BIOREMEDIATION OF WATER CONTAMINATED WITH CRUDE OIL ...

5 downloads 0 Views 2MB Size Report
PETROLEUM HYDROCARBONS USING Pseudomonas aeruginosa –A CASE OF ... dissertation or report submitted to this University or to any institution of the ...... isolates in analytical laboratories in Kampala specifically in the microbiology ...
BIOREMEDIATION OF WATER CONTAMINATED WITH CRUDE OIL PETROLEUM HYDROCARBONS USING Pseudomonas aeruginosa –A CASE OF LAKE ALBERT

BY KIRAYE MICHAEL 2007/HD13/9981U

A Dissertation in partial fulfillment of the requirements for the award of the degree of Master of Science (Chemistry) of Makerere University

September 2015

DECLARATION I Kiraye Michael declare that this dissertation characterizes my own original work, apart from where outstanding acknowledgment is inferred, and that it has not been previously included in, dissertation or report submitted to this University or to any institution of the sot for any academic qualification.

Signed................................................... Date......................................................

KIRAYE MICHAEL

ii

APPROVAL

This is to certify that Kiraye Michael conducted his research at the college of Physical Sciences in the Department of Chemistry of Makerere University under our supervision.

1. Dr. John Wasswa Department of Chemistry Makerere University Sign: ……………………………………

Date: ……………………………………..

2. Dr. Gabriel Kasozi Department of Chemistry Makerere University Sign: ……………………………………

Date: ……………………………………..

iii

ACKNOWLEDGEMENT I would like to express thanks to the Almighty God for his compassions and loveliness upon my life. I highly raise the value of the assistance given to me by different professionals at the College of Natural Sciences particularly those in the departments of Chemistry and Biochemistry. Special thanks go to Dr. Vincent P. Alibu and Mr. John Omara lecturers in the Department of Biochemistry and Dr. Rose Nalwanga of the Department of Biological sciences, Kyambogo University for her kind support in this research.

My sincere appreciation goes to my supervisors Dr. John Wasswa and Dr. Gabriel Kasozi for their time and diligent direction from the time I started on this research project till its completion. Furthermore, I would like to thank my family for their constant support towards my education Finally I acknowledge all my friends, course mates, classmates and relatives for their support and assistance during the entire course of study.

May the Almighty God Bless You entirely.

iv

CONTENTS ACKNOWLEDGEMENT ------------------------------------------------------------------------- iv ABSTRACT---------------------------------------------------------------------------------------- xi 1

CHAPTER ONE: INTRODUCTION ----------------------------------------------------- 12

1.1

Background to the study---------------------------------------------------------------------------------------------------------- 12

1.2

Statement of the problem ----------------------------------------------------------------------------------------------------------4

1.3 Objecti ves--------------------------------------------------------------------------------------------------------------------------------5 1.3.1 General objective -------------------------------------------------------------------------------------------------------------------5 1.3.2 Specific objectives ------------------------------------------------------------------------------------------------------------------5 1.4

Justification of the study------------------------------------------------------------------------------------------------------------5

1.5

Scope of the study ---------------------------------------------------------------------------------------------------------------------5

2

CHAPTER TWO: LITERATURE REVIEW ---------------------------------------------- 6

2.1

Pollution studies in Lake Al bert -------------------------------------------------------------------------------------------------6

2.2

Hydrocarbon structure -------------------------------------------------------------------------------------------------------------7

2.3

Hydrocarbon removal using physical methods -----------------------------------------------------------------------------7

2.4

Concentrati on of the petroleum or hydrocarbons -------------------------------------------------------------------------8

2.5

Petroleum biodegradation by microbial consorti a ------------------------------------------------------------------------8

2.6

Bioremedi ati on reacti on order ---------------------------------------------------------------------------------------------------9

2.7

Hydrocarbon Bi oremediation studies ------------------------------------------------------------------------------------------9

2.8

Bioremedi ati on process of PHC contaminants ---------------------------------------------------------------------------- 10

2.9

Petroleum hydrocarbons (TPH) bi oremediation ------------------------------------------------------------------------- 12

2.10

Using Gas Chromatography-Mass Spectroscopy ------------------------------------------------------------------------ 13

2.11

Evaluati on of decontamination rate ------------------------------------------------------------------------------------------ 13

3

CHAPTER THREE: MATERIALS AND METHODS ----------------------------------- 15

3.1

Water sampling ---------------------------------------------------------------------------------------------------------------------- 15

3.2

Al bertine Graben ------------------------------------------------------------------------------------------------------------------- 15

v

3.3

Culture: Nutrient broth medium: --------------------------------------------------------------------------------------------- 17

3.4

Petroleum Hydrocarbons (PHs) Extraction ------------------------------------------------------------------------------- 18

3.5

Instrumental analysis -------------------------------------------------------------------------------------------------------------- 18

3.6

Bioremedi ati on procedure ------------------------------------------------------------------------------------------------------- 19

3.7

Determinati on of the remedi ation --------------------------------------------------------------------------------------------- 20

3.8

Quality assurance and data analysis ----------------------------------------------------------------------------------------- 20

4

CHAPTER FOUR: RESULTS AND DISCUSSIONS ------------------------------------ 21

4.1

Chromatograms --------------------------------------------------------------------------------------------------------------------- 21

