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1
CHAPTER 1
INTRODUCTION
1.1 Background of Study
Methyldiethanolamine (MDEA) is a clear, water-white, hygroscopic liquid with
an ammoniacal odor. It will absorb carbon dioxide and hydrogen sulfide at lower
temperatures and release the hydrogen sulfide at higher temperatures. It is used for
selectively remove hydrogen sulfide from gas streams containing carbon dioxide.
According to (Kohl and Nielsen, 1997) MDEA selectively removes H2S from
natural gas streams while piperazine acts mainly as a corrosion inhibitor and surfactant.
A corrosion inhibitor is a chemical compound that, when added in small concentration,
stops or slows down corrosion (rust) of metals and alloys. The slower rate of reaction
of CO2 with MDEA is compensated through the addition of small amounts of
rate-promoting agents such as DEA or piperazine.
During the gas sweetening process of absorption and desorption non-reclaimable
contaminants (exhausted amines) tend to accumulate in the system and can cause both
major reductions in efficiency and operational problems due to the closed loop nature of
the system. Therefore, wastewater from gas sweetening units frequently becomes
contaminated with raw amine-solutions, amine degradation products, thermal stable salts,
heavy hydrocarbons and particulates (Furhacker et al., 2003).
2
Figure 1.1 Chemical structure of MDEA and piperazine
When tested with laboratory animals, MDEA is considered slightly toxic by
single oral dose and practically nontoxic by single dermal application.
MDEA is considered moderately irritating to the eyes, but only slightly irritating
to the skin. The product is not corrosive under the conditions of the corrosivity test and
is not regulated as a hazardous material for transportation purposes. Because of the
low vapor pressure of MDEA, exposure to vapors is not expected to pose significant
hazard under normal workplace conditions (Huntsman, 2007).
3
1.2 Problem of statement
MDEA is a compound that is used in oil refinery industry to absorb and strip
hydrogen sulfide and carbon dioxide. Due to technological or human malfunctions,
MDEA may be found in process waters and afterwards transported to the wastewater
treatment plant (Bord et al., 2004). Failure to remove MDEA from the wastewater can
negatively affect the environment and ecosystem. Chemical treatment of MDEA rich
petroleum wastewater requires specialized equipment which increases the financial
burden. Moreover, chemical treatment of MDEA rich petroleum wastewater will
produce other products that may be more harmful to environment. However, a natural
process usually takes a long time to degrade the MDEA. Biological techniques should
be investigated to treat the wastewater. Thus, a research was conducted to enhance the
efficiency of selected bacteria to degrade the MDEA.
1.3 Objectives
The objectives of the study are
1 to isolate and characterize MDEA degrading bacteria from MDEA rich petroleum
wastewater
2 to utilize bacteria to degrade MDEA in solution
4
1.4 Scope of study
Sample was collected from petroleum processing industries at Melaka. This
study was focus on the factors affecting growth of the bacteria that could degrade
MDEA to enhance their growth. In order to evaluate the ability to degrade MDEA,
microorganisms were screen for growth in medium that contain only MDEA as their
sole carbon and/or nitrogen source. The isolated bacterial cultures would identify up to
genus level by their morphological and biochemical characterization. Monitoring of
parameters such as pH, redox potential, concentration of MDEA was determined before
and after the biological treatment of the selected bacteria. Quantitative determination
of MDEA using gas chromatography and ion chromatography also was studied.
5
CHAPTER 2
LITERATURE REVIEW
Methyldiethanolamine (MDEA) is a clear, colorless or pale yellow liquid with an
ammonia odor. It is miscible with water, alcohol and benzene. It is also known as
N-Methyl diethanolamine and has the formula CH3N (C2H4OH)2.
2.1 Characteristics of petroleum wastewater
Petroleum wastewater is usually characterized by color, odor and high
concentration of solid. The color of the wastewater can also be affected by industrial
contributions to the treatment system: color contributed by industry typically is not
removed by the pretreatment system. Unusual odors such as petroleum odors may
indicate abnormal industrial discharges. Wastewater such as petroleum wastewater is
generally somewhat warmer than tap water. An increase in wastewater temperature
will increase microbial activity. However, when wastewater reaches high temperatures,
microbial activity will be inhibited. Determination of the forms and concentrations of
solids present in wastewater can provide an operator with useful date for the control of
treatment processes. Changes in these physical characteristics can indicate unusual
influent (wastewater entering a treatment system) or operating conditions. COD is
widely used to measure the overall level of organic contamination in wastewater. The
contamination level is determined by measuring the equivalent amount of oxygen
required to oxidize organic matter in the sample (Idaho Department of Environmental
6
Quality, 2006). The presence of some anionic species and/or stronger acids (as
compared with hydrogen sulfide and carbon dioxide) in the raw feed gas to amine plants,
leads to the formation of amine salts from which amine is not recoverable through steam
stripping process. The amine salts are called heat stable salts and both organic and
inorganic salts may occur. The inorganic salts such as chloride, sulfate and phosphates
typically are found in produced or cooling waters (Abdi and Meisen, 1992).
7
2.1 The role of MDEA as corrosion inhibitor in petroleum processing industries
MDEA are used in refineries and gas plants around the world to remove both
H2S and CO2 from feed gas. In petroleum processing industries, modern process of
crude oil refining, utilizing catalytic hydrorefining, reforming, and hydrocracking,
reforming, and hydrocracking, result in the formation of large volumes of gases
containing hydrogen sulfide. Under moist conditions, hydrogen sulfide will be
oxidizing to acid sulfuric and cause corrosion to the metals and alloys. On the other
hand, CO2 can cause problems in gas processing plants and refineries alike. It may
cause problems in hydrate formation, and affect specification of products such as
ethylene in gas cracking units. The corrosion in amine plants is not caused by the
amine itself, but is caused by the H2S, CO2, and by amine degradation plants (Rennie,
2006).
In order to remove hydrogen sulfide from the post-refining gases, a corrosion
inhibitor is employed. A corrosion inhibitor is a chemical compound that, when added
in small concentration, stops or slows down corrosion (rust) of metals and alloys.
MDEA is a corrosion inhibitor usually used in petroleum processing industries; it is
usually promoted by piperazine to increase the efficiency of MDEA.
The absorption reaction between MDEA and H2S takes place at ambient
temperature, and is limited to H2S, with a minor quantity of CO2. After that, the
MDEA-rich solution coming from the absorption tower is flashed, raised in temperature,
and stripped in a regeneration tower to free the contained H2S and CO2. Before again
starting the absorption step, the lean MDEA is cooled to ambient temperature, and is
partially treated to remove the heat-stable salts that are not regenerated in the stripping
tower. Accumulation of heat-stable salts, such as oxalates, tyocianites, and formiates,
must be avoided since they reduce MDEA-solution activity (Bressan.L et al., 2000).
