-S-CoA
II
o
t
8
acetyl-CoA
Figure 1.2 Degradation
of alkanes
by Acinetobacter sp. (Hamme et al., 2003)
NAD/NADH= nicotine amide adenine
S-CoA= acetyl
coenzyme
dinucleotide
A
10
Introduction
a
secondary
alcohol
and
alcohol for further breakdown (Figure 1
Rhodococcus,
utilize respiration
electron acceptor
hydrocarbons. They
encoded
by the
pathway for
in
be
the
most
for
degrading aromatics
plasmids
bacteria
catabolic
examined
sp. strain
in
surface
for
several studies.
PP2
degradation
pathways
via a
three-
can
vary among
Secretion
hydrophobicity during
soluble phenanthrene
biodegradation
naphthalene
For example,
(Parales
attention
has
of
a
growth
and
also
were
The
operon encodes
for the
second codes
Molecular
PAHs in Pseudomonas
degraded
by
converged with
into the
postulated
medium
and
the
conversion of
dioxygenase (Hamme
phenanthrene was
surfactant
strains.
hydrocarbons degradation pathway is
The
four-ring
oil
the aromatics in gasoline,
dioxygenase-initiated pathway that
pathway.
Specific
and
in
most of
from Pseudomonas putida. The first
the aromatic nucleus via
found
to adapt to many different
salicylate via catechol meta-cleavage to acetaldehyde and pyruvate.
The
as well
able
hydrocarbons
characterized polycyclic aromatic
NAH7
ubiquitous
degrading
naphthalene conversion to salicylate.
introduced into
primary
Both microorganisms, Acinetobacter
This bacteria is
general.
are responsible
efficiency in
extensively
a
to generate ATP where oxygen serves as the terminal
to
appears
contaminated soils and soil
most
.3).
metabolized to
in electron transport.
Pseudomonas
although the
is further
the subsequent ketone
et
oxygen
is
al., 2003).
putida
were
Pseudomonas
the naphthalene
increased
to increase uptake of
cell-
poorly
Haddock, 2004).
been
of polycyclic aromatic
given
to other dioxygenases associated with the
hydrocarbons (PAHs). These
enzymes represent
11
Introduction
C j 3H27CH2CH2CH3
rubredoxin
02
reductase
NADH
(ox)
(red)
alkane
monooxygenase
H.O
rubredoxin
reductase
(ox)
NAD+
(red)
C13H27CH2CHCH3
OH
NAD+V
secondary
dehydrogenase
alcohol
NADH
O
C j 3H27CH2CH2CCH3
NADH
reductase
rubredoxin
alkane
(ox)
(red)
I
monooxygenase
reductase
rubredoxin
NAD+
(red)
(ox)
Cj3H2yCH2-0-C-CH3
o
H90
w
acetylesterase
C13H27CH2OH
+
CH3COOH
S-CoA
follows
as
same path
Acinetobacter for primary
CH3C-0-S-CoA
alcohol
II
O
Figure 1.3 Degradation
of alkanes
by Rhodococcus sp.
NAD/NADH= nicotine amide
adenine
(Hamme
et
al.,
2003)
dinucleotide
S-CoA= acetyl coenzyme A
12
Introduction
multicomponent systems that
catalyze
for degradation
reactions are required
specific
a
of
dibenzofurans, dibenzo-/?-dioxin
Carbazole dioxygenase activity had been
archetype carbazole
has been recently
A PAH
17484),
,9-dioxygenase,
characterized
and
-
salicylate could
be
study the
substrate
with /?-cresol
mixtures
isolated from
in detail (Pieper
et
Pseudomonas
utilized
Loh, 2002). Some
very
active
dioxygenase
in
The
CA10,
during cell
Both
(ATCC
putida
growth on
p-cresol
carbazole-
sodium
and
the bacteria as the sole carbon and energy sources (Gen-Yu
by
Pseudomonas
other
sp.
were
aerobic
degradation.
hydrocarbon
N-heterocycle
carbazole
(Resnicek
found to
Strains
of
grow
on
aromatic
the Bijerinckia genus are
The
presence
of
biphenyl
benzo(a)pyrene, benzo(a)anthracene
enable these microorganisms to oxidize
and the aromatic
resinovorans
Pseudomonas
salicylate.
constituents of gasoline as a sole source of carbon.
also
strains.
al., 2004).
interactions
sodium
and
and carbazole.
only in Pseudomonas
observed
phenol-degrading microorganism,
was also used to
containing
and
1
dioxygenation. These
regioselective
at
el., 1993).
1.3.2 Anaerobic degradation
In
contrast
to the fact that aerobic
extensively investigated, the
The roles
of
bacteria that
during biodegradation
environments where
lagoons,
stagnant
participate
are
not
hydrocarbons
fresh
is
same
investigated the question,
hydrocarbon
not true about anaerobic
in these
fully
occur
and ocean
microbial
hydrocarbon
has been
metabolism.
processes under anoxic/anaerobic conditions
understood.
(e.g. in
waters
metabolism
and
whether or not the
deep
in
Oxygen
is
not
available
in
all
sediments, flooded soils, eutrophic
oil reservoirs).
biodegradation
of
Several
studies
hydrocarbons is
have
possible
13
Introduction
under
anoxic
It
conditions.
not
was
the
until
late
1980s
that
new
groups
of
microorganisms were found to degrade hydrocarbons under strictly anoxic conditions.
Studies have
biochemical
that
mechanisms
hydrocarbon
metabolism
and
in
employed
aerobic
For example, unsubstituted, methyl-substituted,
ethyl-substituted cyclopentenes, cyclopentanes and cyclohexanes were consumed
methanogenic
field
in
the presence of sulfate
Dimethyl-substituted
(benzene, toluene,
biodegradation
of
toluene
cyclopentanes
compounds
p-xylenes
m,
and
ethylbenzene
these individual
and
degrade
depends
under
in
described
Nevertheless, degradation
reducing
aerobic
as well as
conditions,
(III)
and
aerobic
of this compound
is
under
cyclohexanes
were
the terminal
Benzene
electron acceptor.
as
is
conditions.
site-specific
under
well
as
the other
cleavage
is
anaerobic
hand, it is
highly
site-
Ethylbenzene
nitrate-reducing
conditions.
Fe (III)
and sulfate
site-specific under
Utilization
One typical
conditions.
On
The
compounds.
and sulfate terminal electron acceptor.
under
methanogenic
on
cleaved aerobically.
specific
was
and
isomers)
anaerobic
recalcitrant under nitrate -reducing conditions.
degradation
less effectively
Rabus, 2001). Several laboratory
xylene
usually
the presence of Fe
rather
biodegradation had clearly demonstrated biodegradation
is benzene degradation. Benzene is
example
but
the presence of sulfate (Widdel and
studies conducted on
BTEX
Usually,
lag
conditions.
biodegraded only in
of
those
alkanes, cycloalkanes, and some alkenes have been shown to
under anaerobic conditions.
without a substantial
and
differ completely from
special
(Riser-Roberts, 1992).
N-alkanes, branched
be degraded
by
confirmed that these microorganisms activate organic compounds
of o-xylene
conditions
is
enhanced
(Schreiber
et
by
al.,
2004).
14
Introduction
Hydrocarbons
trimethylbenzenes,
alkanes can
reactions
may
conditions,
reducing
be
in
a
by
metabolized
under
take place under Fe
well
as
anaerobic
m-,
bacteria,
bacteria. Other terminal
in
p-xylene,
and
branched
(Figure 1.4A). These
conditions
or
and
o-,
as n-alkanes
(Ill)-reducing, denitrifying
anoxygenic photosynthetic
and methanogenic
during
used
be
including
and phenanthrene
naphthalene
also
alkylbenzenes
as
such
and
sulfate
reducing
syntrophic consortia of
electron acceptors than
02
proton-
shown
to
this metabolism include manganese oxides, soil humic acids and fumarate
fermentative
oxidation.
These
as electron acceptors grow
in
microorganisms
that use nitrate, ferric iron or sulphate
(Boopathy,
cocultures with other anaerobes
syntrophic
2004).
Most
conditions
recent
at
studies
in
carbon-2
alkylsuccinates
to
fatty
has
not
been
acid metabolism.
enzymatic and genetic characterization
proposed
pathway, fumarate
addition
to form benzylsuccinate. Series of
CoA
benzoyl-CoA
and
cleavage
followed
fatty acids
(Widdel
by
is
reactions
and
denitrifying
to toluene is
(3-oxidation
a central
Benzoyl-CoA
compounds.
aromatic
which
in the
by
intermediate in the
reductive
those
anaerobic
(1-
degradation
of
that these reactions lead
with respect
bacteria Azoarcus
mediated
resemble
the
hydrocarbon
reactions convert
undergoes
that again
of
expected
the most studied
under
fumarate, yielding
to
biochemistry
However, it is
Toluene has been
activated
was
addition
an
The
1.4B).
elucidated.
n-hexane
with
connection
(Figure
methylpentyl)succinate
that
showed
sp.
benzylsuccinate
In the
synthase
benzylsuccinate to
anaerobic
acetyl
degradation
dearomatization
in the (3-oxidation
to
and
of
ring
reactions of
Rabus, 2001).
15
Introduction
~ooc-v^
COO"
|
COO"
^^^COO"
CH,
benzylsuccinate
toluene
xylenes
(methyl (succinates
"OOC
^coo
B
COO"\^^
n-hexane
( 1 -methylpentyl)succinate
"OOC
COO"
coo
N^coo-
ethylbenzene
( 1 -phenylethyl)succinate
OH
H20
2H++2e"
ethylbenzene
1 -phenolethanol
CO?
D
+
naphthalene
COO"
H+
Energy
2-naphtoate
Figure 1.4 The initial reactions during anaerobic degradation
aromatic hydrocarbons (Townsend et al., 2004).
of saturated and
16
Introduction
For ethylbenzene,
dehydrogenase to
ethylbenzene
This
produce
after
by
activation
similar
detected in
in
proceeds,
to
analogy
methylbenzoyl-CoA
PAH
metabolites
yield
metabolic
Azoarcus
well
a
as
toluene,
(1-phenylethyl)
anaerobic
ethylbenzene addition
degradation
pathway for
m-xylene
to
m-methylbenzylsuccinate
m-
Naphtalene degradation
compounds) in
monoaromatic
bacteria (Figure 1.4D). The identification
enrichment culture
as activated
acid)
indicated the further
via subsequent reduction of
appear
investigated
studies
to be activated
in
river
a phenanthrene-adapted anaerobic
sediment
The
current studies
such as sludge source, the presence of
the addition of electron
degradation
of
to
sulfate-
of other
metabolism of
the two rings to
by
a mechanism
toluene (Hamme et al., 2003).
phenanthrene
factors
succinate
to form 2-naphtoate (the central intermediate in a pathway
sulphate-reducing
of
Previous
several
pathway for sulfate-reducing
indicating
via
thiolytic
and
Rabus, 2001; Riser-Roberts, 1992).
denitrifying
(presumably
to that
degrading
sp.
that
decalin-2-carboxylate. Alkylnaphthalenes
similar
of
and
by
Acetophenone is
3-oxo-3phenylpropionyl-CoA
evidence
of
the benzoyl-CoA
in
2-naphthoate
acetophenone.
are also metabolized under anaerobic conditions.
to
as
that
(Widdel
proceeds via carboxylation
analogous
to
oxidation
to that of toluene metabolism. In this case
enrichment cultures of
dehydrogenation
1-phenylethanol (Figure 1.4C).
yields
fumarate (Figure 1.4C). There is
reducing
conditions starts with
to acetyl-CoA and benzoyl-CoA. The
bacteria is
was
and
denitrifying
is followed
reaction
carboxylated
cleavage
oxidation under
rates
donors
on
PAH degradation
for PAHs in
municipal
individual
rates.
sludge
The
group
explore the effects
or mixed
PAHs,
results show
under
capable of
anaerobic
of
pH and
that the order
conditions
is:
17
Introduction
phenanthrene > pyrene > anthracene >
the order
is
fluorene
acenaphthene >
fluorene
> acenaphthene.
In
petrochemical sludge
> phenanthrene > anthracene > pyrene
(Chang et al.,
2001, 2003; Meckenstock et al., 2004)
The
Anaerobic degradation
two
Dechloromonas
isolated for study
transformed
by
mechanism
by
of
which
benzene has
not yet
(RBC
JJ),
strains
aromatic
alcohols,
intermediates
as
that the
hydroxylation
benzene
degradation.
of
benzene to
The
carboxylation of phenol to
with this
of
It is
much
and
benzene
steps
in
to benzoate could then
under
anaerobic
occur
by
the
by the reductive removal
studies
one of the carbons of
be
can
degradation suggesting
initial
one of the
13C-Labeling
been
yet
benzoate have been
suggest
under anaerobic conditions.
of
that the carboxyl
benzene. Latest
However,
degradation
studies show
the
microorganisms
of petroleum and refined products proceeds
the presence of oxygen than under anoxic conditions.
microorganisms
Aerobic
bacteria have
for
are not ubiquitous.
obvious that the
faster in
except
cresols, phenol, demethylation
Phenol
acids.
is
unclear.
to lignin-derived aromatic acids
anaerobic
phenol
is
indicate that benzene
form 4-hydroxybenzoate followed
benzene degradation
ability
studies
conversion of phenol
benzoate is derived from
possibility
and
of
hydroxyl group to form benzoate.
carbon of
no pure cultures of
intermediates, including
aldehydes
occurs
been described in detail because
methanogenic cultures acclimated
consistentiy detected
the
and
degradation
benzene
(Riser-Roberts, 1992). Some
anaerobic conditions with several
products,
anaerobic
degrade
bioremediation
contamination, however
a
larger
range
processes
they
are
are
often
of
hydrocarbon
very
effective
expensive
Furthermore,
compounds
in
treating
(e.g. hydrogen
than
aerobic
anaerobic.
hydrocarbon
peroxide).
For this
18
Introduction
reason
biodegradation
anaerobic
bioremediation technology
and
that can
be
used
contaminated groundwater
1.4 Experimental
The primary
microorganisms
of the
microbial
activity
C02
on
of organic
were
be
et
soil, sediment,
observed that
without oxygen
in
al., 2002).
and aquatic sites
in
western
techniques, (ii) investigate the biodegradation
using
measurement
crude
oil,
as
(iv)
microliter plate-based
an
appropriate
examine
waste and crude oil
applicable
situ
(i) isolate hydrocarbon-degrading
to
by
the
in bioremediation
population
for
New York State
potential of each
assay,
criterion
(iii) determinate
the evaluation of
changes
during
bacterial consortium,
a mixed
the most superior hydrocarbon degraders in
that would
was
degradable
are
2003; Coates
this study
chemical analysis
the utility
identify
al.,
indigenous to the terrestrial
by detailed
degradation
et
of
in
goals
objectives of
selective enrichment
strain
(Johnson
advantageous
hydrocarbons. It
(BTEX)
ethylbenzene, and xylene
and
for the decontamination
ground water contaminated with petroleum
benzene, toluene,
by
cost-effective
provides
order
processes on
to
an
and
prepare a microbial
industrial
the
(v)
blend
scale and with
crude oil spills.