4.2

The relati onshi p between sum corrected area ( 𝑺𝒄𝒂𝟐 ) after bi oremedi ati on and amount of PHs left 22

4.3

The relati onshi p between sum corrected area, 𝑺𝒄𝒂𝟏 ti me in days before bi oremedi ati on ----------------- 23

4.4

The relati onshi p between sum corrected area counts, 𝑺𝒄𝒂𝟐 and ti me in days after bioremedi ation --- 23

4.5

Relati onshi p between ti me and amount removed ------------------------------------------------------------------------ 24

4.6 The relationship between Bioremediation Rate, Reciprocal of Amount removed (PHs dilution) and Reci procal of ti me------------------------------------------------------------------------------------------------------------------------------ 26 4.7

Amount removed by Pseudomonas aeruginosa ( 𝒎𝒑) [g/L] ---------------------------------------------------------- 28

4.8

The control experimental results ---------------------------------------------------------------------------------------------- 30

4.9

Amount left by Pseudomonas aeruginosa (𝒎𝒍) [/L] --------------------------------------------------------------------- 32

4.10

Combined expl anation of bi oremedi ation ---------------------------------------------------------------------------------- 35

4.11

Bioremedi ati on rate ---------------------------------------------------------------------------------------------------------------- 37

4.12

Bioremedi ati on reacti on second order kinetics --------------------------------------------------------------------------- 39

4.13

Reci procal of ti me versus amount left s quared --------------------------------------------------------------------------- 41

Table 4.5: Showing the relati onshi p between the reci procal of ti me [day-1 ] and amount left s quared [ g2 /L 2 ]-- 42 Figure 4.12: A graph of the reci procal of ti me day-1 and amount left s quared i n g2 /L 2 .--------------------------------- 42 4.14

Bioremedi ati on proposed second order mechanism -------------------------------------------------------------------- 43

4.15

Reduction of PHs from the Lake Al bert water surface ---------------------------------------------------------------- 44

4.16

Significance of second order bi oremediation kinetics of contami nated Lake Al bert water --------------- 45

5

CONCLUSIONS AND RECOMMENDATIONS ---------------------------------------- 47 vi

5.1

Conclusions --------------------------------------------------------------------------------------------------------------------------- 47

5.2

Recommendati ons ------------------------------------------------------------------------------------------------------------------ 47

6

REFERENCES ---------------------------------------------------------------------------- 49

7

APPENDICES ----------------------------------------------------------------------------- 56

7.1

Appendi x I: Pseudomonas aeruginosa isolates --------------------------------------------------------------------------- 56

7.2

Appendi x II: Col umn chromatography cleanup stage ----------------------------------------------------------------- 57

7.3

Appendi x III: Brown band vol ume ------------------------------------------------------------------------------------------- 58

7.4

Appendi x IV: Raw data----------------------------------------------------------------------------------------------------------- 59

7.5

Appendi x V: Raw data for control experiment --------------------------------------------------------------------------- 60

vii

LIST OF TABLES Table 4.1: Relationship between time in day of bioremediation and amount removed including the control experiment ............................................................................................................................................................................25 Table 4.2: The variat ion of bioremed iation rate (gday-1) with time in days.......................................................................26 Table 4.3: showing the relationship between amounts of PHs removed and amount left in grams per litre versus time in days ...................................................................................................................................................................................32 Table 4.4: showing the relationship between Bioremediation Rate per gram per day versus (Amount removed)2 [g 2 /L2 ] .................................................................................................................................................................39 Table 4.5: Showing the relat ionship between the reciprocal of t ime [day-1 ] and amount left squared [g2 /L2 ] ..............42 Table 7.1: Showing the relationship between sums corrected area of chromatograms and mass of PHs spread on water (mg/ ml) before and after bioremed iation .............................................................................................................59 Table 7.2: Showing the relationship between sums corrected area of chromatograms and mass of PHs spread on water (mg/ ml) before and after bioremed iation for the control experiment .............................................................60