8
MDEA reactions
with H2S H2S + R’RRN HS- + R’RRNH
+ Fast
with CO2 CO2 + H2O HCO3- + H
+ Slow
H+ + R’RRN R’RRNH
+ Fast
2.2 Effect of MDEA in petroleum wastewater
Due to technological or human malfunctions, MDEA may be found in process
waters and afterwards transported to the wastewater treatment plant. Elevated levels of
MDEA have detrimental effects on the effectiveness of ammonia steam stripping and
biological filter performances. In addition, MDEA have a high contribution to the total
organic carbon (TOC) and the total nitrogen in tail waters (Bord et al., 2004). TOC is
the amount of carbon bound in an organic compound and is often used as a non-specific
indicator of water quality or cleanliness of pharmaceutical manufacturing equipment.
It provides a speedy and convenient way of determining the degree of organic
contamination. Petroleum wastewater that contains high concentrations of nitrogen can
affect public health and have harmful ecological impacts. The principal forms of
nitrogen are organic nitrogen, ammonia (NH4+ or NH3), nitrite (NO2
-), and nitrate (NO3
-).
Ammonia is extremely toxic to fish and many other aquatic organisms and it is also an
oxygen-consuming compound, which can deplete the dissolved oxygen in water. The
depletion of dissolved oxygen in water is a problem in aquatic ecosystem since
maintenance of a high oxygen concentration is crucial for survival of the higher life forms
in aquatic ecosystem. Another ecological impact is eutrophication. All forms of
nitrogen are taken up as a nutrient by photosynthetic blue-green bacterial and algae. The
excessive growth of bacteria and algae due to the increase of the amount of nitrogen
discharged into water, contributes to the reduction of the oxygen level in water.
Although nitrate itself is not toxic, its conversion to nitrite is a concern to public health.
Nitrite is a potential public health hazard in water consumed by infants (Sedlak, 1991).
9
2.4 Aerobic biodegradation of MDEA
A number of studies have demonstrated that macromolecular organic substrates
must be enzymatically hydrolyzed to smaller subunits before they can be taken up and
metabolized by the microbial cell. Several studies have indicated that enzymatic
hydrolysis of organic compounds by activated sludge microorganisms under aerobic
conditions are distinguishably more efficient than under anaerobic and/or anoxic
conditions (Li and Chrost, 2006). Aerobic biodegradation is the breakdown of organic
contaminants by microorganisms when oxygen is present. Aerobic bacteria use oxygen
as an electron acceptor, and break down organic compounds, often producing carbon
dioxide and water as the final product. Aerobic biodegradation is an important
component of the natural attenuation of contaminants at many hazardous waste sites.
The aerobic biodegradability of MDEA was investigated in a standardized batch test and
a continuous flow experiment (40L/d). The results of the experiment based on total
organic carbon (TOC) measurements indicated that the batch test of MDEA-solution
was non-biodegradable during the test period of 28 days, whereas the continuous flow
experiments showed biodegradation of more than 96%. This was probably due to the
adaptation of the microorganisms to this particular wastewater contamination during
continuous flow experiment (Furhacker et al., 2003).
10
2.5 Determination of MDEA
The analysis of alkanolamines in refinery process waters is very difficult due to
the high ammonium concentration of the samples. However, a sensitive, rapid, accurate,
and precise analysis for the quantitative determination of methyldiethanolamine (MDEA)
can be performed using either gas chromatographic or cation exchange chromatography.
2.5.1 Gas chromatographic determination of MDEA
GC is generally used for identification and quantification of volatile and semi
volatile organic compound in complex mixtures, nut it cannot identify their union.
Sample is injected into the port of the GC device. The GC instrument vaporizes the
sample and then separates and analyzes the various components. Each component
ideally produces a specific spectral peak. The time elapsed between injection and
elution is called the ‘retention time’. The size of the peaks is proportional to the quantity
of the corresponding substances.
MDEA was determined by GC fitted with a flame ionization detector. One μL
of the sample is injected into a Hewlett Packard Instrument 5890 Series II equipped with
a chromatographic column HP Ultra 1 (25 m x 0.2 mm x 0.33l m). The oven
temperature is held isotherm at 120 °C. The flow rate of the carrier gas (He) was 0.67
mL/minute and detection temperature 290°C. T detection limit under the above
measuring conditions was 0.l2 g/L MDEA (Furhacker et al., 2003).
The flame ionization detector does not respond to water, nitrogen, oxygen,
carbon dioxide, carbon dioxide, helium or argon. For a specimen contains water, a
flame ionization detector should be used. Column HP ultra 1 designed for hydrocarbon
and drug applications that require precise column to column reproducibility.
11
Specifically, it is used for the analysis of hydrocarbons, amines, and drugs. Temperature
range: -60°C to 325/350°C. Phase composition: bonded and cross linked 100%
dimethylpolysiloxane Column HP ultra 5 is a non- polar column and designed for
alcohol and primary amine applications. Phase composition: Bonded and cross-linked;
solvent rinsable (Agilent, 2008).
2.5.2 Ion chromatographic determination of MDEA
Ion Chromatography (IC) has been a successful tool for the quantification of ions
in many diverse types of industrial and environmental samples. This method has a
greater attraction since it can go for very low detection levels with minimum
environmental release. The consumption of time and chemicals is also reduced
considerably by this technique. Ionic components of a sample are separated into discrete
bands by passing the sample through a separating column filled with a specially
designed ion exchange resin (stationary phase). The charged functional group on the
stationary phase can be exchanged with other ions of the same charge in the mobile
phase.
For strong acids, bases, and electrolytes, ion exchange is the preferred retention
mechanism. The charge of the stationary phase is used to control selectivity, and the
strength of the displacing agent in the mobile phase is used to adjust the retention factor
(Heftman, 2004).
Cation exchangers are employed for cation estimation while anion exchangers
are used for the estimation of anion. Depending upon the function of the separating
column packing material is made with styrene-polyvinyl-benzene copolymer or
polymethacrylate, polyhydroxy alkylmethacrylate or spherical silica gel attached with
specific functional groups. Cation exchangers are produced by the sulphonation of
styrene-divinyl benzene resin. The combination of support material and ion exchange
12
groups is critical in the separation behavior, interference with eluents. Ions of alkali
metals, alkaline earth metals and ammonium radicals are easily adsorbed and eluted in
cation exchange resins. This is an effective tool for the separation of alkanol amines.
In general for mobile phase aqueous eluent is more preferred but mixed
aqueous-organic eluent is also used according to the requirement. Since electrical
conductance is a property to all ionic species in solution, cations thus separated are
detected by conductivity detector (Al-Shawi and Gowda, 2007).
2.6 Identification of methyl diethanolamine degradation products
Partially degraded, aqueous methyl diethanolamine solutions were analyzed by a
gas chromatograph equipped with a Tenax column and flame ionization detector;
nitrogen was used as the carrier gas. To aid in product identification, the gas
chromatograph was coupled to a mass spectrometer operating either in electron impact
or chemical ionization mode. The most important degradation products were found to be:
methanol, ethylene oxide, trimethylamine, ethylene glycol, 2-(dimethylamino)ethanol,
1,4-dimethylpiperazine, N-(hydroxyethyl)methylpiperazine, triethanolamine; and
N,N-bis(hydroxyethyl)piperazine (Chakma and Meisen, 1988).
