Application Of Microbes In Environment

Deep-desulfurization of Dibenzothiophene and Its Derivatives

Present in Diesel Oil by a Newly Isolated Bacterium

Achromobacter sp. to Reduce the Environmental

Pollution from Fossil Fuel Combustion

Prof Dr Wan Azlina Ahmad

1.

Nurulsyifaa' bt. Mustapa

921122075366

2.

Puteri Norathirah bt. Megat Abu Bakar

930515086722

3.

Siti Nur Aishah bt. Mohd Ali

920604106108

4.

Siti Nur Ain Fatihah bt. Abdillah

920513036052

Introduction
Dibenzothiophene(DBT) originated from
fossil fuel air pollution
Burning of fossil fuels containing sulphur
can be important sources of sulfur dioxide.
SO2 it is invisible and has a nasty, sharp
smell.
It reacts with sulfuric acid, sulfurous acid and
sulphate particles to form harmful
compounds.

Desulfurization

Flue-gas Desulfurization (FGD)

To remove sulfur dioxide (SO2) from exhaust flue
gases of fossil-fuel power plants, and from other
sulphur oxide emitting processes.
When these substances are burned, almost all of
the sulfur are converted to sulfur dioxide (SO2).
Further reaction of SO2 will form H2SO4. H2SO4
are difficult to remove and harmfull to
environment.

How FGD works

Uses calcium/sodium based alkaline
reagent.
The reagent will injected in the flue gas
thus, SO2 is absorbed and neutralize
become into solid compound
The solid then remove from waste gas
stream using downstream equipment.

Hydrodesulphurization Process
Remove sulphur (S) from natural gas and from
refined petroleum products such as gasoline or
petrol.
The hydrodesulphurization reaction can be simply
expressed as
Ethanethiol + Hydrogen Ethane + Hydrogen sulfide

C2H5SH + H2

C2H6 + H2S

Effects of Sulfur Dioxide to

Environment
harm crops and trees, textiles,
building materials, animals, and
people
corrodes metal, and causes building
materials and textiles to deteriorate
and weaken.

With combining with other

structure will produce a haze
thus reduce visibility

If there are fine dust particles

in the air, can damage a
person's respiratory system.

Major effect of Acid

Deposition (Acid Rain).

PHYLOGENETIC TREE

Phylogenetic tree
A phylogenetic tree or evolutionary tree
is a branching diagram or tree
showing the inferred evolutionary
relationships among various biological
species or other entities.

PHENETIC
METHOD

CLADISTIC
METHOD

Unweighted pair group

method with arithmetic
mean (UPGMA)
- UPGMA is the simplest
method of phylogeny. It uses
clustering approach and
uncorrected data to build a
tree.

Maximum parsimony (MP)

- Maximum parsimony assumes that trees
with the minimum number of
evolutionary changes are the most
preferable trees. MP bases on the number
of character-state changes to construct
all possible trees and give each a score.

Neighbor-hood joining
(NJ)
- The phylogenetic tree is
constructed from a starlike tree by grouping with
shortest distance of branch
length together.

Maximum likelihood (ML)

- Maximum likelihood use statistical
tool to evaluate a hypothesis about
evolutionary history. It constructs all
possible trees of evolutionary from an
observed data set.

SCREENING, ISOLATION AND PURE CULTURE OF DBT

UTILIZING BACTERIA

The bacteria isolated from contaminated crude

petroleum-oil soil sample.

Use dibenzothiophene(DBT) for isolation

process.
The bacterial dry biomass increase as well as
protein content in culture medium increase.
96hours incubation.
Further confirmed with a concomitant reduction
of DBT content from the culture medium with
respect to time.

TAXANOMIC IDENTIFICATION OF BACTERIAL

ISOLATE NBTU-02

Identification of NBTU-02
Positive test

Negative test

catalase

glucose

oxidase

xylose

urease

mannitol

gelatin hydrolysis

lactose

nitrate reduction

sucrose

indole production

maltose

citrate

REMARKS:
The Strain NBTU-02 give negative for the following test.
Aerobic Gram negative bacterium with a rod-like morphology.
Growth at 42C.

Remarks:
Phylogenetic relationships of strain NBTU-02 and
other closely related Achromobacter species based on
16S rDNA sequencing.
Neighbor-hood joining method.
Sequences from Bacillus sp. HSCC 1649 T (accession
no. AB045097) was considered as out-group.
This method demonstrated the evolutionary
relationship between the strain NBTU-02 and other
closely related Achromobacter sp., suggesting a
distinct phylogenetic position of this strain within the
genus.