viii

LIST OF FIGURES Figure 1.1: French oil giant Total E&P during its oil exploration activities ............................................................................ 3 Figure 2.1: The degradation of straight chained alkanes ........................................................................................................10 Figure 2.2: The degradation of cycloalkanes ............................................................................................................................11 Figure 2.3: The degradation of benzene.....................................................................................................................................11 Figure 2.4: The degradation of methyl benzene .......................................................................................................................12 Figure 3.1: The Lake Albert shore (Kaiso) fro m where a sample o f water was collected ................................................16 Figure 3.2: the map of Uganda, showing Albertine Graben...................................................................................................16 Figure 3.3: Fishermen row next to an oil exp loration site next to Lake Albert at kaiso in the Bulisa district ..............17 Figure 4.1: The chro matograms used to get the sum corrected area (Sca) ..........................................................................22 Figure 4.2: UCM Sca after bioremediation vs PHs removed (g/ L).......................................................................................23 Figure 4.3: The relationship between time in days of bioremediation and UCM total sum corrected area of chromatogram peaks after bioremed iation......................................................................................................................27 Figure 4.4: Prediction curve for the relationship between time in days of bioremediation and UCM total sum corrected area of chro matogram peaks after bioremed iation.......................................................................................28 Figure 4.5: Petro leu m hydrocarbons removal versus time in days .......................................................................................29 Figure 4.6: Petroleu m hydrocarbons removal versus time in days for the control experiment ........................................31 Figure 4.7: A mount of Petroleu m Hydrocarbons removed/left versus time in days ..........................................................33 Figure 4.8: Po lynomial model for PHs removal versus time in days ...................................................................................36 Figure 4.9: Bio remediat ion rate per day versus time in days .................................................................................................37 Figure 4.10: Regression for Bioremediat ion Rate/gday-1 vs (A mount removed)2 /g 2/L2 ..................................................40 Figure 4.11: Regression for Bioremediat ion Rate/gday-1 vs amount removed (g/L)........................................................41 Figure 4.12: A graph of the reciprocal of t ime day-1 and amount left squared in g2 /L2 . .....................................................42 Figure 4.13: The proposed mechanism fo r the boremediation process ................................................................................43 Figure 4.14: Bioremed iation Rate/gday -1 versus reciprocal of A mount removed ..............................................................44 Figure 7.1: The pure cultures A and B of Pseudomonas aeruginosa ...................................................................................56 Figure 7.2: The pure cultures A and B of Pseudomonas aeruginosa ...................................................................................56 Figure 7.3: The sterile cabin for bioremediation and cleanup of the samples during the activity. ..................................57 Figure 7.4: Bro wn band volume appearance during colu mn chro matography cleanup stage ..........................................57 Figure 7.5: Bro wn band volume (ml) versus time (days) .......................................................................................................58

ix

ABBREVIATIONS AND ACRONYMS 𝑅𝑏 = Bioremediation rate. 𝑅𝑒 = Amount removed. 𝑅𝑒0 = Original Amount removed. 𝑆𝑐𝑎 = Sum corrected area of chromatogram peaks. 𝑆𝑐𝑎1 = Sum corrected area of chromatogram peaks before bioremediation. 𝑆𝑐𝑎2 = Sum corrected area of chromatogram peaks after bioremediation. ∆t = model time step size and. µmax = maximum contaminant utilization rate per mass of microorganisms. C = concentration of contaminant (PHs). GC/FID = Gas Chromatography with Flame ionization Detector. GC-MS = Gas Chromatography with mass spectroscopy Detector. KC = contaminant half-saturation constant. LAS1 = Lake Albert Sample location one. LAS2 = Lake Albert Sample location two. Mt = total microbial concentration. PHCs = Petroleum HydroCarbons. PHs = Petroleum Hydrocarbons. PPO = polyphenol oxidase. TPH = Total Petroleum Hydrocarbons. 𝑇 = Time in days. UCMA = Unresolves Complex Mixture area for the chromatograms UCM = Unresolved Complex Mixtures m = mass in grams (𝑚)0 = Initial amount of PHs at the beginning of the bioremediation mp = Amount removed by Pseudomonas aeruginosa mL = Amount left by Pseudomonas aeruginosa

x

ABSTRACT Uganda at the moment is making an effort to exploit and make petroleum products. This is taking place around the Albertine Graben waters at Kaiso in Buliisa District. Lake Albert being the main focus. The concern is that these water bodies are ecological habitants for many aquatic organisms and likewise the main drinking water sources in the region. Irrespective of the point that Lake Albert water points are famous for quite a lot of uses both ecological and economical, they're expected to be badly polluted by crude oil petroleum hydrocarbons (PHs). Consequently they will need to be handled with environmentally friendly methods. Bioremediation is one of such approaches among which Pseudomonas aeruginosa was used. Thus the current study targeted determining the amount and the rate at which Pseudomonas aeruginosa remediates PHs from fresh water of Lake Albert. Lake Albert Water was sampled and brought to laboratory and contaminated with 10% m/v PHs (100g/L). The water was inoculated with Pseudomonas aeruginosa (turbidity of 0.04 absorbance at a wave length of 600nm). This contained approximately 3.0x107 colony- forming unit (CFU)/mL. The samples were kept at room temperatures to imitate the temperature of Lake Albert. Results indicated that the initial rate, 𝑅𝑏𝑖𝑜𝑖 was 33.78 g/L per day for n-hexane soluble PHs. Correspondingly, the highest amount removed at the saturation point was 89.3g per litre. The bioremediation process took the second order kinetics registering a half- life of 3.9 days. This implies that the original amount can reduce to half the initial amount after 3.9 days (about 93.6hrs). On Conclusion, Lake Albert is already contaminated with petroleum hydrocarbons. Pseudomonas aeruginosa significantly (p = 0.03) (p < 0.05) bioremediates petroleum hydrocarbons from Lake Albert water with the highest removal rate between day 1 and day 3. The maximum removal efficiency of petroleum

xi

hydrocarbons (PHs) from water by Pseudomonas aeruginosa is achieved between 5th and 6th day.