13
2.7 Redox potential
The redox potential of water system is a measure of electrochemical potential or
electron availability within these systems Electrons are essential to all inorganic and
organic chemical reactions. Redox potential measurements allow for rapid
characterization of the degree of reduction and for predicting stability of various
compounds that regulate nutrients and metal availability in water. Redox potential is
determined from the concentration of oxidants and reductants in the environment. The
inorganic oxidants include oxygen, nitrate, nitrite, manganese, iron, sulfate, and carbon
dioxide, while the reductants include various organic substrates and reduced inorganic
compounds.
Oxidation and reduction reactions involve transfer of electrons from one
compound to another and play a major role in regulating many reactions in biological
systems. It is a coupled reaction. The tendency of compounds to accept or donate
electrons is expressed as ‘reduction potential’ or ‘redox potential’. The redox potential of
a substance depends upon : affinity of molecules for electron; concentrations of
reductants and oxidants (DeLaune and Reddy, 2005).
Figure 2.1 Electron flow preference as a function of the different electron couples
(Dos Santos et al., 2007)
14
CHAPTER 3
RESEARCH METHODOLOGY
3.1 Growth of bacteria in petroleum wastewater
Wastewater samples collected for the growth of bacteria was inoculated with the
following media: 10% (v/v) nutrient broth, Horikoshi broth, glucose 4% (w/v) and LB
broth. The conical flask was incubated at 30ºC, 40ºC and 55ºC with 200 rpm. A
volume of 500 mL of Horikoshi medium contained 10% (w/v) D-glucose (autoclaved
separately), 425 mL: distilled water with peptone, 2.5g; yeast extract, 2.5g; KH2PO4,
0.5g; MgSO4.7H2O, 0.1g and filter sterilized 10% (w/v) Na2CO3. Glucose 4% (w/v)
was prepared by dissolved 2 g glucose in 50 mL distilled water. The composition of
the LB broth in 500 mL was as follows: tryptone, 5 g; yeast extract, 2.5 g; NaCl, 5g.
The cell growths were monitored periodically over a 12 h period. A volume of
2 mL wastewater was centrifuged at 4000 rpm for 5 minutes. The supernatant was
drained off and the pellet was resuspended with 2 mL of distilled water. The cell
concentration of the cultured media was determined by the cell optical density at 600 nm
with a spectrophotometer (Cecil instrument 1000 series, Cambridge, England). The
blank used was distilled water.
15
3.2 Isolation of microbes
A volume of 10% (v/v) nutrient was added in 20 mL of petroleum wastewater
in conical flask. The conical flask was then incubated in shaking incubator at 30ºC
overnight. One loop of wastewater was taken out and streaked it on nutrient agar,
Horikoshi agar, LB agar and petroleum wastewater agar.
3.3 Preparation of glycerol stock culture
Microbes from plate A and C were inoculated into 20 mL filter sterilized
petroleum wastewater with 10 % (v/v) suitable nutrient in separate flasks. Microbes
from plate B and D were inoculated into 20 mL Horikoshi medium in separate flasks.
The conical flasks were incubated in shaking incubator overnight at 30ºC. The
inoculums was pipetted out from the previous conical flask into new conical flask with
60 mL filter sterilize wastewater or Horikoshi medium separately. A volume of 0.5 mL
60% (v/v) glycerol and 1.5mL inoculum at exponential phase was pipetted into 2.5 mL
microcentrifuge tube. The final concentration of glycerol stock culture was 12.5%. The
microcentrifuge tube then stored at -80ºC freezer.
3.3 Partial identification of isolated bacteria
An unknown sample may contain different bacteria, so a culture was made to
grow individual bacterial colonies. Many different criteria may be employed for
identification, though it is often desirable to employ the easiest techniques possible such
as colony and cellular morphology and biochemical tests.
16
3.3.1 Colony and cellular morphology
Two smears were prepared for each organism. One loopful of inoculum in
Horikoshi medium and wastewater was transferred onto slide and a smear was prepared.
The preparation was heat fixed by passing the slide through a Bunsen Burner flame for
several times. The slides were smear by flooded with crystal violet. The stain was
left on the slide for 1-2 minutes and washed with distilled water. The slide was
blot-dry with scott paper and allowed to air dry. The stained smears were examined
under the light field microscope. Purple stained cells were observed. The shape and
cell arrangements were recorded into a table.
3.4.2 Biochemical tests
The isolated microorganisms were identified with reference to Bergey’s Manual
of Determination Bacteriology (1994) as the primary source. Biochemical test to be
carried out were oxidase test, catalase test, urease test, starch hydrolysis, triple sugar iron,
indole, motility, Methyl red/ Voges-Proskauer test, oxidation fermentation test, simmon
citrate test, nitrate reduction test, MacConkey test, gram staining, spore staining.
Characteristics and features of bacteria were recorded. Analysis was made based on
the results which had been obtained.
17
3.5 Screening for MDEA degrading bacteria
The isolated bacteria were screened in various types of medium to determine
their abilities to degrade MDEA.
3.5.1 Mineral salts medium (MSM)
All cultivations were performed in mineral salts medium which contains (g L-1
):
MgSO4, 0.1 g; KH2PO4, 0.1 g; NaH2PO4, pH 9. Different nutrients sources were
added:
(a) MSM + carbon source + nitrogen source
(b) MSM + carbon source with 50 ppm MDEA
(c) MSM + nitrogen source with 50 ppm MDEA
(d) MSM with 50 ppm MDEA
Strain A, B, C, D and E (10% v/v) were inoculated into 10 mL solution (a), (b),
(c), and (d) separately in a conical flask. The conical flasks were incubated at 30°C
with 200 rpm. The cell growths were monitored by OD 600nm periodically at t0, t12, t24,
t30 incubation period. The MSM were supplemented with trace element solution (0.1%
v/v): Disodium EDTA, 0.5g; FeSO4.7H2O, 0.2g; H3BO3, 0.03g; CoCl2.6H2O, 0.02g;
ZnSO4.7H2O 0.01g; MnCl2.4H2O, 3mg; NaMoO4.2H2O, 3mg; NiCl2.6H2O, 2mg;
CaCl2.2H2O, 1 mg and added distilled water to 1 litre (Atlas.R.M, 2006). Vitamin
solution (0.1% v/v). NADH (0.05% v/v): 0.5 g NADH powder was dissolved in 10ml
distilled water. Nitrogen source was (10% v/v) NH4H2PO4, 0.24 g/L; while carbon
source was glucose, 10 g/L.
18
3.5.2 Petroleum wastewater as growth medium for bacteria
Strain A, B, C, D and E (10% v/v) were inoculate into 40 mL filter sterilised
petroleum wastewater in separate flasks. The conical flasks were incubated at 30°C
with 200 rpm. The growth of the bacteria was determined by the spread plate method.
The sample is serially diluted in sterile media with dilution factor 10-4
, 10-6
and 10-8
and
0.1 mL of the diluents are transferred to sterile Horikoshi plates and spread with a sterile
bent glass rod for every 8 h time interval. A volume of 10 mL of sample was taken out
and centrifuged at 4000 rpm for 15 minutes and keep it at 4ºC for MDEA analysis.
