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 It is used for

temperatures and release the hydrogen sulfide at higher temperatures.

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).

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 Because of the

is not regulated as a hazardous material for transportation purposes.

low vapor pressure of MDEA, exposure to vapors is not expected to pose significant hazard under normal workplace conditions (Huntsman, 2007).

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 Chemical treatment of MDEA rich

negatively affect the environment and ecosystem.

petroleum wastewater requires specialized equipment which increases the financial burden. Moreover, chemical treatment of MDEA rich petroleum wastewater will However, a natural

produce other products that may be more harmful to environment. process usually takes a long time to degrade the MDEA. be investigated to treat the wastewater.

Biological techniques should

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

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 Monitoring of

genus level by their morphological and biochemical characterization.

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.

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. indicate abnormal industrial discharges. Unusual odors such as petroleum odors may Wastewater such as petroleum wastewater is An increase in wastewater temperature

generally somewhat warmer than tap water. will increase microbial activity.

However, when wastewater reaches high temperatures, Determination of the forms and concentrations of

microbial activity will be inhibited.

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

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 The inorganic salts such as chloride, sulfate and phosphates

inorganic salts may occur.

typically are found in produced or cooling waters (Abdi and Meisen, 1992).

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 On the other It may

oxidizing to acid sulfuric and cause corrosion to the metals and alloys.

hand, CO2 can cause problems in gas processing plants and refineries alike. ethylene in gas cracking units.

cause problems in hydrate formation, and affect specification of products such as 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).

MDEA reactions with H2S with CO2 H2S CO2 H+ + + + RRRN H2O RRRN HS- + RRRNH+ H+ Fast Slow Fast HCO3- + RRRNH+

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 TOC is

organic carbon (TOC) and the total nitrogen in tail waters (Bord et al., 2004).

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).

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 Aerobic bacteria use oxygen

contaminants by microorganisms when oxygen is present.

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 L

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). temperature is held isotherm at 120 C. The oven

The flow rate of the carrier gas (He) was 0.67 T detection limit under the above

mL/minute and detection temperature 290 C.

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. flame ionization detector should be used. For a specimen contains water, a

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 The combination of support material and ion exchange

styrene-divinyl benzene resin.

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 30 C, 40 C and 55 C 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. was prepared by dissolved 2 g glucose in 50 mL distilled water. Glucose 4% (w/v) 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. 2 mL wastewater was centrifuged at 4000 rpm for 5 minutes.

A volume of

The supernatant was The cell

drained off and the pellet was resuspended with 2 mL of distilled water.

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. overnight. The conical flask was then incubated in shaking incubator at 30 C

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 30 C. 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 -80 C 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 The slide was

left on the slide for 1-2 minutes and washed with distilled water. blot-dry with scott paper and allowed to air dry. under the light field microscope.

The stained smears were examined

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 Bergeys 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. the results which had been obtained. Analysis was made based on

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. 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 Different nutrients sources were

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. with 200 rpm. The conical flasks were incubated at 30 C
600nm

The cell growths were monitored by OD

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). distilled water. Vitamin solution (0.1% v/v). NADH (0.05% v/v): 0.5 g NADH powder was dissolved in 10ml 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. with 200 rpm. The conical flasks were incubated at 30 C

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 4C 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 The flasks were incubated at 30 C with 200 rpm.

Horikoshi medium with inoculum.

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 The cell

redox potential was determined by Orion 3-Star Plus Benchtop pH Meter.

concentration of the cultured media was determined by the cell optical density at 600 nm with a spectrophotometer and spread plate method. performed for a 12 h interval time. Spread plate method was

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 One l of the wastewater sample is injected into The oven

fitted with a flame ionization detector.

a GC with a chromatographic column HP-5 fitted with splitless injection. temperature is held isotherm at 120 C. mL/min and detection temperature 250 C.

The flow rate of the carrier gas (He) was 0.7

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 m filter.