1 CHAPTER ONE: INTRODUCTION 1.1 Background to the study Uganda is currently making an effort to explore and take part in the exploitation of petroleum products. This is mainly around the natural water bodies in the Albertine Graben. Contrastively, these water bodies in Albertine graben are ecological habitants for various aquatic organisms and also the principal drinking water sources in the area. Regardless of the fact that Lake Albert fresh water bodies are famous for pretty a lot of functions both conservational and economical, they're anticipated to be tremendously polluted by crude oil petroleum hydrocarbons (PHs). Subsequently they will require to be dealt with by environmentally friendly methods. Bioremediation is one of such methods of which Pseudomonas aeruginosa was used. Bioremediation is a restoration intervention method that uses microorganisms such as bacteria and fungi to degrade hazardous organic contaminants or convert them to environmentally less toxic compounds of safe levels in soils, subsurface materials, water, sludges, and residues(Barathi et al., 2001). It is based on the fact that organisms are capable of taking in substances

from

the

environment

metabolism(Chapelle, 1999).

and

use

them

to

enhance

their

growth

and

Bacteria, Protista, and fungi are well known for degrading

complex molecules and transform the product into part of their metabolism (Das et al., 2010). They have long been considered as one of the predominant petroleum hydrocarbon degrading agents found in the environment, which are free living and abundant(McCarthy et al., 2004) . The petroleum hydrocarbons are increasingly becoming pollutants of major concern within the environment(Lee et al., 2001). Due to its complicated composition, petroleum has the potential to elicit multiple types of toxic effects(Orisakwe et al., 2004). It can cause acute lethal toxicity, sub- lethal chronic toxicity, or both depending on the exposure, dosage, and the organism exposed. Some components of petroleum have the potential to bioaccumulate within susceptible aquatic organisms and can be passed by trophic transfer to other levels of the food chain(Barron et al., 2001). Globally petroleum hydrocarbons have been a problem in case of spillage and xii

seepage into water bodies

for example; BP/Deep water Horizon Oil Spill, Gulf of

Mexico’’(Çavaş et al., 2005). Crude oil and refined fuel spills from tanker ship accidents have damaged natural ecosystems in Alaska, the Gulf of Mexico, the Galapagos Islands, France, and many other places(Helton et al., 1999). The quantity of oil spilled during accidents has ranged from a few hundred barrels to several hundred thousand barrels (Nounou, 1980). Smaller spills have already proven to have a great impact on ecosystems especially if the oil spill occurs in remote sites where it is difficult to give a crisis ecological response (Carson et al., 2003). Spills of Hydrocarbons in crude oil that occur in aquatic environment are usually far further harmful than those that occur on land, since this can cover a large area in form of a thin oil film on water surface and are not easily removed (Blumer et al., 1973). The physical and chemical methods like volatilization, photooxidation, chemical oxidation, and bioaccumulation that have often been used in cleaning up these hydrocarbons are rarely successful in rapid removal of these compounds (Roy et al., 2014), In addition, these methods are not safe and cost effective when compared to microbial bioremediation. The success of bioremediation technologies applied to hydrocarbon-polluted environments highly depends on the biodegrading capabilities of native microbial populations or exogenous microorganisms used as inoculants(Brüggemann et al., 2003). The presence of microorganisms with the appropriate metabolic capabilities is the most important requirement for oil spill bioremediation(Das & Chandran, 2010).

Crude oil contamination can prove to bring a pronounced ecological risk and can affect the economy of the country(Beamish, 2001).Unfortunately, more efforts have been placed on the drilling of the crude oil, with limited plans geared towards restoration of the affected water bodies in case of accidents. Lake Albert and its catchment area are susceptible to such accidents(Kathman et al., 2011). The lake supports an important community of fishermen who entirely depend on the maintenance of its ecological inte grity(Karp et al., 2012), while its water catchment area corresponds to a landscape where agricultural activities, deforestation, over stocking of livestock and wildlife are a major feature alongside key protected areas of global significance(Wandera et al., 2010) . Thus, the aquatic ecosystem around Lake Albert may be destroyed in case of crude oil spillage that can take place from the extraction grounds (Kathman & Shannon, 2011). 2

Oil exploration and the potential oil drilling activities in the Albertine region are along national parks and game reserves from which tourism industry earns much of its income (Kathman & Shannon, 2011). Any catastrophic environmental challenge due to oil spillage will not only affect the aquatic life but also the terrestrial life and generally whole scope of climatic factors in the region, with a long term effect on the tourism industry(Smith, 2012) a case in Buliisa district.

Buliisa District is found in the northern portion of the Western Region of Uganda. It is composed of one county with two sub counties and one town council. It has a human population of 76,900, of which 79 % is able to get safe water(Bainomugisha et al., 2011).

Of late oil has been discovered in Buliisa District. The additional expansion of the oil industry drives the reason in an intensification of the need of the safe water demand in the district. The Figure 1.1 shows the exploration grounds for oil in the region.

Figure 1.1: French oil giant Total E&P during its oil exploration activities http://www.redpepper.co.ug/oil-giants-total-ep-splash-shs5bn-on-buliisa-farmers 3

The access rates to safe water varies from 72 % in Buliisa Sub-County to 86 % in Biiso SubCounty. The functionality rate in urban and rural areas is 79 % and 74 % respectively (Bainomugisha et al., 2011).

Buliisa partakes 259 domestic water access points, all have been working properly for over 5 years. The main water supply technologies that people in Buliisa use are the shallow water points and the deep borehole technologies.