19
Material
and
Methods
g/1 and glucose
1.0 g/1,
2. MATERIALS AND METHODS
2.1. Liquid
PC
pH
media
7.2. Bushnell-Haas
(NH4)3P04 g/1, KN03 1
for
used
sterilized
yeast
0.5
12.0
FeCl3 0.05
g/1,
pH
for 20
minutes at
7.2. Peptone broth
viable cell counts.
g/1,
KH2P04 lg/1
(0.1%,
Each
pH
7.2)
was
medium was
C.
1.0
g/1 of glucose and
g/1 of proteose
starch, 0.3
agar)
CaCl2 0.02
g/1,
PCA
and enumeration of total viable cells
yeast extract,
g/1 of
120
2.5
media
extract, 0.5
g/1 of
g/1 and
MgS04 0.2
dilutions to determine bacterial
For isolation
g/1 of
medium contained:
yeast extract
serial
2.2 Solid
2.5
tryptone 5.0 g/1,
medium contained:
peptone, 0.5
g/1 of sodium
were used.
Each
14.0
g/1 of
g/1 of casein
pyruvate, 0.3
agar)
agar
and
R2A
medium was sterilized
g/1 of
agar
hydrolysate, 0.5
K2HP04, 0.05
g/1 of
(5.0
for 20
(0.5
g/1 of
g/1 of
minutes at
peptone,
g/1 of
glucose,
MgS04
120
and
C.
23 Hydrocarbons
Medium
chain
length
pentadecane were purchased
were obtained
oil were purchased
Leepershank
crude
oil
were
at
at
Rochester Institute
provided
of
from
a
and
2,6,10,14-tetramethyl
and samples of motor oil
local
Wegmans. Samples
phenolic waste samples were obtained
(CIMS)
(C10-C17)
from SIGMA. Gasoline
State, Castrol Syntec, Mobil 1
and olive
hydrocarbons
gas station.
of
Corn oil,
Quaker
canola oil
Mexican, Smakover, Alaska,
from INTERBIO Houston, TX. Organic
from Center for Integrated
and
Manufacturing Studies
Technology.
20
Material
and
Methods
2.4 Chemicals
PCA
agar
was
from EM Science, MERCK KGaA, (Darmstadt,
purchased
Germany), Bacto-peptone broth
and tryptone
from DIFCO Laboratories (Detroit, MI),
Bushnell-Haas broth from BECTON Dickinson (Sparks, MI),
yeast extract and glucose
from SIGMA Chemical Co. (St. Louis, MO).
2.5 Microorganisms
Microorganisms
their isolation
and
in
used
all experiments were
from Genesee River
technique
Canandaigua Lake
September
2004.
supplemented with
enrichment
and
sediment
Toomey's Corner
Bushnell-Haas
2 %
methods
v/v
hydrocarbon
represent:
2,6,10, 14-tetramethylpentadecane
mixture
of motor
oil
(Quaker
mixture of organic waste
rotary
shaker at
23C, 1
(CPM
State)
I)
Bushnell-Haas Broth containing
continued
to incubate. Unless
plated after appropriate
incubation,
mixture
of
motor
from primary
the
same
dilution
pure colonies were
on
hydrocarbon
isolated
by using
in
and
equivalent
station; equivalent
incubation
on
transferred to a fresh
primary
culture
and
enrichment, 0.1 ml of media was
incubated
a single
as
in
technique
substrates used
one week of
mix
from
were collected
State);
gas
enrichment was
agar and
Samples
enrichment
(Quaker
After
stated, after
PCA
NY)
the
from local
2nd
otherwise
2004.
hexadecane, heptadecane
oil
and used motor oil.
ml of sample
selective enrichment
The hydrocarbons
gasoline
and
April
in
used
substrates.
(pristane);
by
(East Bloomfield,
was
equivalent
in
obtained
soil
Broth
isolated
at
26
C.
colony isolation
After 48 hour
procedure
21
Material
from
each enrichment.
plates at 3- week
mixed with
40 %
determined
glycerol and stored at
was spread on
PCA
applications
plate -based
(Casey
et
and
absorbance
diversity
to
monitor
biomass
indicator
and
to
pellet
placed
the
in
cells.
recentrifugation
Haas
at
(before enrichment)
dilutions
26C for 48 hours).
developed
major parameter of
The
a
range
of
use of microtiter plates
for
microbial
wide
originally described
this assay is the
method offers
to measure
for
fast, cheap
hydrocarbon
use
and
turbidity
by
or
easy detection.
degrading
ability
by
7.2) in 125
ml
a color change.
were
incubated for 48 hours
aseptically
agar
experiments were
ml of series of
of microbial communities was
growth.
enables
Pure bacterial isolates
flask
(0.1
procedure
been
have
assays
Mills (1991). The
addition of
monitoring
the original sample
incubated
PC A
use.
2004; lones & Dudley, 1997). The
al.,
expressing the functional
The
agar plates and
Methods
Screening of isolated microorganisms by microtiter plate technique
Microtiter
Garland
in
dilution-agar plating
serial
10"2-10"8
2.6
for future
-70C
and replated at
in biodegradable
not used
number of total viable cells
by
4C
colonies were stored at
intervals. Bacterial isolates
The initial
was
Isolated
and
for
inoculated in 10
at
23C
sterile centrifuge
rotary
PC
shaker.
medium
After
(pH
incubation,
cells were
tubes and centrifuge for 10 minutes at 15,000 rpm
After washing the
another
on
ml of
cells
10 minutes, the
in 5
ml
of
Bushnell-Haas
cells were resuspended
in 4
medium
and
ml of Bushnell-
medium.
22
Material
Microtiter plates in
sterilized
Bushnell-Haas
Controls did
days
violet
low
At the
or
For better
plates
|il of
(3
ml of
by
new plates
was used
ml).
1
.0
The
incubated
from
by
for
incubated for 21
were
positive results.
For
after
medium
to
precipitate; crude oil samples
red
at
wells with
containing
5
of crude oil
of
ml of a
a
48-hour
of
kept
final
volume.
The
1
ml
ratio
consistent with original
0.07
ml of
hydrocarbon). 50
hydrocarbon degraders.
biometric
flask
biometric
sidearm of
23
in
ml of
ml of cells and
for determination
culture.
system
(containing
Hydrocarbon
biometric flask
was
filled
48
Bushnell-Haas
ml
substrate was then added
with
10
ml of
0.1 M KOH.
C, non-shaking.
Samples for measuring
periods
indicated
Bushnell-Haas medium, 0.5
consisted of
were
hydrocarbon.
incubated for 24 hours
were
the experimental conditions, was
Bacterial inoculation
Flasks
Plates
microtiter plate was scored
2.7 Biodegradation
to 2% v/v (1.0
Plates
strain.
p.1 of
pi of
precipitate.
well as
INT indicator
medium)
7
pi of cells and
300
manipulation and easier sampling, original microtiter plates with
wells were replaced
additives, as
with each well contained
Methods
this period, 50 pi of p-iodonitrotetrazolium
end of
chain alkanes a positive result was
for brown
up
bacterial
was added to each well.
indicator. Each
were scored
of
(pH 7.2), 50
hydrocarbon
non-shaking.
(INT) indicator
addition of
medium
not contain
26C,
at
our experiment were set
and
carbon
a sidearm of the
dioxide
were
flask. Evaluation
utilization of crude oil and organic waste was
taken
by
a syringe
of carbon
determined
by
dioxide
in
scheduled time
during
a colorimetric
microbial
titration. The
23
Material
amount of
trapped
saturated
barium
solution.
The
chloride and
chloride and
control
0.1
KOH
was subtracted
dioxide
was
growth
diluted in 0.8
against
blank (3
Cell
pump for 15
pre-weighted
1
microbial
in
the amount of
HC1
for
needed
1.0
ml
of
until colorless
1.0
with
needed
g
medium.
ml of
barium
neutralization of
for
into
neutralization
micromoles of
of
Absorbance
dry
weight of cells
Spec21. Sample (0.2ml)
was measured at
600
nm
medium).
in biometric flasks
of
determination
microtiter plates was measured on
was
determined in the initial
filtered through
disks. The disks
paper
2.9 Hydrocarbon
of
fresh KOH
HC1
of
0.05 M HC1
milliliters was converted
minutes and rinsed twice with
filter
of
volume of
of cell growth and
ml of sample was
yield was expressed as
Sets
difference in
Bushnell-Haas
concentration
of experiment.
ml
The
from
Bushnell-Haas
ml of
ml of
10
addition
Methods
during microbial degradation of hydrocarbons.
2.8 Measurement
Microbial
after
ml of phenolphthalein with
sample contained
the unexposed KOH. The
evolved carbon
0.1
accurately titrated
was
ml of phenolphthalein.
the experimental
of
C02 in KOH
and
WOBLR
distillated
were
dried
water.
out at
PISTON
Cells
and
final
phase
pressure/vacuum
were collected on
75C for 12 hours. The
the
cell
dry weight per liter.
analysis
test tubes experiments were designed in order to qualitatively analyze the
hydrocarbon degradation
at
GC-MS. Tube containing
cultures of
bacteria
and
24
Material
hydrocarbons
conditions as
liquid
were
during
(pH 7), 80
extraction
(approximately
was
5 ml)
carried
with
clear vial
for further
microorganisms was
column
using
a
use.
1
out
ml of
by
hexane. This
x
6890, GC
0.2 mm; helium
20:1. The initial temperature
per minute and
and
50
entire
pi of
hydrocarbon. The
volume
of
mixture was emulsified
top layer
pi
one
by
test tube
and
shaking
was recovered and transformed
disk filters. 1
spectra as
to quantify
were performed
direct interface. The instrument
IMS, 60m
Mass
were used
The GC/MS analysis,
Hewlett-Packard Model
Bushnell-Hass
ml of sterilized
to a
Prior to injection to GC-MS, the hydrophobic layer containing
split ratio.
hydrocarbons
mixture of
4
inoculum
mixing the
filtered through 0.2
1:20
contained
pi of microbial
to resettle for five minutes. The
allowed
C
the same time period and in the same experimental
biometric flasks. Each test tube
medium
sample
incubated
Methods
and
the
retention
using
a
MS-5973
conditions were the
flow 1 ml/min;
70C kept for 5
final temperature
of
injected
times
onto
the
of standard
each analyte.
spectrometer coupled
equipped with a cool-on-column
column
was
well
pi was then
inlet
following: capillary
18.5
pressure
minutes with a
280C kept for 10
and
to a
capillary
column HP-
psi and split ratio
temperature
minutes with
total
ramp
run
of
14
time 30
minutes.
A
minutes.
time
The
was
solvent
solvent
delay
solvent
employed
in
order
front reached the detector
approximately
delay. The
was
at
7 minutes,
solvent used
in
so
at
to prolong detector lifetime at 0-4.5
4.0
minutes and
there was no loss of
all analysis was mixture of
initial
analyte retention
resolution
due to initial
hexanes.
25
Material
2.10 Identification
The
classification
in
the
determination
and
Microbiology
Hospital, Rochester, NY. An
Hazelwood, Mo., USA)
detects bacterial
of
hydrocarbon-degrading
Toomey's'
macro-
on
the
in the
and
identification
to general principles of
system.
microbial
The
of
microwells of plastic
were subcultured onto agar and
VHEK
Strong
isolated
soil
was
Memorial
VITEK API 20E (BioMerieux, Inc.
for determination
was used
of
strains
Corner
and
Immunology Laboratory
automated test system
measuring fluorescence. Isolates
according
and
growth and metabolic reactions
37C before testing
Methods
of microorganisms
from Genesee River sediment, Canandaigua Lake
performed
and
isolates. It
test cards
incubated for 24 h
microorganisms
by
at
identified
were
classification, using selective media and
and microscopic examination of morphological characters.
2.11
Laboratory equipment
During
SorvallR
the experiments
following laboratory
equipment and
devices
were used:
RC-5B Refrigerated Superspeed Centrifuge, ENVIRON rotary shaker, LEICA
GAVEN Coumpound Microscope, Low
temperature
incubator Model
2005, VWR
Sheldon thermostat Model 1330GM, Analytical balance Mettler AE 163,
PISTON
pressure/vacuum
pump,
GC/MS Hewlett-Packard Model 6890,
WOBLR
UltraspecR
2000.
26
Results
and
Discussion
3. RESULTS AND DISCUSSION
3.1 Cells
counts and characterization of
Prior the screening
in
populations were estimated
1010
forming
colony
Plate
units
hydrocarbon
each original sample.
(CFU) has been found
to be an order lower
were selected
by
enrichment
increase
significant
Canandaigua Lake
microorganisms, the bacterial
degrading
Appreciable
in
(109
CFU). Indigenous
was observed after
and
fact
that repeated exposure to
isolated in this study
week of enrichment
These
petroleum products at a site will
adaptive capabilities of the microorganisms and though
from
obtained
microorganisms
the first and the second
was
in Table 3.1.1 indicate
results
motor oil mixture and equivalent mixture of gasoline/motor oil.
the
bacteria up to
Tommey's Corner
organisms
culturing technique. As the
hydrocarbon-degrading
number of
to exist in the Genesee river aquifers.
bacteria from the Canandaigua Lake
counts of viable
determined
of
bacterial isolates
a
the
in the
results confirmed
usually increase the
increase the
rate of
degradation
with a new exposure to a compound.
The type
It
was
of enrichment substrate
observed
that
the
existence
significantly
of
organo-phenolic
enrichment medium repressed microbial growth
Comer
after
samples
first
enrichment
hydrocarbon
of
during
utilizers
hydrocarbons,
organism
the first
to
a
week of
fresh
affected
microbial population.
compounds
in Canandaigua Lake
incubation.
medium
the
Yet,
resulted
and
the
Toomey's
the transfer of microorganisms
in
(Table 3.1.1). Generally, the larger
(CIM) in
an
increase
of
and more complex
numbers
of
the structure
the more slowly is oxidized. This may depend upon the type of
involved
and
the
medium,
in
which
it
was
developed.