3.5.3 Horikoshi medium
Strain B, D and E (10% v/v) were inoculate into 40 mL Horikoshi medium
containing 50 ppm MDEA in separate flasks. The conical flasks were incubated at
30°C, 200 rpm. The cell concentration of the cultured media was determined by the
cell optical density at 600 nm with a spectrophotometer.
3.6 Estimation of MDEA tolerance level for isolated bacteria
Tolerance concentrations of the MDEA for strain B and D was determined by
adding various concentrations to Horikoshi medium ranging from 50 ppm to 6000 ppm
MDEA. A stock solution of the MDEA (1000 ppm) was prepared in double distilled
water and was added to the Horikoshi medium in various concentrations which was then
inoculated with 10% v/v organisms. Control was Horikoshi medium without MDEA.
The conical flasks were incubated at 30°C with 200 rpm. The cell concentration of the
cultured media was determined by the cell optical density at 600 nm with a
spectrophotometer.
19
3.7 Degradation study of MDEA by using selected strains
The degradation study of bacteria B and D was carried out by adding the
bacteria (10% v/v) into Horikoshi medium, 200 mL containing 150 ppm MDEA in
separating flasks. In the same way as the test medium, control was prepared only
Horikoshi medium with inoculum. The flasks were incubated at 30°C with 200 rpm.
Samples were taken at 24 h interval time for MDEA concentration analysis by IC while
samples were taken at 6 h interval time for pH, redox potential and cell concentration
determination. The pH was determined by Mettler Toledo Delta 320 pH meter and
redox potential was determined by Orion 3-Star Plus Benchtop pH Meter. The cell
concentration of the cultured media was determined by the cell optical density at 600 nm
with a spectrophotometer and spread plate method. Spread plate method was
performed for a 12 h interval time. A volume of 0.1 mL of the diluted suspension was
spread onto the surface of the plate with a glass rod.
3.8 Determination of MDEA
3.8.1 Gas chromatography
MDEA was extracted by using liquid-liquid extraction method. Equal volume of
ethyl acetate was adding to wastewater sample and separated by separating funnel. The
process was repeated for three times and the sample extracted was evaporated in fume
hood to a ten times concentration. The MDEA was determined by Agilent 6890N GC
fitted with a flame ionization detector. One μl of the wastewater sample is injected into
a GC with a chromatographic column HP-5 fitted with splitless injection. The oven
temperature is held isotherm at 120°C. The flow rate of the carrier gas (He) was 0.7
mL/min and detection temperature 250°C.
20
3.8.2 Ion exchange chromatography
Sample was prepared in 50 times dilution by working eluent. The selection of
the mobile phase was performed by a trial and error method using 4mM/L H2SO4 + 55%
Acetonitrile or 5mM HNO3. Mixed well and filter through 0.2 μm filter.
A Metrohm Compact IC 761 equipped with a conductivity detector, Metrosep
cation column C1, sampling loop of volume of 20 μL were used for chromatographic
investigastions. The cation column C1 is a 125 x 4.6 mm column packed with 5.0 μm
spherical silica gel with polybutadiene maleic acid groups. The IC was operating at 27°C
and 7.6 MPa.
21
The research methodology was summarized as shown in Figure 3.1.
Figure 3.1 Summary of research methodology
22
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Enrichment and isolation of indigenous bacteria from MDEA rich
petroleum wastewater
Optical density 600nm was used to monitor the growth of the indigenous
bacterial. However, there was a limitation by using OD 600nm since precipitate will be
formed when filter sterilized MDEA rich petroleum wastewater exposed to air. The
precipitate formed was contributed to a higher reading. Thus, control was important to
compare the growth of the indigenous bacteria with a nutrient added. Results showed
that the most suitable nutrient for growing the indigenous bacteria was adding 10% (v/v)
LB or Horikoshi at 30 ºC, 200 rpm.
LB was a nutritionally rich medium, including peptides, casein peptones,
vitamins, trace elements, and minerals. Peptide and peptones were provided by
tryptone. Vitamins and trace elements were provided by yeast extract. Sodium ions for
transport and osmotic balance were provided by sodium chloride (Tortora et al., 2007).
Horikoshi medium was a medium rich in amino acids, nitrogen source, vitamin and
contained certain trace elements to support the growth of the indigenous bacteria with
Na2CO3 was added to adjust the medium pH to pH10.
23
Two of the five bacterial strains were isolated on petroleum wastewater agar and
the other three were isolated on Horikoshi agar after incubated at 30ºC for three days.
Only three isolates from Horikoshi agar showed good growth.
4.2 Partial identification of isolated bacteria
4.2.1 Colony and cellular morphology
Five morphologically different aerobic bacterial colonies were isolated from
MDEA rich petroleum wastewaters. Morphological observations of these isolates
demonstrated two of them as Gram positive cocci while the other one is Gram negative
rod.
24
Table 4.1: Colony morphologies for isolated bacterial strains
Isolates
Colony
morphology
A B C D E
Shape Irregular Circular Rhizoid Irregular Irregular
Size <1mm 1mm-7mm <1mm 1mm-4mm 2mm-7mm
Surface Rough Glistening Rough Glistening Wrinkled
Texture Brittle Buttery Brittle Moist Moist
Elevation Flat Convex Flat Convex Raised
Margin Entire Entire Filamentous Wavy Wavy
B D E
Figure 4.1 Colonies morphology for bacteria B, D and E
B D E
Gram negative cocci Gram negative rod Gram positive rod
Figure 4.2 Gram staining for bacteria B, D and E
25
4.2.2 Biochemical tests
Biochemical tests results were recorded for analysis in identifying the genus of
bacteria that were cultured and isolated from the MDEA rich petroleum wastewater.
The results were summarized in Table 4.2.
Table 4.2: Results of biochemical test for bacterial strains B, D and E
Bacterial strain
Test
B D E
Oxidase - + +
Catalase + + +
Indole - - -
Motility + + -
O-F O O -
Simmon citrate - - -
TSI - - -
Starch hydrolysis - + +
Urease + - -
MR - + -
VP - - -
Mac Conkey - - -
Nitrate reduction test - + +
Partial identification
of bacteria
Micrococcus sp. Pseudomonas sp. Corynebacterium
sp.
* Symbols: +( positive reaction), - (negative reaction), F (glucose fermenter), O (glucose
oxidized)
26
Strain B which form irregular clusters in liquid medium, oxidase negative, nitrate
reduction negative, do not ferment glucose, gram negative has been identified as
belonging to the genus Micrococcus. Strain E also formed irregular clusters but differ
from the above isolates in that they are gram positive, oxidase positive, and nitrate
reduction positive. Based on these characteristics, strain D has been assigned to the
genus Cornybacterium. Strain D colony are gram negative bacilli, white in color,
motile, reduce nitrate to nitrite has been identified as genus Pseudomonas (Refer to
appendix C).
4.3 Screening for MDEA degrading bacteria
All the five isolated strains were studied for their ability to grow in three
different types of medium to determine to utilization of MDEA as carbon and/or
nitrogen source.
4.3.1 Mineral salts medium
All of the five isolated strains were unable to grow in MSM that contained only
MDEA as carbon and/or nitrogen source. Refer to Figure 4.3- 4.6, strain A and C are
said to be fastidious microorganism since it was unable to growth in all types of MSM.