The district has two groundwater built pumped piped water source systems used by approximately 20 % of the population who can get to safe water while 80 % of the benefiting population obtain water from point water sources that can be vulnerable to PHs contamination in case of crude oil spills (Vokes, 2012). On this basis, the current research determined the kinetics of the remediation process for these PHs using unresolved complex mixtures approach.

Unresolved complex mixtures: Unresolved complex mixture (also UCM or hump) is a feature frequently Paid close attention to in gas chromatographic (GC) data of crude oils and extracts from organisms that take action to oil. The aim for the UCM hump attendance is that GC does not resolve and recognize a noticeable portion of the hydrocarbons in crude oils(Frysinger et al., 2003).

1.2 Statement of the problem The emergency of oil exploration industry in Uganda makes it susceptible to petroleum related environmental challenges including spillage. Unfortunately, there are no well-documented environmentally friendly methods for remediation of petroleum hydrocarbons spillage in water bodies within the country. The study was aimed at exploring the use of a pure culture of Pseudomonas aeruginosa in reducing such a case of petroleum hydrocarbons spillage into Lake Albert water. 4

1.3

Objectives

1.3.1

General objective

The study was aimed at assessing the ability and extent by which Pseudomonas aeruginosa could remediate water from Lake Albert contaminated with petroleum hydrocarbons.

1.3.2

Specific objectives

1. To determine the amount removed of petroleum hydrocarbons (TPH) from water by Pseudomonas aeruginosa over a period of time.

2. To determine the remediation rate in amount of total petroleum hydrocarbons (TPH) removed per unit time.

1.4

Justification of the study

Crude oil contamination of water bodies by hydrocarbons is a major concern for oil exploration and drilling activities. The Lake Albert region is one such area vulnerable to this concern. For this reason, studies aimed at abating such a challenge are necessary. This was the main issue of this work.

1.5

Scope of the study

The study was laboratory based on water samples collected from Lake Albert that were contaminated with common petroleum hydrocarbon mixture such as that of petrol, diesel, paraffin, and engine oils. Selection of site of the water sample was decided after observing the distance and the infrastructure of the crude oil extraction plant.

In this work, contaminated water sample Inoculation with petroleum-degrading active bacteria such as Pseudomonas aeruginosa was used for bioremediation process.

5

2 CHAPTER TWO: LITERATURE REVIEW 2.1

Pollution studies in Lake Albert

Pollution study that was carried on Lake Albert revealed that, there is an indication of the condition that customarily governs behaviour of the factors of this Lake both physically and nutritiously(Evans, 1997). This being possible if the water is not contaminated to change these factors. The studies on the effect of the factors in water by petroleum hydrocarbons indicate that they do alter the physical and nutrient factors in the aquatic environment (Chandra et al., 2013). This is due to the fact that petroleum hydrocarbon consist of various hydrocarbons like CH3 (CH2 ) n CH3 , C6 H12 , C6 H6 and C7 H8 (Joshi et al., 2014) that can cover a large surface area of the lake and prevent the water from interacting with the atmosphere. It is reported that Bioremediation is important for converting petroleum hydrocarbons into soluble metabolites. One of the most PHC clean up from water bodies and in soils (Howard, 2003). The microbial bioremediation is also associated with a number of environmental and human health risks. Reactions in microbial bioremediation involve evolution of a wide range of metabolites. Metabolites from the microbial bioremediation may pose health concern depending on the amount of PHC contamination dealt with in the process. Degradation of alkanes is a widespread occurrence in nature, and numerous microorganisms, prokaryotic and eukaryotic, capable of utilizing these substrates as a carbon and energy source have been isolated and categorized. Bacterial strategies for accessing these highly hydrophobic substrates are presented, along with systems for their enzymatic degradation and conversion into products of less aquatic environmental effects(Goldman et al., 2015). Furthermore the current knowledge on the regulation of bacterial long-chain n-alkane metabolism and survey progress in understanding bacterial pathways for utilization of n-alkanes under anaerobic conditions was also studied(Latha et al., 2012). The research method of this work will follow the same footstapes to achieve the results of the analysis of water contaminated with petroleum hydrocarbons.

6

While many studies have reported the abilities of bacteria to degrade hydrocarbons with different carbon numbers, most bacteria can consume only a narrow range of hydrocarbons, Geobacillus jurassicus grows only on C6– C16, and Bacillus thermoleovorans degrades n-alkanes up to C23. Indeed, few strains capable of degrading a wide range of hydrocarbons have been identified to date(Song et al., 1990). Some exceptions include Acinetobacter sp which was described as one that degrades long-chainn-alkanes (C13–C44). Since the water extraction procedure in this method is similar to that found in the water portion of semi- volatile petroleum products, i.e. from kerosene through heavy fuel oils), these products maybe characterized

using this

extract(Schumacher, 2002).

2.2 Hydrocarbon structure Studies also revealed that, owing to variable chemical structures and molecular weights petroleum hydrocarbons differ in their liability to microbial attack as follows: n-alkanes > branched alkanes > low- molecular-weight aromatics > cyclic alkanes(Leahy et al., 1990). Biodegradation rates are highest for saturated hydrocarbons, followed by light aromatics with high- molecular- weight aromatics and polar compounds being highly recalcitrant to biodegradation(Leahy & Colwell, 1990). (Atlas, 1991)reported a more rapid degradation of aromatic hydrocarbons compared to n-alkanes. In comparison (Leahy & Colwell, 1990)observed that more naphthalene than hexadecane was degraded in water and sediment mixtures.