RESPONSE SURFACE
METHODOLOGY
(RSM)

Definition of RSM

RSM
RSM identify the reason for changes in output
response by manipulating several independent
variables.
To see the relationships of independent variables
for producing an optimized condition by
generating a predicted value from mathematical
model.

 The general expression for better understanding of

RSM is as follow:
y = f (x1, x2) + e

y output response.
y relies on independent variables of x1 and x2 and
the experimental error, e.
e comes from the experimental errors and other
variations that are not included in response
function, f.
In RSM, the value of f is unknown and hence there
is a need to do some approximation function
model.

Approximation Function
Model

Low-order
polynomials

First-order model

Second-order
model

First Order Model

First-order model is a model where f can be defined by
linear function of independent variables which
expressed as:
y = 0 + 1xi1 + 2xi2 + .......+ qxiq + i
Where:
y is the output response;
x1, x2...xq is the independent variables;
q is the number of design variables;
i is the experimental runs;
N is number of experimental runs;
i is the experimental error.

(i = 1, 2, ......., N)

First Order Model (cont.)

Cannot be used if there is a curvature on the response
surface.
Orthogonal design is an example of first-order model.

Second-order model
This model can estimate linear, quadratic and
interactions effects for all factors.
Overcome the weakness of first-order model
Example : Central Composite Design (CCD).

 General expression

= 0 +

=1

=1

=1.

=1

Where:
y = predicted response;
0 = intercept term;
= linear effect;
ii = square effect;
ij = interaction effect;
xi and x j = variables.

The most popular design for this is Central Composite Design

(CCD).

FINDINGS FROM ARTICLE

Based on the article, several independent variables have been used for
optimization to study the degree of desulfurization;
incubation time (h),
medium pH,
concentration of DBT (% v/v)
inoculum size (% v/v).
Based on the initial screening, these parameters have highly influenced the
bacterial growth.
The optimized condition was determined by observing the bacterial growth
through the measurement of optical density (OD) at 600 nm (response).

 Achromobacter sp.
capable of degrading DBT
within reasonable period.

Higher degree of
desulfurization was done by
Achromobacter sp. in more
basic medium.

 Bacterial growth increase in

higher concentration of DBT
contributing to faster
desulfurization process by
Achromobacter sp.

More basic medium and high

concentration of DBT lead to better
bacterial growth. Better bacterial
growth indicating higher
desulfurization rate.

Better desulfurization rate

occur as inoculum volume and
medium pH increase.

4S PATHWAY

4S pathway

Achromobacter sp. followed sulfur-specific

pathway (4S pathway) for deep-desulfurization.
DBT is sulfurized and converted to 2hydroxybiphenyl (2-HBP) then have been
methylated at the hydroxyl group to form 2methoxybiphenyl (2-MBP).

4S pathways
enzymesenzymes

 Dibensothiophene
monooxygenase (DszC)
convert DBT to DBT-sulfoxide
and finally to DBT-sulfone
(DBTO2) through the addition
of 2 oxygen atoms to the
sulfur atoms.

DBT-sulfone monooxygenase
(DszA) carries out the next
step in the pathway producing
2-hydroxybiphenyl-2-sulfinic
acid (HBPS) through addition
of a final oxygen to
heteroatom.

 HBPS is then converted

to 2-HBP by HBPS
desulfinase (DszB)
producing 2-HBP.
At this point , the sulfur
has been released from
the hydrocarbon in the
form of sulfite.

 Last, the 2-HBP have been methylated at the

hydroxyl group to form 2-MBP.

HO

2-HBP

H3CO

2-MBP

Analysis of Instrument

In GC-MS,

Figure b: mass spectrum of 2-HBP

Figure c: mass spectrum of 2-MBP

Explanation based on graph:

1. Showed a signal at m/z 170 amu (MS) which
was consistent with the occurrence of 2-HBP as
the metabolite .
2. The presence of 2-MBP at m/z 184 amu was
also detected by GCMS analysis.
3. These results suggest that 2-HBP may have been
methylated at the hydroxyl group to form 2MBP.

4. Methylation of the hydroxyl group of 2-HBP by

Achromobacter sp. to produce 2-MBP eliminates
the inhibitory effect of the 2-HBP on the
desulfurization process.
5. The process become more effectives.
6. Since 2-MBP is less toxic compared to 2-HBP it
may significantly reduce environmental pollution
from fossil fuel combustion.

In GC-FID

Figure a-b: chromatogram of (a) control and (b) Achromobacter

sp. Desulfurization diesel oil post 24 hours

 GCFID chromatogram confirmed that

the quality of diesel oil remains
unaffected after treatment with
Achromobacter sp.

THANK
YOU