Results also showed that all the bacteria strains were unable to utilize MDEA as their
carbon source in MSM. Refer to Figure 4.4, strain B was able to utilize MDEA as
nitrogen source for growth. Strain D and E was unable to utilize MDEA as its carbon
and/or nitrogen source in MSM. This probably due to the lack of some growth factor
in MSM thus failed to support the growth for the bacteria.
27
Figure 4.3 Growth of isolated bacterial strains in MSM with carbon and nitrogen
source
Figure 4.4 Growth of isolated bacterial strains in MSM with carbon source and 50
ppm MDEA
28
Figure 4.5 Growth of isolated bacterial strains in MSM with nitrogen source and 50
ppm MDEA
Figure 4.6 Growth of isolated bacterial strains in MSM with 50 ppm MDEA
29
4.3.2 Petroleum wastewater as growth medium for bacteria
Growth of the bacteria in MDEA rich petroleum wastewater was monitored
viable count since precipitate formed will give inaccurate OD600 units. Pure strains of
B, D and E were unable to grow in wastewater after 24 h of incubation at 30°C, 200
rpm.
4.3.3 Horikoshi medium
By refer to Figure 4.7, all the three isolated strains were able to grow in the
Horikoshi medium. When MDEA was added to the Horikoshi medium, both strain B
and strain D give higher OD600nm reading as compared growth in Horikoshi medium.
These probably due to the bacterial strain were isolated from the MDEA rich
environment and able to utilize the MDEA as their nutrient source. Strain E was
unable to utilize MDEA its nutrient source since there was not much different for OD
600nm in Horikoshi medium or growth in Horikoshi medium containing MDEA.
30
Figure 4.7 Growth curves of strains B, D and E in Horikoshi medium with 50 ppm
MDEA
4.4 Estimation of MDEA tolerance level for isolated bacteria
The MDEA tolerance levels of the strain B and D were determined to evaluate
the influence of the MDEA on the bacterial strain. The absorbance reading for OD 600
nm was used to monitoring the growth of both strains.
Figure 4.8 showed that strain B was able to growth in the medium containing
MDEA ranging from 50 ppm to 2000 ppm. Thus, it was estimated that the MDEA
tolerance level for strain B ranging between 1000 to 2000 ppm with the higher
absorbance reading (Appendix E). Tolerance level of MDEA for bacteria D was
around 100 ppm since it was able to grow well around 100 ppm MDEA (Figure 4.9).
31
Figure 4.8 Growth curves of strain B in MSM with different concentration of MDEA
(ppm) supplemented with glucose source
Figure 4.9 Growth curve of strain D in Horikoshi medium with different
concentration of MDEA (ppm)
32
4.5 Determination of MDEA
4.5.1 Gas chromatography
Extraction is a process in which two phases come into contact with the
objective of transferring a solute or particle from one phase to the other. For the
separation of MDEA from sample, the phase (ethyl acetate) was immiscible liquids, and
the solute (MDEA) was in soluble form.
Sample preparation from wastewater using liquid-liquid extraction was failed to
extract MDEA from sample. Peak has been appeared around the same retention time
with standard MDEA (Appendix I). However, it was not MDEA after injection of
sample into ion chromatography with better sensitivity. MDEA cannot be extracted by
organic solvent (liquid-liquid extraction) because MDEA was highly soluble and
strongly polar. Instead water was the most polar solvent that can bind strongly to
MDEA. Besides that, MDEA need a high concentration for quantification by GC.
Refer to Appendix F, the detection limit using GC was 1000 ppm MDEA and not
sensitive compare to ion chromatography.
Analysis of hydrolysis products of MDEA and their degradation products in
aqueous extract and determination by gas chromatography is both challenging and time
consuming since concentration to dryness and derivatisation as part of sample treatment
are required. The presence of extraneous materials may interfere with the
derivatisation resulting in low apparent recoveries.
33
4.5.2 Ion exchange chromatography
A buffered aqueous solution as the mobile phase carries the sample from loop
onto a column that contains some form of stationary phase material. The analytes was
eluted by the protons from the eluent. In the study of MDEA, two types of eluents
were used by trial and error method.
3 4 5 6 min
500
1000
1500
2000
mV
ch3 MD
EA
12
7.4
11
2 3 4
Figure 4.10 Ion chromatogram of 120 ppm MDEA by using 4mM/L H2SO4 +
55% Acetonitrile as working eluent
34
3 4 5 6 7 min
100
200
300
400
mV
ch3
MD
EA
12
0.2
50
Figure 4.11 Ion chromatogram of 120 ppm MDEA by using 5mM HNO3 as
working eluent
Refer to Figure 4.10, 4mM/L H2SO4 + 55% Acetonitrile is fairly satisfactory to
elute the MDEA and separation of MDEA from other components. Refer to Figure
4.11, working eluent using 5mM HNO3 was quite satisfactory to elute the MDEA and
the peaks were comparatively shaper.
Compare to GC, the present results support the fact on the sensitivity and
specificity of IC on the determination of MDEA.
35
4.6 Degradation study of MDEA by using selected strains
4.6.1 Bacterial growth profile
Both bacteria have shown typical growth profile of bacteria except that strain D
did not show any lag phase. This was probably due to strain D consisting of actively
metabolizing cells used as inoculum compared to strain B. By referring to Figure 4.12,
the optical density (600nm) for strain B was much higher yielding up to 20 OD600 units.
This was expected because strain B might caused precipitation of the Horikoshi medium
components. In addition, it was believed that the bacterial growth reached the decline
phase after 45 h of incubation while the decline phase of strain D was reached after 35 h
of incubation (Figure 4.13).
Figure 4.12 OD 600nm for strain B with and without MDEA
36
Figure 4.13 OD 600nm for strain D with and without MDEA
Table 4.3: Generation time for strain B and D (Appendix H)
Bacteria Generation time
(minutes/ generation)
B 115
B with MDEA 114
D 80
D with MDEA 56
Besides that, the generation time which refers to time taken for a single cell to
double itself was also calculated. Generation time is referred to as the doubling time for
the entire population (Ray, 2004). Table 4.3 shows that under optimum temperature of
growth, strain D has the shortest generation time, followed by strain B. In the presence
of MDEA, strain D showed shorter generation time (56 minutes/generation) than the one
without MDEA (80 minutes/ generation). For strain B, there were not many differences
in generation time with and without the presence of MDEA. Ability to divide itself faster
or shorter generation time show the ability of strain D grew well in the presence of
MDEA.
37
4.6.2 MDEA degradation analysis by IC
Horikoshi medium is a complex medium containing compounds with different
chemical and biological functionality including KH2PO4, Na2CO3, MgSO4 and yeast
extract. MDEA required high resolution separations and classes of compounds from
the rest of the complex mixture. (Bord et al., 2004) stated that amine solutions such as
MDEA will undergo thermal and chemical degradation leading to the formation of basic
compounds such as ammonium cations (Kaminski et al., 2002). This caused analysis
of MDEA in Horikoshi medium very difficult due to the factor above and high
ammonium concentration of the samples. Refer to Appendix I, there was a peak of
Horikoshi medium that similar to MDEA peak.