2.3

Hydrocarbon removal using physical methods

Eelier studies in the eighties and nineties asserted that the oil undertakes a series of actions such as evaporation, dissolution, photooxidation, and emulsification with water to form emulsions or mousse and perhaps eventually the formation of tar balls(Payne, 1982). The development of oil mousse increases the surface area of the oil and thus making it more available for microbial attack(Bartha et al., 1977). Tar balls, however, are exceptionally unmanageable to degradation due to their solid state and low surface area to volume ratios(Bartha & Atlas, 1977). And that PHAs are greatly persistent in the environment due to their high hydrophobicity and therefore have a low availability to biodegrading microorganisms(Sabljic, 2001). Biodegradation of low molecular wt. hydrocarbons occurs readily when PAHs are in aqueous phase (Venosa et al., 2003). Consequently, the low solubility and dissolution rates of the larger complex PAHs may limit their availability for biodegradation. Phenanthrene and anthracene have water solubilities of 7

1.29 and 0.07 mg/l respectively(Willumsen et al., 1996). (Willumsen & Karlson, 1996) went on to say that phenanthrene in solution system is more biodegradable than in crystalline systems.

2.4 Concentration of the petroleum or hydrocarbons Microbial study on uptake showed that despite the fact that the rates of microbial uptake and biodegradation of water-soluble compounds are usually related to the concentration of the compound, the matching cannot be the same of compounds with low aqueous solubility and those, which can exert membrane toxicity at high concentrations(Barron & Ka'aihue, 2001). The biodegradation rates of high molecular weight PAHs such as naphthalene and phenanthrene are proportional to their aqueous solubility rather than their concentrations in a given solution(Wong et al., 2004). On the other hand, high concentrations of highly soluble or volatile organic compounds may be detrimental to microbial forms due to their toxicity(Khan et al., 2000). A study on adaptation of microbial populations showed that exposing hydrocarbon contaminants to microbial populations confers adaptation(Madsen et al., 1992), often this lead to enhanced mineralization(Powell et al., 2008). It was documented that 55% pyrene mineralization in environments when the inoculum was grown on pyrene compared to l% mineralization by indigenous population(Grosser et al., 1991). Another researcher also testified complex metabolic activity when the inoculum was grown with crude oil as a substrate compared to nutrient broth(Grishchenkov et al., 2000).GC MSD Temperature programming :It was emphasized that a temperature program of 35 °C for 4 minutes, 35 °-300°C at 10°C /min and 300°C for 3 minutes could be used to generate a good chromatogram using n-hexane as solvent (Veriotti et al., 2000). Another alternative temperature programme can be set from 100°-300°C at 2.5°C/min at a column pressure of 2.0 atm while detector temperature is kept at 150 °C (West et al., 1990).

2.5 Petroleum biodegradation by microbial consortia A study of the bacteria responsible for bioremediation asserted that Numerous biodegradations necessitate more than a single species(Grady, 1985). Distinct microorganisms can metabolize only a partial range of hydrocarbon substrates, so collections of mixed populations with overall broad enzymatic capacities rate are required to bring the rate and extent of petroleum biodegradation further(Foster, 1962). Another study showed that microbial strains belonging to various genera have been detected in petroleum contaminated soil or water (Abed et al., 2002). Went ahead to propose that each strain or genera have their roles in the hydrocarbon conversion 8

processes. More evidence for the mutual aid of mixed cultures in biodegradation is reported by (Harayama et al., 2004). (S. Meyer et al., 1999) who observed a successive change of the composition of oil-degrading bacteria over a period in oil-contaminated sand samples. Related observations were reported in sequential enrichments in medium containing residual crude oil (Venkateswaran et al., 1995).In the case of using Pseudomonas aeruginosa for bioremediation of crude oil contaminants in water bodies, their most important advantage is probably their ecological acceptance(Prince, 2002).

2.6 Bioremediation reaction order It has been stated by(Vincent et al., 2011)that, Bioremediation of water polluted with common hydrocarbons such as diesel is a first order reaction with rate constant of 0.002hour -1 and half- life (t1/2 ) of 346.5 hours. This study considered the kinetics of the bioremediation of diesel and not a mixture of hydrocarbons.