27
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z3S
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v
x
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Q
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oo
x
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on
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o
3
3
<u
C
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c_>
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CA
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c
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oo
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t-
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o
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X
ON
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fi
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CN
rn
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Tr-
Results
For this reason, the longer
toxic
co-metabolites
the
enhanced
apparently
utilization more complex compounds
and
fresh
enrichment period as well as
medium and
in investigated
Discussion
decantation
bacteria
of
proliferation
and
of
capable
of
from Canandaigua Lake
samples
Toomey's Comer.
The morphology
counts
experiments.
and
type
Summary
objectives of
this study was to
determinate
their
conditions.
results
diversity
degradation
is
of
an
from
The
each
and
numerous
of
bacterial
microorganisms
Toomey's Corner
bacteria have
presence of
PCA
hydrocarbons
appear
still
as
in Table 3.1.2. One
potential
of strain was
location
in
done
standardized
after
similar
strains.
of microorganisms
of
of
in
cell
the
order
to
culture
Gram staining
were characterized with a
diversity
is generally
in
apparently
and
As the
very high
the natural
hydrocarbons. Extensive
by
accomplished
mixed
microbial
species.
plates
indicated that
colonies
in
each
after application of enrichment
sample
significantly decreased.
naturally occurring in Genesee
to be limited to degradation of
demonstrated the ability to
solely
investigated in
culturable strains as possible
eliminate
metabolic
pollutants
observations of
technique, the variety
Lake
presented
important factor in the biodegradation
petroleum
Although, many
colonies were also
biodegradation
populations, rather than single microbial
Visual
many
first screening
original samples
of microorganisms.
environments
as
for bacteria to
examination
indicate
a
bacterial
the results is
isolate
hydrocarbon
For this reason,
microscopic
of
of
source of
energy
River, Canandaigua
petroleum
compounds,
survive and proliferate
and carbon.
in the
Results
29
Results
and
Discussion
Table'3.1.2
Microscopic
Name
characterization of
Location
isolated bacterial
Enrichment
Gram
substrate
stain
strains
Morphology
(color/
shape and
organization)
G*
Genesee
Gl
river
C,6: C17: TMPD
Milky/
in
chains of 2-5 with
in
clusters with smooth
cocks
smooth edges
C,6: C,7: TMPD
G2
C
Yellow/
rods
edges
C,6: C17: TMPD
G3
G+
Beige/ individual
rods with smooth
edges
C,6: C,7: TMPD
G4
Light brown/
G
scattered rods with rough
edges
Canandaigua
CL1
G
Gasoline
Beige/clusters 3-4
oval cells
Lake
G"
CL2
Gasoline
CL3
Gasoline
CL4
Gasoline
Brown/chains in 2-4
rods
G"
Light
yellow/individual cocks
G"
Purple/filamentous
G+
Motor
CL5
oils
Light brown/
rods
rods
in
clusters
mixture
Motor
CL6
oils
G
Beige/scattered
cocks smooth edges
mixture
G"
Motor
CL7
Yellow/filamentous
oils
rods
mixture
G"
Mixture
CL8
White/individual
of
oval cells
gasoline/motor
oil
Gasoline
Toomey's
TCI
G
Pink/tiny
scattered rods
Corner
G"
TC2
Gasoline
TC3
Gasoline
TC4
Gasoline
Yellow/coccobacilli in
pairs
G"
Light brown/
G"
Light
yellow/
G"
Motor
TC5
Beige/filamentous
oils
rods
mixture
G"
Mixture
TC6
Beige/individuals tiny
of
cocks
gasoline/motor
^1
on
G+
Mixture
TC7
Brown/
of
rods
in
pairs
gasoline/motor
/^;i
on
G+
Mixture
TC8
Beige/clusters
of
of
3-5
rods
gasoline/motor
oil
Legend: Microorganisms
in 26
C,
single
were plated on
bacterial
PCA
2nd
enrichment.
agar plates after
strains were replated
colony isolation and microscopically
Lake isolates, TC= Toomey's Corner isolates
examined.
on new
GR=
PCA
plates
After 3 days
by
of
incubation
the method of
Genesee River isolates,
CL=
single-
Canandaigua
30
Results
from
Discussion
the microscopical examination demonstrate that the type of enrichment substrate
significantly
but the type
of
collection of
only the
affected not
bacterial
A high
Lake
and
strain as well
diversity
ability to
after a second enrichment resulted
four bacteria from Genesee river sediment,
utilize various
3.2
(Table 3.1.2).
of microorganisms
and eight colonies
(Table 3.1.1),
concentration of microbial population
from Toomey's Corner
soil.
eight colonies
All
in the
from Canandaigua
strains were tested
for their
hydrocarbons.
Screening of bacterial
isolates for
utilization of
hydrocarbons in
shake
flasks experiments
Even though
that
enrichment
have been especially
the
characterize
isolated
degradable
capability
more
of
a
monitored
only those indigenous
to degrade
potential
hydrocarbons
of
determining
hydrocarbons, it
isolates to
substrate
regularly
both
utilization.
chemical
hexadecane, heptadecane
for preliminary
by
measuring
The
structures,
and
screening.
optical
density
of
structure
straight
order
linear
a
The
growth
of
for
compound
chain
to
each
experiment
alkanes
is
are
investigate the
as well
branched,
2,4,6, 10-teramethylpentadecane
microorganisms
of experimental medium.
weight of cells was also used as a second parameter
potential of
necessary to
was
preliminary batch flask test
its biodegradability. Generally,
utilize
microorganisms
for individual isolates. For this reason,
readily than branched hydrocarbons. In
equivalent mixture of
dry
selected
microbial strain was submitted to a
important in
as
acclimated
biodegradation
detailed investigation
used
culturing
an
was
was
Additionally,
for evaluating the biodegradation
isolated bacteria.
31
Results
As the Figure 3.2.1 (above) indicates
growth of
bacterial
characterized
the
fastest
isolates
grown
solely
source of
limited. This
energy
be
the
after
and
same
degradation
144 hours
indicating
a
by
of
sediment.
enrichment
Growth
GR1
of
incubation. When GR 2, 3
initiated 60 hours
was
In
growth rate.
in the
survive on
concentration represents a
and
spite of
presence of
or
the
presents
for
increase
production of cell
above
0.5
isolates indicates
The
g/1.
The
biomass. The
initial
the
and
same tests
for hydrocarbon
isolated from Canandaigua Lake
experiments are presented
much
of
initial
GR1
dry
decreased
dry
in the
from lysis
weight
of
clone
was
1.34
weight.
This
result
in Figures 3.2.2
Toomey's
and
the
remaining
cell yield of
lower degradation
degradability
and
as
this substrate is significantiy
final
concentration of
slower growth and
that these strains have a
after
hydrocarbons
g/1.
The
3.2.3. When CL5, 6
this
did
not
and
4
than GR1.
results
and
by
GR2, 3
were conducted with all
Corner.
This
indicates
clones
potential
in
cells
that a significant amount of carbon obtained from hydrocarbon utilization is used
strain
4
GR4.
concentration
6 fold increase to
is
the fact that
the occurrence of mutualistic relationships
GR1, GR2
The final
experiments.
clone
in
clone exhibited
strain might thrive on metabolic products or products
(below)
Discussion
was observed
incubation. This
of
growth
lower
carbon, its ability to
of other microorganisms such as
Figure 3.2.1
48 hours
substrate,
3-weeks
explained
GR3
enrichment process.
during
lower slope,
isolated
might
phase within
growth
on
with much
strain was
initial
a rapid
highest
and
were
inoculation
GR3
by
isolated from Genesee river
strains
difference
a significant
and
16
from
8 isolates
strains
these
were
32
Results
and
Discussion
Figure 3.2.1
Growth and
of
cell yield of
bacteria isolated from Genesee
river sediment on mixture
hydrocarbons
Q
O
.c
2
CD
100
80
60
Time
(hours)
144
0
Time
Legend: Bacteria
were
incubated
at
23C,
120
(days)
non-shaking in 2%
v/v of equivalent mixture of
hydrocarbons
(hexadecane: heptadecane: 2,6,10,14-tetramethylpentadecane). Growth of isolated clones was
measured on UltraSpec2000 at 600 nm at designated intervals. Dry weight was determined in 0
and
144 hours
of
incubation. GR1, GR2, GR3
and GR4=
Genesee River isolates
33
Results
Figure 3.2.2
Growth and
cell yield of
bacteria isolated from Canandaigua Lake
and
Discussion
on mixture of
hydrocarbons
-#-
1.0
CL1
-
-D-
-A-
CL2
CL3
CL4
1,
0.8-
-o CL5
o
o
^
' s^
CL6
Q
-Ar-
0.6
/\
CL7
-
CL8
I
O
(3
0.4
0.2
/
-
>
mAff-^-^
-
0.0*
/
I
20
^
-i
40
1
1
i
1
1
60
80
100
120
140
Time
'
(hours)
C
o
2
*-*
0
o
c
o
o
a>
O
144
0
Time
(hours)
Legend: Bacteria were incubated at 23C, non-shaking in 2% v/v of equivalent mixture of hydrocarbons
(hexadecane: heptadecane: 2,6,10,14-tetramethylpentadecane). Growth of isolated clones was
measured on UltraSpec2000 at 600 nm in designated intervals. Dry weight was determined in 0
and
144 hours
of
incubation. CL1-CL8= Canandaigua Lake isolates
34
Results
Figure 3.2.3
Growth and cell
hydrocarbons
yield of
bacteria isolated from Toomey's Corner
60
Time
80
100
120
and
Discussion
on mixture of
140
(hours)
144
0
Time
(hours)
Legend: Bacteria were incubated at 23C, non-shaking in 2% v/v of equivalent mixture of hydrocarbons
(hexadecane: heptadecane: 2,6,10,14-tetramethylpentadecane). Growth of isolated clones was
measured on UltraSpec2000 at 600 nm at designated intervals. Dry weight was determined in 0
and
144 hours
of
incubation. TC1-TC8= Toomey's Corner isolates
35
Results
grown
hydrocarbon mixture,
on the
inoculation. However,
is
characterized
Among Toomey's
and TC4 even
these isolates
that
were
longer (90
appear to
0.6
g/1
by
very
potential and
run
rapid
density
in batch
(60 hours),
slope,
indicating
hours)
growth of
weight
needed more
microorganisms on
dry
weight of
These
(above 0.5 g/1)
than 60
to initiate their growth on hydrocarbons.
be the fastest growing
CL2, CL3
a rapid growth rate.
dry
and maximum
Comer isolates, TCI bacteria
growth of
isolated
and
branched
strains on single
type of
indicated that the isolates
previous growth experiments
linear
after
at
hours
Yet, both
of
hydrocarbons
mixture
TCI
reached
and
TC4
that is 6 times increase above the initial concentration.
Considering
mixture of
less than 20 hours
observed
phase
isolated from Toomey's Corner. The final
more then
from
long lag
spite of
clones reached the maximum optical
144 hours.
was
Discussion
these strains did not reach as high growth as the rest of the
Canandaigua Lake isolates. In
and CL7 clones
growth
and
alkanes.
In
order
were are able
the results
to
grow on
to further investigate the biodegradation
their dependence on hydrocarbon structure, the
experiments with one type of
hydrocarbon,
hydrocarbon
as
most successful strains were
the sole source
of carbon and
energy.
As
the
results
degradable hydrocarbon for
hours
after
inoculation)
was reached
utilizing the
all
isolates,
was observed
in 110 hrs. TCI, TC4
observed after
48 hours
substrate.
3.2.4, hexadecane
indicate in Figure
of
except
for GR1, CL1
and
CL7
incubation. TC6
Considering
the
CL2
and
and
exhibited a
and
fact that
GR4
CL3. The fastest
TC8. Their
longer
required at
some of
represented
these
lag
an
easily
growth
(20
maximum growth
phase with growth
least 72 hours starting
microorganisms
have been
36
Results
Figure 3.2.4
Growth of isolated bacteria on hexadecane
as sole carbon and
120
100
Time
were
incubated
was measured on
at
GR= Genesee River
isolates,
at
600
nm at
source
140
140
(hours)
23C, non-shaking in 2%
UltraSpec2000
120
100
80
Time
Legend: Bacteria
Discussion
(hours)
60
40
20
energy
and
v/v of
hexadecane. Growth
designated intervals
CL= Canandaigua Lake
isolates,
against
TC=
of
isolated
Bushnell-Haas
clones
medium.
Toomey's Corner isolates
37
Results
Figure 3.2.5
Growth of isolated bacteria
on
heptadecane
as sole carbon and
energy
and
Discussion
source
0.8
Q
O
%
o
O
0.0
Time
Time
were
incubated
clones was measured on
medium. GR=
at
100
80
60
Legend: Bacteria
(hours)
(hours)
23C, non-shaking in 2%
UltraSpec2000
at
600
nm at
v/v of
heptadecane. Growth
designated intervals
of isolated
Bushnell-Haas
Toomey's Corner
against
Genesee River isolates, CL= Canandaigua Lake isolates, TC=
isolates
38
Results
Figure 3.2.6
Growth of isolated bacteria
energy
on
2,6,10,14-tetramethyIpentadecane
and
Discussion
as so]e carDon and
source
20
40
60
80
Time
(hours)
Time
(hours)
100
120
140
o
CD
Legend: Bacteria were incubated at 23C, non-shaking in 2% v/v of 2,6,10,14-tertramethylpentadecane.
Growth of isolated clones was measured on UltraSpec2000 at 600 nm at designated intervals
against
Bushnell-Haas
medium. GR=
Genesee River isolates, CL= Canandaigua Lake
isolates,
TC= Toomey's Comer isolates
39
Results
not exposed to
hydrocarbons
that the adaptation
is
high degree
a
compounds and chemicals that
on
many factors,
degradation
play
such
as
they
degradation
exhibited the
was
CL7
compared to
highest
shorter
initial
biodegradation
optical
density
heptadecane.
TC8
growth on
48 hour
after a
phase.
The
this
naked eye at
on
in
comparison
specific
existing
branching
with
to
in
the
for
the
catabolic
tested bacteria
by direct
and
contact
isolates. Figure
TC4
clones.
between the
growth
significantly
influenced
and most clones reached a
This
lower
generally decreases
of cells was visible with
suggests
cells and
the
to hexadecane and
comparison
Clustering
TC6
and
CL7 bacteria
exhibited
structure
chemical
microbial attack.
96 hours for TC2, C12
TC4
and
other
structure and addition of aliphatic side-chains
substrate was achieved
adapt to
2,6,10,14-tetramethylpentadecane. The
2,6,10,14-tetramethylpentadecane in
Tertiary
low
was
phase, except CL7 clone that
the susceptibility of compounds to
very strong
of enzymes
GR1, TCI, TC8
Still
substrate
bacteria
lag
heptadecane
clones.
potential of most of the
with
on
growth
and
represents the growth of
initiated
There
to before. Adaptation depends
derepression
or
process.
community to
of a microbial
possible
of novel compounds.