It was believed that the MDEA or MDEA degradation product were able to form
reaction with the Horikoshi medium since the medium is an undefined medium. It can
be proved by the increasing of the ‘MDEA’ concentration along the time for a control
medium. This estimation needs to be supported by further experiments to separate
MDEA peak areas with other compounds. Chua et al., 2006 showed that LC-MS
method, based on the mixed mode column, offers a quick and effective of screening of
MDEA and their degradation products. There were no matrix interferences although
numerous compounds were present in the sample.
For strain B and D in MDEA degradation studies by refer to Figure 4.14, the
MDEA concentration decreased dramatically after 24 h. At t=24, strain B degraded
41% MDEA as compared to strain D that able to degrade 65% MDEA. Thus, it was
estimated that strain D was a better strain compared to strain B in degradation of
MDEA.
38
Figure 4.14 Degradation study of MDEA (ppm) using strain B and D
4.6.3 pH analysis
Generally, pH for both strains B and D in Horikoshi medium showed a decrease
within the first 15 h and then increased back to pH slightly lower than the original pH of
medium (Figure 4.15).
For strain B, the pH was decreased within the first 12 h from ±9.35 to ±8.28.
For strain D, the pH was significantly decreased within the first 12 h from ±9.35 to
±7.65. However, pH for both strain B and D become similar after 18 h.
The pH continued to decrease for both of the strain before 30 h may be due to the
accumulation of the acidic byproduct. After 30 h, the pH increased back probably due
to the accumulation of toxic waste such as conversion of NH4 +-N into ammonia. The
microorganisms were classified as alkalotolerant microorganisms with their ability to
survive in pH shift in environment ranging from pH 7.5 to pH 9 (Luo et al., 2002) .
39
Figure 4.15 The pH changes in sample for strain B and D with and without MDEA
4.6.4 Redox potential analysis
The variations of redox potential for strain B and D are presented in Figure 4.16.
For strain B, the redox potential keeps to increase until ± 65 h from ±200 mV to ±500
mV. For strain D, the redox potential only increased until 24 h from ±200 mV to ±450
mV. For both strain B and D, the redox potential increased dramatically up to about
400 mV for the first 10 h.
The initial increase in redox potential could be due to the build-up of oxygen in
the liquid prior to the activation of aerobic growth, while the subsequent decrease in
redox potential might indicate the acceleration of aerobic growth that consumed oxygen
at a faster rate (Luo et al., 2002). High redox potential level in the liquid was required
to achieve high nutrient removal efficiency. Both of the bacterial were strict aerobes
and only active at positive reduction potential value.
40
Figure 4.16 Redox potential status in sample for strain B and D with or without
MDEA
41
CHAPTER 5
CONCLUSION
5.1 Conclusion
The optimum temperature for MDEA degrading bacteria is 30°C and growth
well when supplemented with 10% v/v Horikoshi medium. Five types of microbes can
be isolate from the wastewater. Based on the colony morphologies and biochemical
tests, three bacterial strains were partially identified as genus Micrococcus,
Pseudomonas and Corynebacterium. In this study, Pseudomonas sp. was able to
degrade 65% MDEA compare to Micrococcus sp. that degrades 41% MDEA. MDEA
tolerance level for Pseudomonas sp. was within 100 to 200 ppm while MDEA tolerance
level for Micrococcus sp. was 2000 ppm. Ion chromatography using HNO3 as eluent is
preferred method for screening of MDEA compare to gas chromatography due to better
sensitivity. However, analysis of MDEA in complex sample very difficult due to
numerous interferences in the sample and high ammonium concentration of the samples.
Both of the bacteria strains were strictly aerobic microorganisms since them active at
positive reduction potential (Eh) value and classified as alkalotolerant microorganisms
with their ability to survive in pH shift in environment ranging from pH 7.5 to pH 9.
42
5.2 Future work
Further studies are needed so that the Pseudomonas sp. is able to survive in the
MDEA rich petroleum wastewater either by acclimatizing or adapting bacteria in
suitable concentration of MDEA, grow as mix culture and fulfill the nutrient
requirement in MDEA rich petroleum wastewater. Molecular analysis using 16 sRNA
is suggested for further confirmation of the genus and species of bacteria isolated from
wastewater. A monitoring of parameter during treatment such as chemical oxygen
demand (COD), biological oxygen demand (BOD), total suspended solid, total organic
carbon, ammonical nitrogen and color is required. Besides that, enzyme assay can be
developed to study the effectiveness of treatment.
Screening of MDEA and their degradation product require high resolution
separations equipment with a proper working eluent. By using IC for example the
resolution of MDEA was not sufficient to ensure well separated peaks and thus affecting
quantification of MDEA. Further study using mixed mode column such as HPLC with
IC are needed to develop a rapid and suitable method for the quantification of MDEA in
petroleum wastewater.
43
REFERENCES
Abdi, M. A. and Meisen, A.(1992).Amine Degradation: Problems, Review of Research
Achievements, Recovery Techniques. Research Institute of Petroleum Industry Iran
Agilent technologies, Inc. Column HP Ultra 1. United States (2008)
Al-Shawi, A. W., . and Gowda, N. Ion Chromatographic Determination of Organic
Amines in Scrubbing Solutions of Ammonia Plants. 19th AFA Int’l Fertilizer
Technical Conference & Exhibition. 18- 20 April 2006 Four Seasons Hotel: Doha-
Qatar. (2007).
Atlas.R.M (2006). Handbook of Microbiological Media for the Examination of Food. 2
ed. New York, CRC Press.
Bord, N., Crétier, G., Rocca, J. L., Bailly, C. and Souchez, J. P. (2004). Determination of
diethanolamine or N-methyldiethanolamine in high ammonium concentration
matrices by capillary electrophoresis with indirect UV detection: application to the
analysis of refinery process waters. Analytical and Bioanalytical Chemistry.380,
325-332.
Bressan.L, Ubis, T. and O'Keefe, O. Power from Petronor Refinery: The 800 MWe
IGCC project. Gasification San Francisco, USA: (2000).
Chakma, A. and Meisen, A. (1988). Identification of methyl diethanolamine degradation
products by gas chromatography and gas chromatography-mass spectrometry.
Journal of Chromatography A.457, 287-297.
Chua, H.-C., Lee, H.-S. and Sng, M.-T. (2006). Screening of nitrogen mustards and their
degradation products in water and decontamination solution by liquid
chromatography-mass spectrometry. Journal of Chromatography A.1102, 214-223.
44
DeLaune, R. D. and Reddy, K. R. (2005). Redox potential. USA, Elsevier Ltd.
Difco (1998). Difco Manual. 11 ed., Difco Laboratories, Sparks, MD.
Dos Santos, A. B., Cervantes, F. J. and van Lier, J. B. (2007). Review paper on current
technologies for decolourisation of textile wastewaters: Perspectives for anaerobic
biotechnology. Bioresource Technology.98, 2369-2385.