2.7 Hydrocarbon Bioremediation studies Isolation of hydrocarbon degrading microorganisms study showed that in nature, there are many bacterial communities that degrade petroleum hydrocarbons which are found at relatively higher densities in petroleum contaminated sites. The most common sites are around estuaries, oceans and water sediments, fresh water, deep sea and thermal rents. For this reason these areas are explored for isolating hydrocarbon degrading microorganisms(Pérez Silva et al., 2009). Among those isolated from aquatic habitats, Pseudomonas, Vibrio, Achromobacter, Arthrobacter, Micrococcus, Corynebacter, Acinetobacter, Nocardia and others are the predominant hydrocarbon utilizers, while Aureobasidium, Candida, Rhodotorula and Sporobolomyces are the most common fungi and yeasts isolated from marine environments (Meza Vargas, 2013). For this purpose, Pseudomonas aeruginosa was chosen to be used to clean up an oil contaminated water obtained from L. Albert. This is because, its bioremediation does restore the normal conditions to almost the same level as it was before petroleum hydrocarbon contamination without introducing other contaminants to water(Zheng et al., 2012).A study on bioaugmentation showed that the addition of microbe cultures to a contaminated area to increase the number of microbes can degrade the oil and hydrocarbons(Sublette et al., 1995). On the other hand, a study on biostimulation showed that the addition of nutrients to the contaminated area 9

stimulates microbial growth, such as bacteria, to enhance them to remove environmental pollutants from soil, water, or gases(Vidali, 2001). Specific study on active bacteria of species Pseudomonas aeruginosa showed that it uses a wide range of organic material for food and is able to decompose hydroca rbons like CH3 (CH2 ) n CH3 , C6 H12 , C6 H6 and C7 H8 and has been used to break down oil from oil spills(Das & Chandran, 2010). In the current research, effective microbes (Pseudomonas aeruginosa) were obtained from isolates in analytical laboratories in Kampala specifically in the microbiology Laboratory in the school of veterinary, Makerere University.

2.8 Bioremediation process of PHC contaminants O2 NADH2

H2 O NAD+

CH3(CH2)nCH3

H OH

CH3 (CH2)n-1CHCH3 Alkane hydroxylase

O

Alcohol dehydrogenase

CH3(CH2)n-1CCH3 O2 NADH 2

keto monooxygenase H2 O NAD +

O

CH3(CH2)n-1-O-CCH3 H2O Esterase O

NADH2

CH3COH

NAD+

O

O

CH3 (CH2)n-2C-OH

CH3(CH2 )n-1 -OH

CH3(CH2)n-2CH H2O

2H

oxidation

Figure 2.1: The degradation of straight chained alkanes

10

Degradation of C 6 H12 is as follows: O2 NADH2

H2O NAD+

cyclohexane

OH

NAD+

NADH2

O2 2H

O

O H 2O

8-caprolactone

cyclohexanone

cyclohexanol

O

H2O

O O HO-C-(CH2 )4-C-OH hexanedioic acid (adipic acid)

2H

H2 O

2H O O HC-(CH2)4 -C-OH

O HOCH2(CH2)4-C-OH 6-hydroxyhexanoic acid

6-oxohexanoic acid

Figure 2.2: The degradation of cycloalkanes Degradation of C 6 H6 is as follows: O2 NADH2

NAD+

H

NAD+

NADH2

OH

OH OH

OH

H benzene

benzene dihydrodiol

Figure 2.3: The degradation of benzene Degradation of C 7 H8 is as follows:

11

catechol

CH3

toluene

O2 2H

H2 O

CH2OH

O CH

2H

benzyl alcohol

H 2O

2H

COOH

benzoic acid

benzaldehyde

O2 HADH2 NAD+

OH OH

NADH2

NAD+

HOOC

OH OH H

CO2 benzene carboxyhydrodiol

catechol

Figure 2.4: The degradation of methyl benzene (Rittmann, 1994)

The pyoverdine chromophore is made in a response linking a two-electron degradation, an addition reaction followed with oxidation. This oxidative flow can be conceded with polyphenol oxidase (PPO), MnO 2 , and extracts without cell from Pseudomonas aeruginosa (Dorrestein et al., 2003).It is reported that Pseudomonas aeruginosa

crude extract is associated with

polyphenol oxidase (PPO)(Sanchez-Amat et al., 2001).

2.9 Petroleum hydrocarbons (TPH) bioremediation On the study of on Pseudomonas aeruginosa

for the carbon source revealed that,

Bioremediation process of converting non-decomposable petroleum hydrocarbons into soluble metabolites that are usually decomposable in a short term(Mukherjee, 2002), is usually done with active microorganisms that use them as the carbon source for their metabolism(Jawhari, 2014). The point at which the current work viewed microbial bioremediation as(Kato et al., 2000; A. Meyer et al., 2008). In this evaluation, we condense recent advances in the accepting of bacterial metabolism of long-chain n-alkanes(Okoh, 2006) and (Das & Chandran, 2010).. For example, Bacillus stearothermophilusis only capable of growth on C15–C17(Tani et al., 2001; X.-B. Wang et al., 2011), and Rhodococcus strains capable of degrading n-alkanes up to C36 (L. Wang et al., 2006). Degradation of a wide range of hydrocarbons and crude oil is of crucial importance for bioremediation of oil contamination and microbial enhanced oil recovery(Atlas, 1991). Unfortunately, relatively few bacteria strains have been isolated and studied (Leadbetter 12

et al., 1958). In another study, a Pseudomonas aeruginosa strain is reported for the first time that was capable of utilizing a wide range of hydrocarbons (C6–C40), aromatic compounds, and crude oil with different patterns over time (X.-B. Wang et al., 2011).

