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