Faddin, J. F. M. (1980). Biochemical tests for identification of medical bacteria. USA,
Waverly Press, Inc
Furhacker, M., Pressl, A. and Allabashi, R. (2003). Aerobic biodegradability of
methyldiethanolamine (MDEA) used in natural gas sweetening plants in batch tests
and continuous flow experiments. Chemosphere.52, 1743-1748.
Heftman, E. (2004). Chromatography: Fundamentals and techniques. 6th ed., Elsevier.
Huntsman.(2007). Technical bulletin: Methyl diethanolamine(MDEA). Woodlands:
Huntsman Corporation
Kaminski, M., Jastrzebski, D., Przyjazny, A. and Kartanowicz, R. (2002). Determination
of the amount of wash amines and ammonium ion in desulfurization products of
process gases and results of related studies. Journal of Chromatography A.947,
217-225.
Kohl, A. L. and Nielsen, R. B. (1997). Alkanolamines for Hydrogen Sulfide and Carbon
Dioxide Removal. Gas Purification (Fifth Edition). Houston, Gulf Professional
Publishing.
Li, Y. and Chrost, R. J. (2006). Microbial enzymatic activities in aerobic activated sludge
model reactors. Enzyme and Microbial Technology.39, 568-572.
45
Luo, A., Zhu, J. and Ndegwa, P. M. (2002). SE--Structures and Environment: Removal
of Carbon, Nitrogen, and Phosphorus in Pig Manure by Continuous and Intermittent
Aeration at Low Redox Potentials. Biosystems Engineering.82, 209-215.
Ray, B. (2004). Fundamental food microbiology. IN 3rd (Ed. USA, CRC Press.
Rennie, S.(2006).Corrosion and materials selection for amine service. Institute of
Materials Engineering Australasia Ltd
Sedlak, R. (1991). Phosphorus and nitrogen removal. 2nd ed. United States, CRC Press.
Tortora, J. G., Funke, R. B. and Case, C. L. (2007). Microbiology, An introduction. 9 ed.
San Francisco, Pearson Education,Inc.
46
APPENDIX A
Biochemical tests for bacteria identification (Faddin, 1980), (Difco, 1998)
Oxidase test
The cytochromes are iron-containing hemoproteins that acts as the last link in the
chain of aerobic respiration by transferring electrons (hydrogen) to oxygen, with the
formation of water. The cytochrome system is found in aerobic, or microaerophilic,
and falcutatively anaerobic organisms, so the oxidase test is important in identifying
organisms that either lack the enzyme or are obligate anaerobes. The test is used to
screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in
identifying colonies suspected of belonging to other genera such as Aeromonas,
Pseudomonas, Neisseris, Campylobacter, and Passteurella.
Gram staining
This is a differential stain which distinguishes all bacteria as Gram positive or
Gram negative according to whether or not hey resist decolorization acetone, alcohol or
acetone-iodine after staining with a para-rozaniline dye such as crystal violet, methyl
violet or gelatin violet. The gram positive bacteria resist decolorization and remain
stained a dark purple color while the gram negative bacteria are decolorized and then
counter stained a light pink by either basic fuchsin, safranin, neutral red or dilute carbol
fuchsin.
In gram positive bacteria the dye complex is trapped in the wall following
ethanol treatment which causes a dimunition in the diameter of the pores in the
peptidoglycan layer of the cell wall. The pores in the inner peptidoglycan layer of the
47
gram negative bacteria are thought to be larger and allows the dye to be extracted.
Hydrogen Sulphide Production Test
Some bacteria decompose sulfur-containing amino acid to form hydrogen sulfide
among the products. The hydrogen sulfide is usually tested for by demonstrating its
ability to form a black insoluble ferrous salt.
Bacteria + amino acid with sulfurs H2S + metal Black color
Simmon citrate agar test
Organisms able to utilize ammonium dihydrogen phosphate and sodium citrate
as the sole sources of nitrogen and carbon, respectively, will grow on this medium and
produce an alkaline reaction as evidenced by a change in the color of the bromthymol
blue indicator from green (neutral) to blue (alkaline).
Methyl Red
It is a quantitative test for acid production, requiring positive organisms to
produce strong acids (lactic, acetic, formic) from glucose through the mixed acid
fermentation pathway. Since many species of the Enterobacteriaceae may produce
sufficient quantities of strong acids that can be detected by methyl red indicator during
the initial phases of incubation, only organisms that maintain this low pH (<4.4) after
prolonged incubation (48-72 h), overcoming the pH buffering system of the medium,
can be called methyl red positive.
48
Voges-Proskauer Test
Organism, such as those belonging to the tribe Klebsiellae, which have the
butylene glycol fermentation pathway, yield a large quantities of neutral products, such
as butylene glycol and ethanol, and small amounts of acetoin and ethanol, and organic
acids. In the presence of air and potassium hydroxide, acetoin, the precursor of
butylene glycol is oxidized to diacethyl, which yields a red color complex.
Urease test
Urea is a diamide of carbonic acid. All amides are easily hydrolyzed with the
release of ammonia and carbon dioxide. Urease is an enzyme possessed by many
species of microorganisms that can hydrolyze urea to produce ammonia and carbon
dioxide. The ammonia reacts in solution to form ammonium carbonate, resulting in
alkalinization and an increase in the pH of the medium.
Oxidation/ fermentation of glucose test
Oxidative organisms can only metabolise glucose or other carbohydrates under
aerobic conditions ie. Oxygen is the ultimate hydrogen acceptor. Other organisms
ferment glucose and the hydrogen acceptor is then another substance eg. Sulphur. This
fermentative process is independent of oxygen and cultures of organisms may be aerobic
or anaerobic. The end product of metabolising a carbohydrate is acid.
The method described, sometimes referred to as the Hugh and Leifson test, and is
a semi-solid medium in tubes, containing the carbohydrate under test (usually glucose)
and a pH indicator. Two tubes are inoculated and one is immediately sealed to produce
49
anaerobic conditions. Oxidising organisms, eg. Pseudomonas species, produce an acid
reaction in the open tube only. Fermenting organisms, eg Enterobacteriaceae, produce
an acid reaction throughout the medium in both tubes. Organisms that cannot break
down the carbohydrate aerobically or anaerobically, eg Alcaligenes faecalis, produce an
alkaline reaction in the open tube and no change in the covered tube.
Indole test
Indole, a benzyl pyrrole, is one of the metabolic degradation products of the
amino acid tryptophan. Bacteria that possess the enzyme tryptophanase are capable of
hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid, and
ammonia. When Kovac’s reagent, which is an acidic solution of
p-dimethylaminobenzaldehyde, is added to a medium rich in tryptophan, indole
combines with the aldehyde to from the aqueous medium with xylene, xylol or
chloroform is required before adding the aldehyde.