A Metrohm Compact IC 761 equipped with a conductivity detector, Metrosep cation column C1, sampling loop of volume of 20 L were used for chromatographic investigastions. The cation column C1 is a 125 x 4.6 mm column packed with 5.0 m 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 The

formed when filter sterilized MDEA rich petroleum wastewater exposed to air. precipitate formed was contributed to a higher reading.

Thus, control was important to Results showed

compare the growth of the indigenous bacteria with a nutrient added.

that the most suitable nutrient for growing the indigenous bacteria was adding 10% (v/v) LB or Horikoshi at 30 C, 200 rpm.

LB was a nutritionally rich medium, including peptides, casein peptones, vitamins, trace elements, and minerals. Peptide and peptones were provided by Sodium ions for

tryptone. Vitamins and trace elements were provided by yeast extract.

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 30C 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 Shape Size Surface Texture Elevation Margin Irregular <1mm Rough Brittle Flat Entire Circular 1mm-7mm Glistening Buttery Convex Entire Rhizoid <1mm Rough Brittle Flat Filamentous Irregular 1mm-4mm Glistening Moist Convex Wavy Irregular 2mm-7mm Wrinkled Moist Raised Wavy A B C D E

B Figure 4.1

D E Colonies morphology for bacteria B, D and E

B Gram negative cocci Figure 4.2

D E Gram negative rod Gram positive rod 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 Oxidase Catalase Indole Motility O-F Simmon citrate TSI Starch hydrolysis Urease MR VP Mac Conkey Nitrate reduction test Partial identification of bacteria + + O + Micrococcus sp. + + + O + + + Pseudomonas sp. + + + + Corynebacterium sp. B D E

* 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 Strain D colony are gram negative bacilli, white in color,

genus Cornybacterium.

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 This probably due to the lack of some growth factor

and/or nitrogen source in MSM.

in MSM thus failed to support the growth for the bacteria.

27

Figure 4.3 source

Growth of isolated bacterial strains in MSM with carbon and nitrogen

Figure 4.4 ppm MDEA

Growth of isolated bacterial strains in MSM with carbon source and 50

28

Figure 4.5 ppm MDEA

Growth of isolated bacterial strains in MSM with nitrogen source and 50

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 MDEA

Growth curves of strains B, D and E in Horikoshi medium with 50 ppm

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.
nm

The absorbance reading for OD

600

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 However, it was not MDEA after injection of MDEA cannot be extracted by

with standard MDEA (Appendix I).

sample into ion chromatography with better sensitivity.

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. eluted by the protons from the eluent. were used by trial and error method. The analytes was

In the study of MDEA, two types of eluents

mV 2000

MDEA 127.411

1500 ch3

1000

500

min

Figure 4.10

Ion chromatogram of 120 ppm MDEA by using 4mM/L H2SO4 +

55% Acetonitrile as working eluent

34
M D E A 1 2 0 .2 5 0

mV

400

300

200

100

ch3 3 4 5 6 7 min

Figure 4.11 working eluent

Ion chromatogram of 120 ppm MDEA by using 5mM HNO3 as

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 B with MDEA D D with MDEA 115 114 80 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 (Bord et al., 2004) stated that amine solutions such as the rest of the complex mixture.

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 Chua et al., 2006 showed that LC-MS

MDEA peak areas with other compounds.

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 Thus, it was

41% MDEA as compared to strain D that able to degrade 65% MDEA.

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 The to the accumulation of toxic waste such as conversion of NH4 +-N into ammonia. survive in pH shift in environment ranging from pH 7.5 to pH 9 (Luo et al., 2002) .

microorganisms were classified as alkalotolerant microorganisms with their ability to

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. mV. For strain D, the redox potential only increased until 24 h from 200 mV to 450 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 Both of the bacterial were strict aerobes

to achieve high nutrient removal efficiency.

and only active at positive reduction potential value.