As Figure 3.2.5 shows,
3.2.6
in the degradation
were never exposed
induction
the
role
pathways of a particular compound or an adaptation of
enzymes to the
isolates
important
an
variability in the ability
of
Discussion
only in very low concentrations, it is very
at all or
process might
and
that the uptake of
the hydrocarbon due to the
adsorption.
These
experiments showed
isolates. Because
promising hydrocarbon
our ultimate goal was
to select isolates
degrading
with the
ability in
ability to
certain
utilize not just
40
Results
a mixture of
industrial
hydrocarbons, but eventually
investigation. Seven isolates
as well
microtiter plate-based
number of
different
economic and
easy
substrates.
In
experiments.
very
industrial
isolates for further
successful
These
clones were
efficient method
further tested in
for screening large
to flask experiment microtiter test was
comparison
and could address
most
due to their increased ability to degrade linear
that provided
assay
range of hydrocarbons, oils,
seven
were selected
branched hydrocarbons in batch
Discussion
much complex substrates such as crude oil and
waste, we chose the
organic
and
fast,
the demand for rapid degradation screening of wide
waste and other products.
3.3 Screening of bacterial isolates for utilization
of
hydrocarbons in
microtiter plate experiments
Being
number of
able
to rapidly test
hydrocarbons is important in
industries. Screening
inexpensive
Microtiter
method
plates
potential clones
strains
compounds.
provided a
chain
et al.1998).
that will
amount of
Two different
linear
using
a microtiter plate
growth
have been already extensively
be
This technology
large
bacterial isolates for
seen
We
data rapidly,
assays were
used
during
allowed
utilization of a
large
developing a commercial blend for use in various
for testing bacterial
(Muyzer 1998, Smalla
bacterial
potential
us
to
used
allows
diverse group
a
on
assay
in
applied
screen
a
to
a
large
of
organics.
ecological
this method to select the
exposure
for the rapid,
best growing
diverse type
number
research
of
organic
of substrates
and
inexpensively and reproducible.
developed in this
hydrocarbons from do to Cn
study.
was performed
First,
to
a set of experiments with
evaluate
the effect of chain
41
Results
length
flask
bacterial degradable activity (Figures 3.3.1-3.3.3). Based
on
from Genesee River
Lake (CLl,
CL7)
highest bacterial
range of
sediment
(GR1, GR2, GR4),
growth was observed
which
2000, Siddiqui
seen when
and
decane
directly
Adams 2001,
in the
and
water than
contained
flask
presence of
increase
a
However,
at chain
molecules
with
lower
that
with
surface area of
longer
found that the
hydrocarbons in the
-Cis) are more
Slightly
In
lower
the
of
rates of evaporation are
concentration of
growth would
be the solubility
GR1, GR2, GR4
14
and
more
and
carbon
substrate as well as
substrates
Therefore,
suppressed as well as microbial attachment to
droplets
fact that
decreased the
Ci0 and Q2, these
chain.
et al.
growth was
spite of the
the molecule becomes less soluble in
hydrocarbon
length
was
that our incubation temperature
gaseous phase and
experiments
isolated
biodegradation (Nocentini
though their
points and
for
preliminary
clones
hydrocarbons (C10
Bartha 1990).
to increase the affinity for hydrophobic
absorption.
that
short chain
explanation
increases,
additional
microbial surfactants
surface
in the
likely
the alkane chain
emulsifiers
boiling
amount of alkane
during
medium
(TCI, TC4). It
hexadecane, it is unlikely
to
the liquid alkane. A more
factor. As
Wang
tested; three
and undecane were used as the carbon source.
comparison
increased the
observed
in the
on the
Discussion
two clones isolated from Canandaigua
correlates to their ease of
hydrocarbons have lower
higher in
were
two from Toomey's Corner soil
and
C12-Q7 (Table 3.3.1). Typically,
bioavailable,
these
isolates
experiments the seven most successful
and
TCI
It
excrete
atoms.
These
modify the
thus
was
cell
facilitate their
n-alkanes are more adjusted
the
of
and
water.
the
formation
alkane.
of
surfactants
Application
in
is
of
42
Results
Figure 3.3.1
Growth of isolated
clones
from Genesee River
sediment on various
and
Discussion
hydrocarbons
0.00
C11 C12
C10
C14
C-15 C16 C17
TMPD
C1
C2
Hydrocarbons
Legend: Isolated bacteria
were
incubated in
plate contained
2 %
(pH 7). After 3
weeks of
Hass
medium at
600
microtiter plates
vv of organic compound and
nm.
incubation
After
optical
non-shaking at 26C. Each well of microtiter
v/v of inoculum in Bushnell-Haas medium
2 %
density
Bushnellof each strain was measured against
addition of p-iodonitrotetrazohum
indicator
wells were scored
for
TMPD=
positive results. Cl= control with no carbon source; C2= control with no microorganism;
2,6,10, 14-tetramethylpentadecane,
GR=
Genesee River isolates
43
Results
Figure 3 3.2
Growth of isolated
clones
from Canandaigua Lake
on various
and
Discussion
hydrocarbons
1.00
0.80
o
o
CD
Q
5
o
0.60
0.40
O
0.20
-
0.00
C-I0
Legend: Isolated bacteria
Cn
were
incubated in
plate contained
2 %
(pH 7). After 3
weeks of
Hass
medium at
600
C12
microtiter plates
vv of organic compound and
nm.
positive results. TMPD=
incubation
After
C15
C14
optical
C16
C-17
TMPD
non-shaking at 26C. Each well of microtiter
v/v of inoculum in Bushnell-Haas medium
2 %
density
Bushnellof each strain was measured against
addition of p-iodonitrotetrazolium
indicator
wells were scored
for
2,6,10, 14-tetramethylpentadecane, CL= Canandaigua Lake isolates
44
Results
Figure 3.3.3
Growth of isolated
clones
from Toomey's Corner
soil on various
and
Discussion
hydrocarbons
1.00
0.00
C-10
Cn
C-12
C-14
C-15
c16
C17
TMPD
Hydrocarbons
Legend: Isolated bacteria were incubated in
plate contained 2 % v/v of organic
microtiter plates
compound and
non-shaking at 26C. Each well of microtiter
vv of inoculum in Bushnell-Haas medium
2 %
(pH 7). After 3 weeks of incubation optical density of each strain was measured against
Bushnell-Hass medium at 600 nm. After addition of p-iodonitrotetrazohum indicator wells were
scored
for
positive results. TMPD=
2,6,10, 14-tetramethylpentadecane,
TC=
Toomey's Comer
isolates
45
co
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S
<u
CO
o
O
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5
o
+
c
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F
5
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CD
to
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+
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o
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l|6
e h
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U
>,
X
-a
CO
'
a
c
a
CO
'
;
to
^
3
_r
i:
c
o
&
ca
W
4
CO
u
-a
CO
o
o
o
5u
3
"o
o
-a
-a
E^
a
1=
c
ex
U
CN
as
Results
tetrazolium indicator
chain
hydrocarbons in
surfactants
and
showed
by
bacterial
GR1
the range of
isolated
Cm to Cn to
TCI isolates significandy
and
growth.
that all tested
Final
from
results
strains possess the
ability to
hydrocarbon
enhanced the
Discussion
utilize
the formation of
Yet,
some extend.
and
utilization
microtiter plate experiments after addition of
tetrazolium indicator are presented in Table 3.3.1.
Next,
of
the ability of isolated strains to utilize larger and more
hydrocarbons
Samples
of
and other organic compounds was tested
highly
oil, and common brands
goal of
of motor oil were
this study was to
bioremediation
industrial waste,
aromatic
prepare
processes on an
biodegradability
of
four
summarizes the results of
crude
The figures 3.3.4-3.3.6
days
of
indicate,
incubation
minimal
Tetrazolium test
were
scored
for
on
bacterial
growth
chain
substituted
hydrocarbons,
commercially
bacterial isolates. Final
with various
that would be applicable in
cocktail
investigated
was
the final
of these compounds
as
growth of seven
compounds.
As the
Therefore,
well.
in
observed
(GR1, CL7
and
TCI) from
on samples of organic
industrial
optical
waste
seven
waste.
density
substrates.
tested isolates
The GC-MS
very high
presence of saturated and unsaturated
alicyclic
hydrocarbons
as
significantly
contribute
3.3.2
microtiter plates.
for
industrial
on
Table
the
tested strain after 21
values
well
polycyclic aromatic compounds with substitution of sulphur and
features
available natural
scale and with crude oil spills.
was
that only three
analysis of the waste confirmed a
microtiter plate assay.
growth on more complex organics
organic
positive results
tested
samples
represent
various
showed
oil
bacterial
samples of
a microbial
industrial
using the
complex structures
high
presence
halogen. The
to their high
long
toxicity
of
structural
and
low
degradability.
47
Results
Figure 3.3.4
Growth of isolated
clones
from Genesee River
and
Discussion
sediment on various organic
compounds
1.00
0.00
CI
CM
Clll
UO
SQ
OL
PN
CN
CA
QS
Q
ML
Syn
AL SM
LP MX
C
Substrates
Legend: Isolated bacteria
were
incubated in
plate contained
2 %
(pH 7). After 3
weeks of
Hass
medium at
600
nm.
incubation
After
non-shaking at 26C. Each well of microtiter
2 % v/v of inoculum in Bushnell-Haas medium
microtiter plates
vv of organic compound and
optical
density
of each strain was measured against Bushnell-
addition of p-iodonitrotetrazolium
indicator
wells were scored
positive results. CI-CHI= organic and phenolic waste; UO= used motor oil; SQ=
olive
oil; PN= peanut oil; CN=
Syntec;
MX=
ML= Mobil
Mexican
crude
1;
AL=
com
Alaska
od; Ci6=
oil; CA= canola oil; QS= Quaker State
crude
hexadecane;
od; SM=
GR=
Smakover
crude
20W-30;
oil; LP=
for
squalene; OL=
QS=
Leeper
Castrol
crude
oil;
Genesee River isolates
48
Results
Figure 3.3.5
Growth of isolated
clones
from Canandaigua Lake
and
Discussion
on various organic compounds
I.W
J
0 80
Q
0.60
JL1
-
1 CL7
1
-
O
2
0.40
-
CD
0.20
-
_l
Legend: Isolated bacteria
I
1
were
incubated in
plate contained
2 %
(pH 7). After 3
weeks of
Hass
medium at
600
1
microtiter plates
v/v of organic compound and
nm.
incubation
After
optical
density
non-shaking at 26 C. Each weU of microtiter
v/v of inoculum in Bushnell-Haas medium
2%
Bushnellof each strain was measured against
addition of p-iodonitrotetrazolium
indicator
wells were scored
for
SQ= squalene; OL=
positive results. CI-CIII= organic and phenolic waste; UO= used motor oil;
QS=
CA=
CN=
Quaker
State
PN=
canola oil;
20W-30; QS= Castrol
com od;
peanut oil;
olive oil;
SM= Smakover crude oil; LP= Leeper cmde od;
ML=
AL= Alaska crude
Syntec;
MX=
MobU 1;
Mexican
crude od; C16=
oil;
hexadecane;
CL= Canandaigua
Lake isolates
49
Results
Figure 3.3.6
Growth of isolated
from Toomey's Corner
clones
and
Discussion
soil on various organic compounds
1.00
HB TC1
0.80
-
I
I 04
I
o
o
CD
o
0.60
-
o
-i
o
0.40
-
O
0.20
-
Lin
0.00
CIM
I
CIM
CIM
II
HI
UMO SQ OL PN
CN
QS
CA
Q
ML
AL
SM
LP
MX
C16
Syn
Substrates
Legend: Isolated bacteria
were
incubated in
plate contained
2 %
(pH 7). After 3
weeks of
microtiter plates
vv of organic compound and
incubation
optical
non-shaking at 26 C. Each well of microtiter
v/v of inoculum in Bushnell-Haas medium
2%
density
Bushnellof each strain was measured against
medium at 600 nm. After addition of p-iodonitrotetrazolium indicator weds were scored for
UO= used motor oil; SQ= squalene; OL=
positive results. CI-CHI= organic and phenolic waste;
Hass
QS=
olive oil; PN= peanut oil; CN= com od; CA= canola oil;
Syntec;
MX=
ML=
Mobd 1;
Mexican
crude
AL=
Quaker State 20W-30; QS= Castrol
Alaska crude od; SM= Smakover crude oil; LP= Leeper crude od;
od; Q6=
hexadecane;
TC= Toomey's
Comer isolates
50
CD
1/1
l-
CO
I*
a>
P
ft "w
o
o
b-
u
iCO
.
a
W
U
S-
1
re
0_
o
+
u
+
+
i-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-=
o
+
+
+
CJ
CO
zz
o
a
iz
3
.SP
'ca
zi. CO
r-
U
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
c
f-
CO
c
CO
C
CO
u
^ E
J
"?
<U
CO
O
u
CO
i-
s
c
u
+
+
+
=-
o
>,
^-v
C
O
d>
T
CO
1/1
c
c
.c
L.
a:
"o
+
+
a
+
+
+
.
5
o
8.2
u
w
o
CO
o.
E
E
co
ii
g
b
3 -a
m
5
in
p
.2
fc
cr
1/1
TJ
C
3
C
a
1/1
o
CN
t-
a:
+
+
a
+
+
+
+
1/1
si
CO
+
+
_i
o
o
c
S
J3
c
(1)
CA
c
3
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o
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i:
c
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c~
o
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CN
a
03 -5
c
-
II
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c
+
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L.
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+
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c
c
a
u-
-*<-
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t
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+
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s-
1
c
c
o
12
CO
c
o
o
c
c
O
c
'5
o
TD
e
c
CO
o
i-
o
o
U
<r
to
CO
C CN
k.
O
CO
3
CA
VI
E
re
3
5
a
00
c
3
in
c
u
c
o
U
t-
-a
CJ
o
3
-c
CN
3
-a
E
3
c
C
U
C/J
o
-
=
E
"
3
o
re
E
c^
CJ
*-
U
U
U
D
c
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co
C
>
g
C
S
u
-
o
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o
c
-*
a>
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O
CO
)-
a.
U
U
O U
<
c/3
_)
I
U
U
u
II
CA
CO
U
c
CO
T3
3
u
CO
o
C
UJ
c
CJ
T5
a>
o
CA
-O
CJ
6X
c
c
5
c
c
>
u
to
c
o
T3
5
c
"o
u
o
C
-C
c
CN
CO
O
CO
o
E
+
+
a
c
i-
O
II
c
o
CA
CA
o
'^
JS
'5
o
.5
CA
c
c
u
Results
As the figures further indicate,
enhanced in the
is
canola oil
fatty
elucidated
easy target for
and
oxygen
for
mono-
or
Slightly
motor oil samples.