50
APPENDIX B
Biochemical tests for strain B, D and E
Urease test Voges Proskauer test
Methyl red test Triple sugar iron test
Simmon citrate test Starch hydrolysis test
51
APPENDIX C
52
53
APPENDIX D
Calculation of concentration MDEA (ppm)
Stock MDEA
1.04g/mL = y mg
1 mL
y = 1.04g
ppm MDEA = 1.04 g x 103 mg
0.001L
= 1040000 ppm
MDEA, 100ppm
100 mg/L = __y mg
0.011 L
y = 1.1 mg
100 ppm = 1.1 x 10-3
1.04
= 1.058x 10-3
mL
= 1 μL
1 μL of stock MDEA added in 0.011 L solution yield 100 ppm MDEA in solution
54
APPENDIX E
Calculation of MDEA tolerance level for isolated bacteria
Table 4.4: Calculation of MDEA tolerance level for strain B
Concentration of MDEA
(ppm)
Growth percentage (%)
at t=23
Control (0 ppm) 0
50 86
500 86
1000 87
2000 100
3000 86
6000 80
Table 4.5: Calculation of MDEA tolerance level for strain D
Concentration of MDEA
(ppm)
Growth percentage (%)
at t=23
Control (0 ppm) 0
50 86
500 86
1000 87
2000 100
3000 86
6000 80
55
At t= 23,
OD 600nm for strain B growth with 0 ppm MDEA is 1.218
OD 600nm for strain B growth with 2000 ppm MDEA is 3.312.
Difference of percentage between medium with 0ppm MDEA and medium with
2000 ppm MDEA is (3.312-1.218) /1.218 X 100%=171%
At t= 23,
OD 600nm for strain D growth with 0 ppm MDEA is 1.7
OD 600nm for strain D growth with 100 ppm MDEA is 3.524.
Difference of percentage between medium with 0 ppm MDEA and medium with 100
ppm MDEA is (3.524-1.7) /1.7 X 100%=107%
56
APPENDIX F
Gas chromatogram and calibration curve for MDEA
Signal Retention
Time [min] Type
Width
[min] Area [pA*s] Area %
1 3.380 PB S 0.056 285752.58113 99.78401
1 6.131 PB 0.104 618.52362 00.21599
Figure 4.17 Gas chromatogram for 2500 ppm MDEA
57
Figure 4.18 Calibration curve for MDEA
58
APPENDIX G
Ion chromatogram and calibration curve for MDEA
Figure 4.19 Ion chromatogram of 30, 60, 90 and 120ppm MDEA
Coefficient=0.99992
Co
nc
en
tra
tio
n
150.00
5 10 15 20 25 30 35 40 45 50 55 60
Area
E+02
1
2
3
4
Figure 4.20 Standard calibration curve of MDEA
59
APPENDIX H
Bacterial growth profile in MDEA degradation study
Colonies forming unit
Table 4.6: Number of strain B and D colonies (CFUs) in plate with a serial dilution
Time
(h)
Flask
Number of bacterial colonies (CFUs)
t0 t12 t24 t36 t48 t65
B 10-8
=TNTC
10-10
=151
10-18
=TNTC
10-20
=224
10-18
=TNTC
10-22
=156
10-16
=TNTC 10-16
= 298 10-14
=178
B +
MDEA
10-8
=TNTC
10-10
=148
10-18
=153
10-20
-288
10-18
=TNTC
10-22
=TNTC
10-16
=TNTC 10-17
=145 10-14
=298
D 10-8
=TNTC
10-10
=122
10-12
=14
10-18
=TNTC
10-20
=TNTC
10-18
=168
10-22
=8
10-14
=33
10-16
=0
10-8
=104 10-6
=76
D +
MDEA
10-8
=TNTC
10-10
=158
10-12
=20
10-16
=298
10-20
=TNTC
10-18
=34 10-16
=118
10-14
=
TNTC
10-10
=67 10-8
=31
E.g. calculation:
Number of 151 colonies on a plate10-10
dilution, then the count is
151/0.1 mL x 1010
=1.51x 1013
bacteria per mL
60
Table 4.7: Number of strain B and D colonies (CFUs) in Horikoshi plate
Time
(h)
Flask
Number of bacterial colonies (CFUs) per mL
T0 T12 T24 T36 T48 T65
B 1.51x 1013
2.24x 1023
1.56x 1025
TNTC 2.98 x 1019
1.78 x 1017
B +
MDEA
1.48x 1013
2.88x 1023
TNTC TNTC 1.45 x 1020
2.98 x 1017
D 1.22x 1013
TNTC 1.68x1021
3.3x 1016
1.04x 1011
7.6 x 108
D +
MDEA
1.58x 1013
TNTC 3.4 x 1020
1.18 x 1019
6.7 x 1012
3.1 x 1010
Calculation of generation time for strain B and D
No. of generations =
[log number of cells (end) – log number of cells (beginning)]/ 0.301
(60 min/h x h) / (Number of generations) = minutes/generation
For B without MDEA,
No. of generations = [log (2.24 x 1023
) – log (1.51x 1013
)]/0.301=33.8
(60 min/h x h) / (Number of generations) = minutes/generation
(60 min/h x 65) / 33.8 = 115 minutes/ generation
For B + MDEA,
No. of generations = [log (2.88 x 1023
) – log (1.48x 1013
)]/0.301=34.2
(60 min/h x 65) / 34.2 = 114 minutes/ generation
For D without MDEA,
No. of generations = [log (1.68x 1021
) – log (1.22x 1013
)]/0.301=27
(60 min/h x 36) / 27 = 80 minutes/ generation
For D + MDEA,
No. of generations = [log (6.5 x 1024
) – log (1.58x 1013
)]/0.301=38.6
(60 min/h x 36) / 38.6 = 56 minutes/ generation
61
APPENDIX I
Cation exchange chromatogram in MDEA degradation studied
3 4 5 min
100
200
300
400
500
mV
ch3
Figure 4.21 Ion chromatogram of Horikoshi medium
62
3 4 5 6 7 min
50
100
150
mV
MD
EA
14
7.2
03
3 4 5 6 7 min
100
200
300
400
500
600
700
mV
MD
EA
1
97
.23
3
3 4 5 6 7 min
200
400
600
800
mV
MD
EA
3
07
.98
0
3 4 5 min
200
400
600
800
1000
mV
MD
EA
3
89
.10
5
Figure 4.22 Ion chromatogram of control at t=0, t=24, t=48, t=65
t=0
t=24
t=48
t=65
63
3 4 5 min
200
400
600
800
1000
mV
MD
EA
1
52
.50
0
3 4 5 min
10
20
30
40
50
60
mV
ch3
MD
EA
9
0.3
61
3 4 m i n
200
400
600
800
mV
ch3
MD
EA
1
14
.63
0
3 4 5 m i n
20
40
60
80
mV
MD
EA
1
95
.32
7
Figure 4.23 Ion chromatogram of sample strain B at t=0, t=24, t=48, t=65
t=0
t=65
t=48
t=24
64
3 4 m i n
20
40
60
80
100
120
mV
MD
EA
1
53
.05
3
0 1 2 3 4 5 m i n
20
40
60
80
100
mV
ch3
MD
EA
54
.22
1
3 4 5 m i n
50
100
150
200
mV
MD
EA
9
4.1
52
3 4 m i n
20
40
60
80
100
120
mV
ch3
MD
EA
1
43
.83
5
Figure 4.24 Ion chromatogram of sample strain D at t=0, t=24, t=48, t=65
t=0
t=24
t=48
t=65