40

Figure 4.16 MDEA

Redox potential status in sample for strain B and D with or without

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. be isolate from the wastewater. Five types of microbes can

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 Intl Fertilizer Technical Conference & Exhibition. 18- 20 April 2006 Four Seasons Hotel: DohaQatar. (2007). Atlas.R.M (2006). Handbook of Microbiological Media for the Examination of Food. 2 ed. New York, CRC Press. Bord, N., Cr tier, 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 Fermenting organisms, eg Enterobacteriaceae, produce Organisms that cannot break

reaction in the open tube only.

an acid reaction throughout the medium in both tubes.

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. ammonia. When Bacteria that possess the enzyme tryptophanase are capable of reagent, which is an acidic solution of

hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid, and Kovacs

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 L 1 L 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) Control (0 ppm) 50 500 1000 2000 3000 6000 Growth percentage (%) at t=23 0 86 86 87 100 86 80

Table 4.5: Calculation of MDEA tolerance level for strain D Concentration of MDEA (ppm) Control (0 ppm) 50 500 1000 2000 3000 6000 Growth percentage (%) at t=23 0 86 86 87 100 86 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 1 1

Retention Time [min] 3.380 6.131

Type PB S PB

Width [min] 0.056 0.104

Area [pA*s]

Area %

285752.58113 99.78401 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

C o ncentra ti o n

Coefficient=0.99992
150.00 4 3 2 1 Area 50 55 E+02 60

10

15

20

25 30

35

40

45

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 B 10-8=TNTC 10-10=151 B+ MDEA D 10-8=TNTC 10-10=148 10-8=TNTC 10-10=122 10-12=14 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 10-18=TNTC 10-20=224 10-18=153 10-20-288 10-18=TNTC 10-20=TNTC 10-18=TNTC 10-22=156 10-18=TNTC 10-22=TNTC 10-18=168 10-22=8 10-14=33 10-16=0 10-8=104 10-6=76 10-16=TNTC 10-17=145 10-14=298 10-16=TNTC 10-16= 298 10-14=178 t0 t12 Number of bacterial colonies (CFUs) t24 t36 t48 t65

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 B B+ MDEA D D+ MDEA T0 T12 Number of bacterial colonies (CFUs) per mL T24 T36 T48 2.98 x 1019 1.45 x 1020 1.04x 1011 6.7 x 1012 T65 1.78 x 1017 2.98 x 1017 7.6 x 108 3.1 x 1010

1.51x 1013 1.48x 1013 1.22x 1013 1.58x 1013

2.24x 1023 2.88x 1023

TNTC TNTC

1.56x 1025
TNTC

TNTC TNTC 3.3x 1016 1.18 x 1019

1.68x1021 3.4 x 1020

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

mV

500 400 ch3 300 200 100

min

Figure 4.21

Ion chromatogram of Horikoshi medium

62

150

M D E A 1 4 7 .2 0 3

mV

t=0

100

50

min

mV 700 600

M D E A 1 9 7 .2 3 3

500 400 300 200 100

t=24

min

mV

800

600
M D E A 3 0 7 .9 8 0

t=48

400

200

3 mV 1000

min

800

M D E A 3 8 9 .1 0 5

600

t=65

400

200

min

Figure 4.22

Ion chromatogram of control at t=0, t=24, t=48, t=65

63

mV

M D E A 1 5 2 .5 0 0

1000

t=0

800 600

400

200

min

mV 60 50 40 30 ch3 20 10

M D E A 9 0 .3 6 1

t=24

min

mV 800

M D E A 1 1 4 .6 3 0

t=48

600 ch3 400

200

3
mV 80

min

M D E A 1 9 5 .3 2 7

t=65

60

40

20

min

Figure 4.23

Ion chromatogram of sample strain B at t=0, t=24, t=48, t=65

64

mV

120 100 80 60 40 20

M D E A 1 5 3 .0 5 3

t=0

M D E A 5 4 .22 1

min

mV 100

t=24

80

60

40

20

0 ch3
mV

min

200

M D E A 9 4 .1 5 2

t=48

150

100

50

3 mV

min

M D E A 1 4 3 .8 3 5

120 100 ch3 80 60 40 20

t=65

min

Figure 4.24

Ion chromatogram of sample strain D at t=0, t=24, t=48, t=65