Castrol Syntec in
with used motor
used motor
isolates
oil,
possess a
themselves to
incubation
Additional
protection
and
is generally
optical
Mobil 1
oil, the
and
optical
microbial growth
diverse
resistance
ion toxicity. It is
contribute
to
studies might
density
heavy
possible
elimination
of these
and
isoprenoid
and
requires
insertion into
the
towards
molecular
to metabolize
GR1, TCI
Considering
effected
metals.
for GR1
and
and
CL7
a presence of
by
these
all
metals
heavy
metals
uptake.
in the isolated
strains
which mechanism
is
on
cells were
cultures was
heavy
elements.
The best growing
tested
TCI
metals
Bacterial
clones adapted
that TCI cell's clustering, observed
of
were
these enzymes in their metabolic
were observed
of
CL7
molecule, the
Quaker State 20W-30. When the
is apparently
to
isoprenoids
strain were able
be necessary to clarify
heavy metal
of
one of
densities
make them an
considered as recalcitrant
catalyzed oxygen
least
(C30)
triterpene
GR1, GR2, TCI, TC4
metabolism
CL7 isolated
higher
for transformation
from
an acyclic
growth on unused motor oil.
metal
might
mechanism
Squalene,
contain at
comparison to
twice as low as the
in
likely
(9-32%)
percentage of polyunsaturated
Because the
GR1, GR2, TCI
apparatus.
incubated
lower
di-oxygenase
above mentioned strains
growth on olive oil and
their composition; with a high percentage of monounsaturated
our experiments show that
to utilize squalene.
tested clones was significantiy
The highest bacterial
a chemical structure that
degradation. However,
able
by
microbial utilization.
has
compound
microbial growth of all
presence of natural oil.
(62-77%)
acids
the
Discussion
and
Yet,
is
during
the exact
not
responsible
known.
for
cell
toxicity.
52
Results
The ability
using
of
isolated
composition and viscosity.
on
Leeper
lowest viscosity
were
composition
The
GR1
of
of
with
and
CL7,
able
growth was observed
tetrazolium indicator
very
diverse
were scored
oil resulted
oil was characterized
dark brown
for
in five
Smakover
and
and
following
black
chapter.
positive results on
positive scores.
metabolic
activity
to utilize the Smakover
crude oil.
After 21 days
in any
of
the inoculated
wells and color
on
this
Only
wells
crude oil.
to
TCI,
substrate.
None
of
detection
The
color.
Alaska
by
crude oil
Additionally
CL1 indicated the
and
study.
The
determined
on
by
be discussed in
use of
that the
for screening
linear
the chain hydrocarbons
complex compounds was more
chain
of
incubation
no
after addition of
hydrocarbon degraders
hydrocarbons
isolates GR1, CL7
range of organic molecules.
activity
the greatest bacterial growth was
Mexican, Alaska
characterized
CL7
the sample differed in the
CL1 isolates. This
yellow color.
will
show
of
by
was also negative.
in this
organic compounds
metabolic
and
and
use of microtiter-based assays
efficient
utilize a
oil
Mexican
GR2
also
for TL7
were
TCI, GR1
the isolates was
The
oil
particular
experiments with
and
Figures 3.3.4-3.3.6
very light
and
viscous
very
inoculated
crude
Discussion
to degrade petroleum products was assessed
from different locations. Each
crude oil samples
detected
strains
and
and
and natural
as well the use of
TCI have
Although, GR4, CL1
was
and
a
high ability to
TCI
showed the
oil, their ability to degrade more
limited.
53
Results
3.4 Biodegradation
of crude oil
in biometric
and
Discussion
flasks, evolution of carbon
dioxide
During
as
study the degradation
measure the
compounds.
amount of carbon
Respiratory
pure cultures were measured
strains exhibited the
alkanes as
Because
cumulative
Leepershank
with
dioxide
as well
after
of carbon
production rates.
day
growth
as
the
dioxide
of
C02
C02
during incubation
in
that could be
of
and was
of medium.
incubation. Its
followed
achieved maximum
CQ2
by
a
and
CL7)
on medium chain
in
length
isolates for their ability to
during
began
about
clones.
The
the
were used as
and
mineralization
GR2
4 days
by
rapid
exhibited
evolution
decline
inoculation for
production remained rapid
significant changes
C02
of
exhibited similar
after
21 days for GR2. This
maximum
production
and
biometric flask. These
a
from different locations
CL7 isolate
very
of organic
utilization
microtiter plate experiments.
successful
evolved
GR2
observed
is to
microorganisms
evolution.
production
and
by
using
in Figure 3.4.1. GR1, TCI
later for TCI
turbidity
first 12 days
18 days
isolates
activity
isolated bacteria. Another
isolates (GR1, GR2, TCI
growth on crude oil
first 12 days for GR1, 19 for TCI
bacterial
during
selected
weight of cells were used
during
evolved
samples of crude oil
amounts
of
hydrocarbons
to test the most
crude oil are shown
clone and one
during
four
metabolic
hydrocarbons,
for determination
The
GR1
during
our ultimate goal was
substrates
of
well on more complex organic compounds
utilize crude oil
carbon
highest
potential
dioxide that is
activities of
dry
growth and
indicators for evaluating biodegradation
effective method to
oil
bacterial
previous experiments
within
period coincides
in the
lower
surface of
the
production rates
(987 u-mol)
following
(1234 fxmol) generally in the
was reached
six
same
days. TCI
time
54
Results
Figure 3.4.1
Production of carbon dioxide during Leepershank crude
bacteria
oil
and
Discussion
degradation by isolated
1400
1200
1000
T3
a>
>
o
>
CD
800
CD
600
X
o
"O
C
400
o
o
l_
CO
O
200
20
Time
Legend: C02
and
production was monitored
during
CL7 isolates. Bacterial isolates
v/v od
in
a
biometric flask. Incubation
microorganisms.
(days)
utilization of
were
40
Leepershank
inoculated to 48
was performed at
GR1, 2=Genesee River isolates,
ml of
crude od
by GR1, GR2, TCI
Bushnell-Haas
medium with
23C. Control flask did
TC1=
Toomey's
not contain
Comer isolate,
2%
any
CL7=
Canandaigua Lake isolate.
55
Results
frame
days
as
of
CL7 (18 days), but C02
incubation. Table 3.4.1
for
crude oil
composition of
represents
isolates. GC
all
hydrocarbons
very low
branched
chain
TCI
length
and
alkanes.
CL7)
percentage
As the
oil consists of medium chain
results
with
spite of
use aliphatic
Leepershank
increasing
utilization of
as
significantly
their
of
source
solubilized oil
for GR1
of
the
80 % for
The degradation
Branching
of carbons.
all
and
and
carbon.
production of
access
due to
the
these hydrocarbons
tested bacteria
incubation. The
oil
and
compounds was seen with strain
to efficiently
the Leepershank
carbons.
utilization of
energy
by
during
GR1
were able
Biodegradation
bacterial
production
by
to
of
surfactants
bacterial
of
hydrocarbons. Picture 3.4.1
surfactant production
by
GR1
TCI.
Respiratory
activities of
crude oil are represented
evolution of
by
numbers
these
crude oil was also characterized
shows the changed surface of
and
10-14
with
detected
and a
to degraded the medium
were able
(approximately 90 %
hydrocarbons
that was observed visually after 14 days
surfactants
isolates
all
slightly lower degradation for GR2,
hydrocarbons
length hydrocarbons
aromatic compounds were
significantly influenced the
the various isolates. The highest
TCI. In
No
indicated,
on
slightly decreased
addition of side chains
and
alkanes.
The best degradation
was observed
Leepershank
of
differences in the
significant
showed
the last 10
during
degradation
Discussion
the end of the experiment (Figure 3.4.2). As Table 3.4.1
at
crude
analysis of this oil.
the percentage of
chromatographs
indicates, Leepershank
percentage of
decreased continuously
production
and
all
C02
isolates
was
over
isolated bacteria
in Figure 3.4.3. The
very
62 days
obvious
of
in this
incubation
measured
utilization of
The total
amount of
significantly lower than that
Mexican
the oil on the
effect of composition of
experiment.
was
during
C02
produced
produced
56
Results
and
Discussion
Table 3.4.1
Degradation
of
Leepershank
Hydrocarbon
crude oil
by
isolated bacteria
Retention
Percentage
of
degradation
time
GR1
GR2
Decane
10.29
90.32
82.10
86.86
84.31
Undecane
11.63
92.81
78.16
85.57
85.37
Dodecane
13.03
93.37
67.89
83.65
83.01
Tridecane
14.27
94.76
65.70
82.09
76.89
Tetradecane
15.41
89.11
57.86
79.00
75.69
Pentadecane
16.46
85.43
60.64
74.50
66.56
Hexadecane
17.45
84.90
58.30
67.62
71.34
Heptadecane
18.36
82.81
56.39
63.18
65.18
TCI
CL7
Octadecane
19.25
71.30
50.43
53.48
66.02
Nonadecane
20.13
69.15
42.87
41.45
63.15
Eicosane
21.01
61.14
37.49
33.16
50.77
5-ethyl-hexadecane
12.43
35.39
15.67
22.4
32.14
4-methyl-undecane
12.50
60.83
17.89
28.3
37.4
2-methyl-undecane
12.56
59.40
13.12
21.54
32.12
3-methyl-undecane
12.65
62.40
23.4
27.5
27.65
2,6-dimefhyl-
13.24
48.62
12.34
34.98
29.76
undecane
3 -methyl -tetradecane
16.17
34.55
11.45
31.98
32.67
2,6, 10,1 4-tetramethyl-
17.92
42.10
19.87
34.56
12.45
hexadecane
Legend: The
percentage of
experiments
after
degradation
35
days
Canandaigua Lake isolate
of
of
Leepershank
incubation
and TC=
crude oil
at
23
C.
by 4
microbial
GR1=
isolates in biometric flasks
Genesee River isolates, CL7
Tommey's Comer isolate.
57
Results
and
Discussion
Figure 3.4.2
Gas
chromatographic
analysis
of
Leepershank
crude
before
oil
and
after
degradation
!J
ISM
11.00
12.00
1100
1(00
15.00
16-00
hii
17.00
1100
1500
20.00
a.)
i
g),JiJ^^J-
1S00 1650 1700 17K 1SK ll
m m mis na ttoc m 1U 1W M00 W0 M 89
UH
Legend: The GS/MS
If10 II*
lifl lino * ISOO HO
analysis was performed
Model 6890. The
IMO lito ISH jub M
using
a
MS-80
E0[,
M
c)
spectrometer coupled to a
showing the medium
oil a.) 0 hour b.) 18 days c.) 32 days after microbial
inoculated with Genesee River 1 isolate.
chromatograms
b.)
chain
hydrocarbons
utilization
of
Hewlett-Packard
Leepershank
in biometric flask
crude
experiments
58
Results
Picture 3.4.1
View on Leepershank
crude oil
Legend: The
bacterial
production of
microbial utilization of
medium, 1.0
ml of a
filled
ml of 0.1
with
10
River isolate #
Discussion
biodegradation in biometric flasks
surfactants
Leepershank
48-hour
and
during
measurement of carbon
dioxide
evolution
during
in biometric flask containing 48 ml Bushnell-Haas
2% vv substrate. The sidearm of biometric flask was
crude oil
culture and
M KOH. Flasks
were
incubated
2; Right flask: Genesee River isolate #1
at
23
after
C, non-shaking.
22 days
of
Left flask: Genesee
incubation.
59
Results
during
the degradation
bacterial
strain
GR1
and
There
CL7
was no
in Leepershank
C02 is
evolution of
crude oil
by
caused
this amount was even
less,
Leepershank. Evolution
maximum
low
activity
of
percentage
TCI bacteria
degradation
lower ability
or
1
did
not occur
,4,6-trimethyl
The
evaluate the
evolution
of
remained
by
pattern
possible that
3
weeks of
was
by
not
fluctuating
the
The total
amount of
the quantity that was
bacteria. For CL7
the total
was
clone
C02 formed
more
incubation. It
can
linear
be
and
seen
on
the
that in
to GR1 or CL7 strains, respiratory
for 20 days
stable
of
oil.
of
same
TCI bacteria
comparison
from the
by the
only 39.12%
dioxide
C02 in
area peak of
without
any fluctuation. The
individual hydrocarbon indicates
to utilize aromatic compounds such as 2,3-dimethylnapthalene
hydrocarbons
biodegradation process,
as a tool
above
increase followed
increase. This
only 50%
crude oil
and represents
analysis of crude oil
data
C02
naphthalene.
and can
by
play
GC-MS is
an
a powerful measurement to
important role in validating the C02
for evaluating hydrocarbon degradability. Table 3.4.2
differences in efficiency
discussed
Leepershank
calculated
of this strain
rapid
in Leepershank
represents
was reached after
concentration
exhibited
the inoculated samples.
very
of second
TCI,
each
sequential utilization of more complex compounds, such as
of carbon
(160.30 umol)
spite of
of
of
Discussion
fluctuates for
evolution
degradation. It is very
dioxide (526.11 [xmol) for GR1
formed during degradation
in any
exhibited a similar pattern;
aromatic and cyclic molecules that
carbon
C02
oil.
lag period
decrease that lead to formation
significant
observed
crude
the entire incubation period. Each strain except
during
more than one maximum.
evolution of
Leepershank
of
and
of
degradation for
that the lowest evolution of
each
bacterial isolates. It
C02 by TCI
reveals the
was
already
correlated with the results
60
Results
Figure 3.4.3
Production of carbon dioxide during Mexican
bacteria
crude oil
degradation
and
Discussion
by isolated
400
Time
Legend:
C02 production
was monitored
(days)
during utilization of Mexican
crude od
hydrocarbons
by GR1,
CL7 isolated bacteria. Bacterial isolates were inoculated into 48 ml of BushnellHaas medium with 2% v/v oil in a biometric flask. Incubation was performed at 23C. Control did
not contain any microorganisms. GR1, 2=Genesee River isolates, TC1= Toomey's Corner isolate,
GR2, TCI
CL7=
and
Canandaigua Lake isolate.
61
Results
obtained
from
chromatographs.
length
medium chain
alkanes
This
all
(47%-54%)
were obtained on alkanes with
addition to
its higher degradation
63% for undecane),
aromatic
for
be
3.4.2 After 62 days
medium
inoculated
consisted of
became
more
of
C02
caused
by its
the medium
chain
mineralize
GR1
and
the
length
The
easily
aromatics as
identified
only 5%
of
changes
were
and
significant
C02 fluctuation
shown
from Table
degraded in
mass
and
to
the
spectrometry
1
its color, lost its
changes of oil
previous
during
viscosity
and
collectible.
amounts
C02
evolved.
not exceeded
the amount
comparison to control
on
evolved
Among
During
60
produced on
observed
C02
of
in Figure 3.4.5.
amount of
did
(up
the Mexican crude oil significantly changed
changed
slightly
is
by
incubation. Picture 3.4.2 illustrates the
oil
cumulative
evolved
of
alkanes
and
percentage of n-alkanes were
major residues
rates
branched hydrocarbons
evolution
on
GR1 bacteria
chain.
the growth of CL7 isolate.
on
ability to degrade
incubation, high
of
with
crude oil are shown
the lowest
illustrated
also
period of
period.
The
in the
carbon atoms
branched-chain alkanes, like 2,3-dimethyl-napthalene
increasing
incubation
10-17
ability to
(Figure 3.4.4). The consistency
napthalene
with
is
utilization
this strain might
highest degradation
The relationship between high C02
compounds.
hydrocarbon
tested bacteria. Its
potential on
also exhibited the
Discussion
lowest degradation capability
strain exhibited
among
and
jimol
during
mineralization
of
Smakover
the tested crude oil samples, Smakover had
68 days
of
for GR2
Leepershank
incubation the
and
TCI strains,
crude oil.
the surface of the oil
maximum amount
During
inoculated
which represents
this period no visual
with
GR2
and
CL7 in
flask.
62
Results
and
Discussion
Table 3.4.2
Degradation
of
Mexican
Hydrocarbon
crude oil
hydrocarbons
Retention
isolated bacteria
by
Percentage
time
GR1
TCI
of
degradation
CL7
GR2
Undecane
11.64
63.24
52.14
76.58
62.34
Dodecane
13.03
63.70
54.12
72.03
60.21
Tridecane
14.28
59.44
51.98
76.45
61.76
Tetradecane
15.41
58.01
48.97
73.35
56.43
Pentadecane
16.46
57.52
46.65
63.64
54.11
Hexadecane
17.45
56.26
49.78
69.64
58.97
Heptadecane
18.36
46.06
47.32
69.25
51.23
Octadecane
19.25
43.18
37.89
68.09
49.67
Nonadecane
20.13
45.48
34.78
59.19
44.26
Eicosane
21.01
41.30
26.30
52.98
31.43
Heneicosane
21.94
40.22
16.87
52.12
27.45
Docosane
22.94
35.90
18.98
ND
21.38
Tricosane
24.10
35.84
12.98
ND
23.44
2,3-dimethyl-napthalene
15.69
13.51
9.07
14.32
12.11
4-methyl -undecane
12.5
40.54
12.34
17.93
25.65
21.26
2-methyl-undecane
12.56
42.02
15.63
27.44
2,6-dimethyl-undecane
13.23
38.43
11.34
20.81
19.87
4-methyl-dodecane
13.79
36.14
16.73
36.69
21.56
4-methyl-tetradecane
16.03
58.34
12.54
40.98
18.76
2,6, 10,1 4-tetramethyl-
18.46
28.51
11.23
19.76
32.11
16.83
15.60
4.52
13.62
7.62
19.39
43.64
12.45
18.76
25.67
4
isolates in
pentadecane
1
napthalene
2,6, 10,1 4-tetramethylhexadecane
Legend: The
percentage
biometric flasks
CL7=
of
degradation
of
experiments after
Canandaigua Lake isolate
Mexican
35 days
and TC=
of
crude
oil
hydrocarbons
incubation
at
23 0C.
by
GR1=
Tommey's Corner isolate.
ND=
microbial
Genesee River isolates,
not determined
63
Results
Figure 3.4.4
Gas chromatographic
analysis of
Mexican
crude oil
before
and after
and
Discussion
degradation
^^kL ^-^a.
24.00
.00
26.00
28.00
a.)
9000000
aoooooo
7000000
6000000
soooooo
4000000
3000000
2000000
wft^^i^^vL-
'000000
14.00
Legend: The GS/MS
16.00
analysis was performed
Model 6890. The
b.) 68 days after
River # 1 isolate.
chromatograms
microbial
using
b.)
20.00
18.00
a
MS-80
the chain
spectrometer coupled to a
hydrocarbons
showing
in biometric flask
utilization
of
Mexican
experiments
Hewlett-Packard
crude oil
inoculated
a.) 0 hour
with
Genesee
64
Results
and
Discussion
Picture 3.4.2
View
on
Mexican
crude oil
biodegradation in biometric flasks
^| W
Legend: Change
of
consistency
of
the Maxican crude oil
during
measurement of carbon
dioxide
evolution
ml of
in biometric flask containing 48 ml Bushnell-Haas medium, 1
substrate. The sidearm of biometric flask was filled with 10 ml of 0.1
vv
a 48-hour culture and 2%
M KOH. Flasks were incubated at 23 C, non-shaking. Picture above: Comparison of control
flasks and inoculated flask; Picture below: Closer view on a control flask (left) and inoculated
during
flask
microbial utilization
with
Genesee River isolate #1
.0
(right)
after
20 days
of
incubation.
65
Results
Unlike the
respiratory activity
reached
The
99.12
strains,
GR1
this oil. After 50 days
for GR1 isolate
(112.28 nmol)
of
and was
and
continuously
day
was reached on
produced on
15 days
after
chromatogram
oil composition after
decreases in branched
no significant
68 days
of
indicated that majority
incubation
alkanes such as
degradation
After 50 days
of
the oil, started slightly
to red; the
droplets
and
of
with
GR1
2,3-dimethyl
bacteria
It
C02
next
inoculation. This
Smakover
evolved
12 days.
amount
exhibited
after
first
39 days.
crude oil consists
high
substitution of
chromatographic analysis of
and
CL7
pentane and
strains showed
only
3-methyl hexane. Yet,
of aromatic and cyclic compounds was seen.
of
GR1
inoculation,
changing.
could
be
collection.
was obvious
the oil droplets into
higher
was accompanied
by
with
significant visual changes.
GR1 in
comparison
the color of the medium, as well the
At the
end of
to the
consistency
of
the experiment, the colorless medium
smooth and viscous surface of oil was modified and small clusters of
that facilitated easier
small colonies.
for
increase (199.90 fxmol)
Picture 3.4.3 depicts the biometric flask inoculated
oil
amount of
Leepershank. CL7 isolated
and second
Discussion
exhibited
increasing
after
(Figure 3.4.6, Table 3.4.3). The
The respiratory activity
changed
isolates
complex organic compounds with aromatic character with
alkyl chains and even sulfur
control.
CL7
incubation the
62nd
(211.65 u.mol)
The GC-MS
very
two
20.15% from C02
represents
of
on
[xmol
maximum
maximum
previous
and
smaller
seen on
the
surface.
Cells become
tightly
that bacteria produce a
units,
thereby producing
Oil
changed to a new
attached
consistency
to oil droplets
dispersing agent(s),
which
forming
broke up
new surface area.
66
Results
and
Discussion
Figure 3.4.5
Production
bacteria
of carbon
dioxide
during Smakover crude oil degradation by isolated
300
20
40
Time
Legend: C02
production was monitored
GR2, TCI
Haas
and
medium with
not contain
any
during
CL7 isolated bacteria.
2%
v/v od
in
microorganisms.
CL7= Canandaigua
a
utilization of
(days)
Smakover
Bacterial isolates
were
biometric flask. Incubation
crude od
hydrocarbons
inoculated into 48
was performed at
GR1, 2=Genesee River isolates,
TC1=
by GR1,
ml of Bushnell-
23
C.
Control did
Toomey's Comer isolate,
Lake isolate.
67
Results
The
data for
gas chromatographic
GR1
neither
nor
TL1 brought
about
and
Discussion
significant
any
changes in the profiles of the crude oil hydrocarbons. It was elucidated in studies of Adas
(Atlas, 1981)
that microorganisms
extensively degrade the hydrocarbon in
both GR1
and
TCI incubation
microorganisms
their
ability to
did
the
positively
affect
previously
utilize various
of
of
which
is
and
of
substances
by
experiments
oil
the
degradation
effectiveness
TCI
and
of
of
in
fact that these
on
GC analysis,
rates
have
that secrete large quantities of
active agents might
microorganisms
indicated that the C02
very
that could
not
be
for
strains could
used
hydrocarbons,
biodegradation
by
evolution rates correlate with
time-consuming
to
for evaluating
biodegradation. In
interaction between individual bacterial
during
not
applied
In fact, biodegradation
other
do
their substrate specificity.
somewhat complex and
crude
often
oil
spite of the
generated surface
evolution rates provided an essential criterion
determination
of
degradation ability based
not negligible.
TCI
In
hydrocarbons. For these reasons, both
bioemulsifiers for
data,
crude oil.
use of microorganisms
GR1
uptake
Biometric flasks
the GC
by
enhanced
such surface active agents.
isolation
Smakover
biosurfactants is
emulsification
the oil. This phenomenon might be
not exhibit a significant
produce
been significantly
on
of
capable
strains
order
and
to
Thus
the
C02
the optimal methods
gain
examine
produce.
for
better insight into the
the population changes
the next set of experiments
the bacterial mixtures of four
investigated the
isolates, GR1, GR2,
CL7.
68
Results
Figure 3.4.6 Gas
chromatographic analysis of
Smakover
and
Discussion
crude oil
-aillp^*^
a
I
I
|
I
i
20.00
Table 3.4.3 Composition
Organic
of
Smakover
time
Cyclohexane
cyclopentane
l-ethyl-2-methyl1,2,-dimethyl-
cyclohexane
ethyl-
1
I
|
:
I
22.00
i
i
,2,4-trimethyl
l-ethyl-2-methyl-
5.30
41,56,69,84
5.61
41,56,70,83,98
7.27
41,55,70,83,97,112
7.41
41,55,69,83,97,112
7.02
41,55,67,83,99,112
8.11
41,55,69,84,97,111,126
8.51
41,55,70,83,97,112,126
41,55,69,79,91,105,120
41,55,77,91,105,120
1,3-diethyl-
12.30
41 57,69,79,91,105,119,134
2-ethyl-1,4-dimethyl-
13.56
41,57,69,81,109,119,134
|
26.9
41,57,69,81,91,109,119,134
tetramethyl-
1 -methyl
M
m/z
10.75
1
|
(min)
1
benzene
i
crude oil
10.59
1-ethy]-2-methyl-
|
24.00
Retention
compound
1
I
naphthalene
16.31
41,57,71,81,97,115,128,142
2,3-dimethyl-
17.63
41,57,69,83,95,115,128,141,156
1
18.98
43,57,71,85,97,119,133,155,170
3-methyl-dibenzothiophene
21.98
43,57,71,85,105,119,133,147,165,179,193
tricosane
16.70
43,56,71,85,97,111,133,145,159,174,196,211
1
17.55
41,57,69,83,97,109,123,145,159,173,190,207,224,281,294,306
1-eicosene
18.41
43,57,71,83,97,119,133,147,161,173,187,207,224,243,258,281
1
20.32
43,56, 69,84,99,109,120,145,163
-hexacosene
-chloro-nonadecane
Legend: Gas
chromatographic analysis of original sample of
with retention
column of
times and
m/z ratio.
Sample
was
Smakover
crude oil
diluted in hexane
(above)
and
lul
and composition
was
injected
onto
GC-MS.
69
Results
and
Discussion
Picture 3.4.3
View
on
Smakover
Legend: Measurement
crude oil
of carbon
biodegradation in biometric flasks
dioxide
evolution
during
microbial utilization of
Smakover
crude oil
in
ml of a 48-hour culture and 2% vv
biometric flask containing 48 ml Bushnell-Haas medium, 1
substrate. The sidearm of biometric flask was filled with 10 ml of 0.1 M KOH. Flasks were
.0
at 23 C, non-shaking. Picture above: Comparison of control (left) and inoculated flask
Genesee River isolate #1 (right) after 46 days of incubation; Picture below: Formation of
significant red pigmentation in the flask inoculated with Genesee River isolate #1 (right); control
incubated
with
flask (left).
70
Results
3.5 Biodegradation of crude
and
Discussion
by the bacterial
oil and organic compounds
consortium
Biological treatment
methods
for
relies upon the cooperation of more than a single
true
pure
when complete mineralization of
bacterial
culture
compounds or to
may
enzymatic
may be
capacities
individual isolates in
This is particularly
and
H20 is desired. A
compounds
achieve
total
certain
rapidly
enough
with
overall
of mixed populations
to
degradation
the
of
directed towards understanding the
experiments were
a mixed culture and
generally
capability to readily degrade
consortiums
required
remediation
species.
hydrocarbons to C02
the metabolic
Therefore,
For this reason, further
petroleum.
roles of
have
the
bacterial
have the biomass necessary to degrade the
to meet treatment criteria.
broad
not
hydrocarbon
petroleum
to
develop
a
formulation that
can
be
directly employed into a contaminated area.
In
order
to gain
insights into
the interaction between
growth of each strain was examined on
Mexican
Respiratory
mixtures
GR1/GR2
activities
were measured
experiments.
isolates
inoculation
during
highest
growth
CL7
strain
by
a
production of
and remained rapid
[imol was reached after
107
microbial
As Figure 3.5.1 indicates,
exhibited the
growth of
of
20 days
during
of
the
crude oil and sample of
bacterial
weeks.
incubation. This
within this period
mixture
method as
The
in
containing GR1
C02. C02 production began
first 3
industrial
continued
previous
CL7
immediately
after
amount, 954.34
period coincided with rapid
tenfold and
and
and
maximum
concentration of
the
waste.
GR1/TC1, GR1/CL7, TC1/CL7
using biometric flask
(Table 3.5.1). The initial
CFU/ml) increased
oil-degrading bacteria,
bacterial
the bacterial cells (2.70 x
increasing up
to
71
Results
Figure 3.5.1
Production of carbon dioxide during Mexican degradation
bacteria
and
Discussion
by mixtures of isolated
1200
o
1000
td
CD
>
800
o
5
CD
600
g
"X
g
TD
400-
c
o
-Q
CO
o
200
-
30
20
Time
(days)
Legend: C02 production was monitored during utilization of Mexican crude od by mixtures of isolated
bacteria. Bacterial isolates were inoculated into 48 ml of Bushnell-Haas medium with 2% v/v od
in
a
biometric
microorganisms.
flask.
GR1,
Incubation
2=Genesee
was
performed
River
isolates,
at
23
TC1=
C.
Control did
Toomey's
not
contain
Corner isolate,
any
CL7=
Canandaigua Lake isolate.
72
Results
36 days. In the later
phase of
reached the value of
3.5.1,
7.8
108
CFU/ml (Figure 3.5.2). As
the concentration of CL7
isolate
was tenfold
during the majority of incubation period.
its ability to form
attributed to
substrate
evolution of
produced
C02 fluctuating
by
single
GR1
(respectively
32 % for
consortium of
GR1/CL7.
Bacterial
after
16 days
GR1/TC1
and
of
to
as well with
in
clone
incubation,
8 days before
GR1
and
GR1ATC1
indicates,
TCI isolates
which was
represents
for this
its
40.6%
the bacterial
the entire
on
maximum evolution of
(6.5xl08
evolution of
of
C02
concentration
PCA
plates
rapidly
and
by
highest
bacterial
production of
C02
GR1/GR blend. This
The
by
and
amount of
C02
produced
might
by
be direct
their individual
C02
produced
by
GR1/CL7 blend. As the Table 3.5.1
for GR1/TC1
was
was represented
increasing
CFU/ml)
C02 between 28
produced
produced
clones
dioxide
approximately 34 %
maximum of
between individual
the
strains,
tenfold lower in comparison to
showed, that a majority of the colonies in this mixture
49 days incubation
mixture started
maximum
4 days before
be
access of cells to
amount of carbon
amount
exhibited
particular petroleum components.
GR1/CL7. Colonies
during
total
GR1
strain could
single
with
experiments
of
GR1
facilitate better
bacterial. The total
isolate)
evidence of cometabolic relationships
ability to degrade
concentration of
and
from the Table
seen
concentration of
previous experiments represents
CL7
single
mixture of
respiratory
be
can
lower than
surface active agents that
Similarly
molecules.
The higher
Discussion
gradually decreased
the experiment, cell concentration
x
and
at
day
from
day
48. This
45 days. GR1
by
24
GR1
of
clone.
incubation
period
coincides
clone exhibited
The total
cell yield
period and reached
with
a
significant
the fastest growing
73
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J
Results
Figure 3.5.2
Growth patterns in CFU/ml
6.50E+8
of
bacterial
mixture cultivated on
Mexican
and
Discussion
crude oil
GR1/TC1
4.70E+8
9.20E+7
#
4.20E+7
?
TC1/CL7
?
?
4.50E+7
E
?
2.30E+7
?
o
?
7.80E+6
c
o
o
6.70E+9
"53
A
A
GR1/CL7
O
4.30E+8
A
A
A
A
2.70E+7
8.90E+8
?
?
GR1/GR2
3.20E+8
^>
?
?
4
1.30E+7
24
12
Time
Legend: Growth
PCA
of
individual
species of
plates and cultivated
Corner
isolate,
CL7=
bacterial
for 48 hours
at
36
49
(days)
by plating 0.1 ml of medium on
2=Genesee River isolates, TC1= Toomey's
mixture was monitored
26
C.
GR1
,
Canandaigua Lake isolate.
75
Results
ability among
observed
all tested clones.
in flasks inoculated
interacting role
unknown,
of
results
a
broad
TCI
with
lower respiratory activity
growth as well as
and
CL7
clone.
In
Discussion
spite of the
fact,
was
that the exact
these two clones in the decomposition of Mexican crude oil sample
indicate that
particular compounds.
with
Reduced
and
This
bacterial
each
be
might
strain
attributed to a
specificity attacking the
substrate
posses
lack
or
lower ability to
lower activity
hydrocarbon
complex
is
utilize
of enzymes
molecules
and
metabolizing it.
As Table 3.5.2 indicates,
medium
inoculated
with
GR1
proportion of the aliphatic
indicated in
fraction
substitution
along
GR1
medium
and
significantly
affects
of
Mexican
(Picture
crude
oil
13
branched
linear
alkanes.
compounds.
hexadecane
This
The
of
bacterial
occurrence
resulted
blend
of
4
confirms
by
aliphatic
was
degraded
mineralized as
branched
alkanes with
chromatogram
and
3
less readily
and simple
branched
mixture
3.5.3)
in
51%-65%
of
in
was
slightly
than
comparison
the
of
alkanes.
with no more
pronounced
substitution
(Figure
promising ability
hydrocarbons
less
is
the fact that the degree of
GR1/GR2
about
methyl-groups
of
completely
very high
inoculated
alkanes
the experiment and a great
carbons was attacked
hydrocarbons
in 42% respectively 45%
of
structure of
show
detected for
was
Degradation
3.5.1)
aliphatic
lower. Complete degradation
carbons.
This
alkanes.
detected in the
alkanes were
crude oil was
biodegradation. The GC
CL7 isolates to degrade
The degradation
Mexican
49 days
length longer than 14
linear
with visual observation
and
of
clones at
(Figure 3.5.3). The
substituents and those with chain
than those of
CL7
and
chromatographs
length
no medium chain
to
branched
pentadecane
and
degradation. The significantly
76
Results
and
Discussion
Table 3.5.2
Degradation
of
Mexican
crude oil
hydrocarbons
GR1/TC1
time
2
mixture of
Percentage
Retention
Hydrocarbon
by
isolated bacteria
of degradation
GR1/CL7
GR1/GR2
TC1/CL7
Undecane
11.64
61.24
ND
46.38
ND
Dodecane
13.03
63.71
ND
42.03
ND
Tridecane
14.28
59.23
ND
46.35
ND
Tetradecane
15.41
58.01
ND
43.34
76.43
Pentadecane
16.46
57.54
ND
42.61
84.11
Hexadecane
17.43
56.21
ND
45.24
78.94
Heptadecane
18.36
56.03
ND
39.15
81.23
Octadecane
19.24
53.12
ND
38.09
79.61
Nonadecane
20.13
48.43
ND
39.19
74.2
Eicosane
21.00
41.39
ND
32.78
71.4
Heneicosane
21.94
43.15
ND
22.12
67.4
Docosane
22.94
38.98
ND
ND
61.3
Tricosane
24.10
37.81
ND
ND
63.4
Tetracosane
25.38
36.81
ND
ND
61.2
9.01
ND
ND
19.87
ND
9.50
ND
ND
15.67
62.34
,6-di
methyl -octane
2-methyl-nonane
4-methyl-undecane
12.5
24.5
ND
14.93
65.33
2-methyl-undecane
12.56
22.13
ND
17.44
61.26
2,6-dimethyl-undecane
13.24
18.83
ND
12.81
59.67
4-methyl-dodecane
13.79
26.54
ND
16.69
51.57
7-methyl-tridecane
13.97
23.48
ND
12.87
54.76
2,6, 1 0-trimethyl-dodecane
15.19
16.98
56.43
9.89
49.65
2,3-dimethyl-napthalene
15.70
6.5
11.43
6.1
12.15
4-methyl-tetradecane
16.03
28.34
ND
14.9
42.73
4-methyl-pentadecane
16.10
25.43
64.67
11.45
45.78
16.83
6.61
ND
2.4
15.61
2,3,6-trimethyl-napthalene
17.02
5.76
13.67
2.6
8.98
9-butyl-docosane
17.92
9.87
58.79
4.6
39.58
2,6, 10,1 4-tetramethyl-
18.45
18.52
61.23
11.67
42.91
19.39
14.61
62.45
12.43
44.63
20.11
12.34
57.98
8.12
16.98
21.75
ND
ND
5.68
18.65
1
,4,6-trimethyl-napthalene
pentadecane
2,6, 10,1 4-tetramethylhexadecane
1 1
-decyl-heneicosane
Squalene
Legend: The
percentage of
biometric flasks
CL7=
degradation
of
experiments after
Canandaigua Lake isolate
Mexican
48 days
and
TC=
crude
of
oil
hydrocarbons
incubation
at
Tommey's Comer
23
C.
by
GR1
isolate,
=
4
microbial
mixtures
Genesee River
ND= not
in
isolates,
determined.
77
Results
Figure 3.5.3
GC chromatograms
of
Mexican
crude oil
inoculated
i
i
h
J
j|
I OO
1-*
OC:
1S.OO -lO OO
i
T OO
i
4 0.00
-if-
bacterial
Discussion
mixtures
|
' !
;
|"
j
with
and
*
i
jjj
!
OCTO t
Z0.OO 2S.OO
.OO
a.)
I 8& *$ C>A^v
*'
i*A yvW
^,A*,'.-
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S.OO
6-OC
1D.OD
12. OC
14. OO
18. CO
Ifi.OO
20.00
22 OO
24 OO
28 OO
26. OC
b.)
V>*-a.'
f
Ir'1
tjJiMM
JIMjHa#ifeii^^
,
12.00
in oo
rod
or.
ji
^Jla
20.00
1B.00
16.00
^4.oc
*A^p,
22.00
24.00
26.00
26.00
30.00
c)
..Uv^NN..l\vr,vv_
;
v-
**?**?*
^'^
8.00
1CMKJ
.K.--
T"
r
12.00
00
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16 OC
18.00
20.00
22.00
24.0C
22-00
24.00
26.00
26.00
d.)
w
exo
10:00
.
Aw
u.oc
1200
16.0C
18.00
:cc:0
26.00
28.00
.
e.)
Legend: GC
chromatograms
bacterial
days
of
mixture
of
Mexican
(control):
incubation
at
23
crude
oil
b.) GR1/GR2;
c.)
by hexane after inoculation with a.)
TC1/CL7; d.) GR1/CL7 and e.) GR1/TC1 after
extracted
no
48
C, non-shaking.
78
Results
lower degradation
resistance of
degradation
on
these molecules
of alkylnapthalenes
the molecule.
molecule.
In
microbial
depends
on
is
the aromatic
in the
bacterial blend
each
It
attack.
was
Discussion
confirmed
oxidation rates seem to
needed to understand and
GR1
the
the position, number and type of substituents
be
rings (Roberts, 1992). It is
presence of
high
that
elucidated
cases, substituents hinder the initial enzymatic attack
same
of alkyl side chains on
research
against
However, in another cases,
possibilities occurs
for
of substituted naphthalene
and
and
GR2
clones.
enhanced
not
known,
Therefore
clarify the importance
of
in
this
on
the
the presence
which of
these
considerable more
phenomenon
for the
isolates.
The
already
indicated,
potential
among
illustrates,
degraded
mixtures.
all
the
of
The higher level
length
with
heneicosane
of
the crude
oil
TCI
cell yields at
and
hydrocarbon
chromatograph
most of
percentages
removal
in
the end of experiment
CL7 have the lowest degradation
constituents
in Table 3.5.2,
alkanes compared
or
low
blends. As the GC
significantly lower
22.12% for heneicosane
seen
mixture of
tested
results show
with
the medium chain
evolution and
the bacterial
the majority
incubation. As the
were
CO2
small rate of
were
detected
in Figure 3.5.3
at
comparison to the rest of
(46.38% for undecane)
and squalene where
bacterial
was seen with
to the longer and substituated alkanes
methylcyclohexane,
TC1/CL7 did
of
the medium chain length alkanes
10% for 2,6,10-trimethyl-dodecane). Minimum
alkylnaphthalenes,
49 days
reduction was
9-butyl-docosane,
not removed more then
(only
10%
11-decyl-
of
these
compounds.
79
Results
and
Discussion
Pictures 3.5.1
Visual
observations of biodegradation
S005
5
6
b.)
a.)
(].)
Legend: Visual
observations of
concentration
Mexican
crude oil
crude oil surface
flasks
at
bottom
23
biodegradation
by bacterial mixtures a.) monitoring bacterial
by GR1/CL7, bacteria growing on 2 %vv of
bacterial culture (left); b.) visual changes of Mexican
experiments
in CFU/ml in test tubes inoculated
(right),
control with no
inoculated
C (left),
of biometric
with mixture of
control
flask
after
GR1/TC1
after
12 days
of
c.) bacterial growth of GR1/CL7
8 days of incubation and d.) after 35 days
(right);
incubation in biometric
mixture observed on
of
the
incubation.
80
Results
In
order to expand our
degradation
experiments on
further investigate
substrate.
and
A
biometric flasks
highest
in
then
over the
by
incubation
of
maximum
mixture
(935.5 umol)
CO2
represented
CIM I for
(6.70xlO8
CL7 isolate
Mexican
GR1/CL7
of
by
more rapid growth
of
in
incubation followed
third week of the experiment.
comparison to
GR1,
the
general
of
in this
GR1
by
In
bacterial
this substrate
exhibited
35 days
the
of
the amount produced
cell
yield was
in
population
patterns
consortium.
GR2
Similar
clones.
GR1
patterns
colonies
to the GR2 isolate. As Figure
and reached
a significant
spite of
of
The GR1 isolate dominated
and
comparison
C02 fluctuated
Similar
crude oil.
the entire experiment
the evolution of
Umol) after 9 days
umol) in the
during
as on
in the bacterial blend consisting
were characterized
indicates,
CFU/ml) (Table 3.5.3).
CIM I
each
was reached after
96%
to
different
Rochester Institute
at
on
were used
the bacterial blends
respiratory activity for
Bacterial
crude oil.
v/v of
from CEV1S
average of
total amount of released
GR1/GR2
were observed
in
Mexican
C02. The
changes were observed on
clone
Overall
2%
with
from the
strains on a
the same incubation period on Mexican crude oil. The highest
obtained
3.5.4
inoculated
were
individual
Discussion
received
oil, the same bacterial blends
C02 during 51 days
case of
production of
incubation. The
during
of
in Figure 3.5.4.
shown
lower
crude
amount of organic waste obtained
Technology. Evolution
was
Mexican
interactions
about microbial
population changes and the role of
set of
the same
blend is
understanding
and
its first
maximum
(324.56
secondary increase (657.32
the tenfold lower yield of GR2
trend showed significant
increase in GR1
81
Results
and
Discussion
Figure 3.5.4
Production of carbon dioxide during CIM I degradation by mixtures of isolated
bacteria
1000
O
E
800
=t
T3
CD
"5
600
>
CD
CD
T3
X
o
400
-
x:
c
o
-Q
i_
CO
200
O
20
30
Time
Legend: C02
production was
Bacterial isolates
monitored
were
during
ml of
was performed at
GR1, 2=Genesee River isolates,
TC1=
CIM I by mixtures of isolated bacteria.
Bushnell-Haas medium with 2% v/v od in a
23C. Control did not contain any microorganisms.
utilization of
inoculated into 48
biometric flask. Incubation
(days)
Toomey's Corner isolate, CL7= Canandaigua Lake isolate.
82
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Results
Figure 3.5.5
Growth patterns in CFU/ml
of
bacterial
mixture cultivated on
CIM I
and
Discussion
organic waste
3.1 E+8
GR1/TC1
6.7E+7
1.2E+7
6.7E+8
D
?
GR1/CL7
D
5.6E+8
LL
O
?
3.4E+7
D
?
C
3
O
A
o
6.7E+7
"53
O
A
A
TC1/CL7
A
4.3E+7
A
A
2.3E+7
#
GR1/GR2
?
7 8E+8
?
6.7E+8
?
4.5E+8
?
?
3.7E+7
10
22
Time
Legend: Growth
PCA
of
individual
species of
plates and cultivated
Corner
isolate,
CL7=
bacterial
for 48 hours
at
34
51
(days)
by plating 0.1 ml of medium on
2=Genesee River isolates, TC1= Toomey's
mixture was monitored
26
C. GR1,
Canandaigua Lake isolate.
84
Results
and
Discussion
population (Figure 3.5.5). For this reason, the degradable ability of this isolate cannot be
neglected. GR2 also
observed that
for the three bacterial
concentration of cells
explained
difficulties
experienced
of
others
with
be
more
generated.
spite of
sulphur
this
measurements and plate counts confirmed the
GR1/CL7
and
Screening
from terrestrial
of
of
species.
general principles of microbial
not yet
been described.
TCI
characterized
bacteria,
as
be
might
halogen.
degradation
with
Due to
for
rates
the data obtained
from
very promising degradation
genera
north
New York State
and were
microorganisms
resulted
collection
identified to the
in the
isolated
collection of
20
were chosen as the most
specific
level according to
classification, using VTTEK API 20NE system. Bacterial
Acinetobacter, Serratia
isolated
Serratia
in
strains
(HC)-degrading
Four isolates from this
hydrocarbon degraders
belonged to the
and
fact,
hydrocarbon-degrading isolated
indigenous hydrocarbon
strains
This fact
the
GR1/GR2 bacterial blends.
and aquatic sites
distinct bacterial
TC1/CL7),
60 compounds; many
chromatographs and
In
and
was
and the accumulation of more
than
of
substitutions
It
production.
(GR1/TC1, GR1/GR2
detected
GC analysis,
during
3.6 Identification
superior
analysis
compounds could not
respiratory
ability
and
CO2
the end of the experiment decreased.
The GC
character
individual
at
mixtures
and though
by the depletion of easily degradable compounds
toxic metabolites.
aromatic
in the degradation
participated
from
marcescens
Toomey's
and
Pseudomonas. One
Corner
in
September
(Picture 3.6.1a). This bacteria
was
has
species
2004
was
scored
as
85
Results
positive
the
for glucose
Enterobacteriaceae
from this taxonomy
is
not
able
to
characteristics of
is the
are gelatinase
use
malonate.
Serratia
pigment
formation
as well as
to
during
in
our experiments
medium and
PCA
in biometric flasks. Therefore,
we assume
that that
growth of
Serratia is
is
experiments showed
Serratia
cultures and
not considered
It has
Satish,
and
not
showed
very intense
result
was
at room
red pigmentation.
oil polluted soil
by
that this bacteria
Yet,
no significant
microtiter plates
formation is
related
elucidated that the
temperatures
(Bartelt,
very strongly
spite of
the fact that
hydrocarbon degraders, this
degradability (Ijah, 1998; Bindu
marcescens exhibited
supports
In
and groundwater contaminated with
good crude oil and gasoline
and though the strongest adherence to the oil
This
held
main representative of
1996). In addition, Serratia
our research.
characterized
hydrocarbons in
pigment
marcescens
that incubation at 26C on PCA plates resulted in rapid
to be a
very
plates.
hydrocarbons. It
more pronounced when cultures are
bacteria has been isolated from
in
was shown
on
2000). Our
ability
Serratia that is
genus
growth
and
in Table 3.6.1. Serratia
incubation
of glucose
representative
biochemical
of
during
pigmentation
and
from
incubation in PC
was observed
degradation pathway
gasoline.
It
description
summarized
species
production of typical red pigment.
produced pigment
is
most of
The tests further indicated that this bacteria
Detailed
marcescens
Discussion
fact that
spite of the
negative, Serratia marcescens as a
showed positive results.
commonly isolated
most
for lactose fermentation. In
and negative
and
from
all
the highest emulsification
investigated
microorganisms
investigations from Ijah's
studies.
86
Results
Table 3.6.1 Biochemical
characterization of
TCI
CL7
GR4
0
LAC
0
0
ONPG
H2S
IND
MR
+
0
0
0
0
0
0
0
0
0
0
0
VP
+
0
0
CIT
+
0
0
PHE
0
0
0
ARG
0
0
0
LYS
ORN
+
0
0
+
0
0
UREA
0
0
0
MOT
0
0
0
GLC
0
+
+
K/NC
KIA
K/A
K/K
MAC
ND
+
+
NR
ND
0
+
OXD
ND
0
+
and
biochemical
Discussion
isolated bacteria
Parameter
Legend: Identification
and
reaction was performed on automated
test system
VTTEK 20API
isolate, CL7= Canandaigua Lake isolate,
ARG=
GR4= Genesee River isolate;
arginine dihydrolase, CIT= citrate, H2S= sulfate production,
IND= indole production, LAC= lactose utilization, LYS= lysine decarboxylase, MOT= motility,
MR= methyl red, ONPG= orthonitrophenyl galactopyranoside, ORN= ornithine decarboxylase,
PHE= phenylalanine deaminase, UREA= urease activity, VP= Voges-Proskauer, GLC= glucose
(A= acid, K=alkaline, NC= no change), MAC=
MacConkey
utilization, KIA= Klingler iron agar
NE.
0= negative, +
agar, NR=
=
positive; TC1= Toomey's Corner
nitrate reduction, OXD= oxidase.
87
Results
CL7 bacterial
strain
isolated from Canandaigua Lake in September 2004
and
was
Discussion
identified
as
Acinetobacter baumannii (Picture 3.6.1b). The GR4 isolate collected from Genesee River
sediment
in April 2004
Both bacteria
able to
belong
break down
was
contamination.
are
not
restricted
Pseudomonas
sp.
bacteria screening for
motility
was negative.
in
the present
to
oil
polluted
sp. oxidized
These bacteria have been described
terrestrial
study
same
Pseudomonas
as
well
aquatic
revealed that crude oil
areas
only,
The last isolate GR1 has been
of
areas
degrading
Acinetobacter
as
in this study have been isolated from
crude oil pollution.
(Picture 3.6.1c).
activity for Acinetobacter. For the
isolated
Yet,
species
The identification test
nitrate reduction and
bacteria
Pseudomonas
carbohydrates under anaerobic conditions.
reduced nitrate and was oxidase positive.
common
unnamed
to the group of nonfermentative gram-negative bacilli and are not
showed positive catalase
oxidase,
identified to
natural
not yet
habitats
glucose,
as the most
hydrocarbon
microorganisms
baumannii
with no
and
history
of
identified (Picture 3.6. Id).
88
Results
and
Discussion
Picture 3.6.1
View
on
final hydrocarbon degraders
a.)
b.)
Legend: The final isolates
VITEK API
Pseudomonas
selected as
20 NE
sp. and
system
d.)
the superiors hydrocarbon degraders
were
identified
by
automated
a.) Serratia marcescens; b.) Acinetobacter baumannii c.)
identified
as
not yet
89
Conclusion
4. CONCLUSIONS
The ability
of
indigenous bacteria,
various
from
especially those isolated
contaminated sites, to metabolize crude oil or aliphatic hydrocarbons is well known. In
this study, bacteria that are able to growth on
were
isolated from the terrestrial
and aquatic
isolated bacteria
compounds
and
in
in
medium
1.4
individually
was
energy
source
in Western New York State. The
length hydrocarbons
demonstrated the
in isolated bacterial
activities
chapter
chain
based-assay
microrotiter plate
oil-degrading
addressed
on
sites
oil as a carbon and
for selecting hydrocarbon degraders. Growth
selective enrichment technique was used
the
heavy
strains.
evaluated
various
presence of
Each
and
and
of
of
organic
hydrocarbon
the research goals,
successfully
executed.
The
following conclusions have been reached:
1 .) Of the 20 bacterial
Lake
and
Toomey's Comer
promising degraders. Three
Acinetobacter baumannii
not
surprising based
ability.
was
to
a
soil
their
isolated from Genesee River sediment, Canandaigua
(East Bloomfield,
Serratia
marcescens.
frequency
Nevertheless, isolation
NY), 4
were selected as
the most
these bacteria typed and found to be Pseudomanos sp.,
of
and
surprising for Serratia
be
and
in the
discovery
soil as well as
apparently
marcescens.
The
their
a good growth on
This bacteria has been
of
Pseudomonas
was
frequent biodegradable
hydrocarbon
not
previously
substrates
considered
strong hydrocarbon degrader.
2.)
different
This
on
clones
Four isolates
crude oil
oil consisted
were
tested for their
samples, the
mainly
highest
ability to degrade
degradability
was
of aliphatic, medium chain
crude oil.
detected
on
From three
Leepershank
oil.
hydrocarbons. After 35 days
of
90
Conclusion
incubation,
C19 was
all
90%
hydrocarbons in
of
GR1 isolate. This
by
removed
tested isolates. Significant
inoculated
the oil
with
GR1
clone
70%
crude oil resulted
samples, the
from degradation
changes
any
GR1
of
in
results
composition of this
the rest of the isolates.
clone
significantly
well
as
capability
of the
of crude oil
complex
biodegradation
combination with
simpler
of
baumannii. The
medium
chain
crude
Acinetobacter baumannii
(CL7)
Mexican
alkanes
were
microbial
blend
and
60%
of
GR1
No
a
by Acinetobacter
crude
oil
minimum
for
growth was observed
very
dispersing
as
agents
by
or
GR1
consortium.
and
Pseudomanos
percentage
of
of
In
containing only 2
(unidentified
Pseudomanos
consortium
biodegradation
spite
CIM I industrial waste,
well
consortium
as
good
by bacterial
by
length hydrocarbons. The
incubation,
indicated
oil
sp.
crude oil, most of the medium chain
utilized
of
clone on previous
production of
oil
bacterial
a
After 35 days
Smakover
waste
such
catabolic
on
GR1
(TCI). Higher
the oil.
of
industrial
by
marcescens
GR1 isolate
capacities,
incubation
branched
efficiency.
by
consistency
accomplished
the surface of
the presence of alkylnapthalenes in
aromatic substrate.
Mexican
highest efficiently among
was observed on
study has demonstrated
of
to
as
well
experiments on
and
C14
Serratia
Nevertheless, strong
hydrocarbons
be
complementary
49 days
this
composition
could
highly
of
as
a good growth of
changed the
3.) Furthermore,
with
as
of medium chain alkanes were removed
Considering
from 89.11 to 69.15%
biosurfactants
in lower degradation
baumannii (CL7).
and
amount represented the
production of
concentrations of substituted alkanes
Mexican
C10-Q3
range
GR1
sp.
strains
clone)
(GR2).
During
hydrocarbons
and
in
and
Acinetobacter
degraded up to 80 %
degradation for substituted
91
Conclusion
compounds
depending
varied
substituents. Generally, the
to
of
62.34%
3
effectiveness,
more groups
one
group
which was
the highest growth and
GR1
4.) The least
marcescens
small
degradation
bacterial
CO2
rates
Nevertheless,
Serratia to
Our
restricted
to
collectable
did
of
produce
10%
industrial
at
the end of incubation.
waste
CIM I
of
Overall,
was exhibited
of
GC
and
oil
lowest
by
the
in
both
comparison
CIM I
Serratia
and
substrates.
A
significantly lower
to
other
consortia.
this bacterial consortium in
is demonstrated in
a
high ability
emulsification agents.
crude oil
finding
degrading
supports the
widely distributed in the environment,
sites with no apparent
(CL7)
chromatographs revealed
mineralization
demonstrated that
This
crude oil and
growth on
prospective application of
hydrocarbon
sites.
the
hydrocarbons
highly effective
oil polluted
Mexican
Acinetobacter baumannii
consortium exhibited
crude
experiments
from
not exceed
mixture of
the effectiveness and
microorganisms are
decreased degradation to 10-15%. Degradation
evolution on
evolution
the optimization process
of
54.76
Addition
case of pure cultures.
successful was the attempt to utilize
(TCI). This
amount of
with
Acinetobacter baumannii.
and
organic waste with
C02
of
number
degraded
substituted compounds were
3 times higher than in
on carbon chain
cyclohexane and alkylnapthalens
mixture of
the length of the carbon chain and the
on
history of crude
microorganisms are not
fact that
crude oil
and therefore can
oil pollution as
it
was
degrading
be
in
"easily"
our case.
92
Future Prospects
5. FUTURE PROSPECTS
This
research
illustrates
work
how
contribute to significandy advance our
hydrocarbon-degrading
by
contaminated
challenging
knowledge
indigenous bacteria in
involved in
industrial
detail
the
the
investigation
and
characterization of
feasibility
degradation,
complexicity
highly
and new
application
of
bacterial isolates
our
on
of the
of
this
component
of
degradation
ability.
bacteria
study primary focused
Yet,
no research
such as nutrient
has been
nutrition and oxygen requirements.
bacterial isolates
as possible commercial strains,
of
on
In
performed on
order
future
hydrocarbon
microbial
to
increase
studies need
and crude oil
concentration, optimum temperature range, oxygen content,
and physical state of the oil.
One
serving
of the
not considered
bioremediation processes, many
to clarify the factors affecting the ability and efficiency
salinity
is
scale.
The bioremediation
isolation
Because
implementing
opportunities exist to further elucidate
methods
chemistry
of a wide occurrence of effective
environment that
crude oil and petroleum products.
methodologies
and
microbiology
of
the
future
proposals
as an example of an
treatment
system
contaminants.
In
most
order
is to apply
a laboratory- scale
15 1
aerated container
industrial lagoon treatment. Aerated lagoons
commonly
employed
settings, this system
petroleum
hydrocarbons, industrial
will allow
the type of
to treat industrial waste and petroleum
to stimulate the industrial
laboratory
are
conditions
as
performing degradation
waste and other contaminates
much
as possible
in
of complex mixture of
by bacterial blend
of
93
Future Prospects
Serratia
levels
marcescens, Acinetobacter baumannii
sp.
with
different
of process control and optimization.
Furthermore,
neglected.
Many
biodegradation
the
formation
chemical oil
are
toxic
in
petroleum
bacteria. For
by
In
microorganisms.
large
quantities of
bacterial isolates
our
used
spite
of
for
the
fact
biosurfactants have been
general
for
use
in
of
emulsifying
dispersing
oil
agents produced
and
cleanup
of
by
must
be
not
enhancement of oil
that
mutant
shown
to be
trend is to favor indigenous
this reason an attempt might be made to isolate and
efficiency
materials,
biosurfactants
hydrocarbon degradation, the
potential and
possible
of
dispersants that have been
to
microorganisms that produce
useful
Pseudomonas
and
characterize
the
isolated bacteria in this study
land-based
spills
of
hydrophobic
such as crude oil and refined products.
94
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