2.10 Using Gas Chromatography-Mass Spectroscopy Studies in PHs degradation were using the gas Chromatography method as a qualitative and semi-quantitative procedure(Schumacher, 2002). In most studies It was used for groundwater or surface water, and soil/sediment from sites where the petroleum products are unknown and/or when multiple types of petroleum products are suspected to be present(Falatko et al., 1992). This method was used to identify petroleum products containing components from C7 to C30 range, as well as heavy oils, with specific product confirmation by pattern matching ("fingerprinting") employing capillary gas Chromatography with flame ionization detection (GC/FID) (Reddy, 1999). EPA method 3510 has been adapted as the extraction procedure for the water portio n of this method(Sauer et al., 1991). While this method is anticipated to be primarily qualitative, it was used to remove the need for further analyses for those samples, which demonstrate TPH levels significantly below the regulatory limits(Huesemann, 1994). It was also reported that if the sample comprises toluene to dodecane (gasoline range), dodecane through tetradecane (diesel range) and/or an unresolved chromatographic cover greater than tetradecane (e.g. motor oils) beyond the reporting limits of this method, then the final quantitation must be done by methods specific for these mixtures(Einhorn et al., 1992; Llompart et al., 1998).

2.11 Evaluation of decontamination rate Another study showed that biodegradability of petroleum hydrocarbons (PHCs) is usually inversely proportional to the number of fused benzene rings (Cermak et al., 2010). Half- lives in water/soil and sediment of the three-ring phenanthrene molecule being in a range from 16 to 126 days while for the five-ring benzo-pyrene may range from 299 to more than 1400 days(Howard et al., 2005). The tenacity of higher molecular weight PHCs being due to largely their low water solubility and resonance energy of their structures(Tian, 2005).

13

14

3 CHAPTER THREE: MATERIALS AND METHODS 3.1 Water sampling Water samples for this study were collected from Lake Albert, located in the Albertine Grebin. This place was selected because the oil exploration facility is in its surrounding areas. The samples were collected from two locations from namely LAS1 with coordinates (01 °32.042N, 03°57.939E) and LAS2 with coordinates (01 °32.032N, 03°57.958E). The samples were mixed to form one laboratory sample. Two (2) litres of water samples from Lake Albert were obtained and kept in sterile flasks. .The water samples in the sterile bottles were placed on Ice in cooler boxes and transported to Makerere University Chemistry pesticide laboratory. The samples were then refrigerated for 10 hours.

3.2 Albertine Graben Albertine Graben stretches from southwestern Uganda to northwestern districts as shown on the figure 3.2.

Albertine Graben covers almost 500 kilometres stretch to 45 kilometres

across(Anderson et al., 2011), making Uganda border with the DRC and runs from Lake Edward southwards up to the border with South Sudan in northern portion as seen in Figure 3.2. The term ‘graben’ may be defined as a depressed crust of the earth’s surface lying between two geological fault lines. The graben is the best studied and prospected part of Uganda’s sedimentary basins (Martini et al., 2013) even though the studies under looked remediation.

15

Experimenter collecting water from Lake Albert shore

Figure 3.2: the map of Uganda, showing Albertine Graben http://www.geoexpro.com/articles/

Figure 3.1: The Lake Albert shore (Kaiso) from where a sample of water was collected

2012/01/uganda-key-to-futurehotspots-in-landlocked-africa

This location was chosen because of being in the vicinity of the oil e xploration and extraction plant as in Figure 3.1 showing the establishment. The water nearby this facility is expected to have future contamination of various petroleum fractions either by mechanical failure of the machines or by incidental spill from the pipelines. Ten percent (10%) (100g/L) contamination was considered because this has potential to form a film on water causing it deprived of gaseous exchange hence affecting aquatic environment including bird impairments. The oil grounds in Albertine graben at the moment are projected to hold deposits mounting to 2.5 billion barrels of crude oil all seen to be extracted by the facility in Figure 3.3, so posing high chances of contamination of the lake. The 1 billion barrels the government is set to extract from what has been concealed to date may result into the contamination being talked about, worse still though it is likely that more oil may be discovered in future in this location. 16

Figure 3.3: Fishermen row next to an oil exploration site next to Lake Albert at kaiso in the Bulisa district http://www.theguardian.com/global-development/poverty- matters/2014/may/12/kenya-ugandaeast-africa-first-oil-producer The concentration of petroleum hydrocarbons was determined using Chromatography-Mass Spectroscopy following the unresolved complex mixtures approach for quantification method. Samples of the petroleum hydrocarbons were made by mixing 40g of the common hydrocarbons in the market ranging from solids to liquids making a large sample weighing 240g of the mixture. Those used were grease super power (MP3), Petrol, diesel, kerosene, petrol engine oil and diesel engine oil. The solid hydrocarbons were mixed in liquid hydrocarbons before applying them to form a film on water surface. Approximately ten percent (10%) contamination was intentionally made for this Lake Albert water sample selected using various amounts of PHs of known amounts.

3.3 Culture: Nutrient broth medium: Nutrient broth medium composed of peptic digest of animal tissue (5.0g/l), sodium chloride (5.0g/l), beef extract (1.5g/l), yeast extract (1.5g/l), final pH at 25 °C (7.4 ± 0.2), ingredients was used to boost the growth of Pseudomonas aeruginosa 17

Cryopreserved culture: This was achieved following a procedure described by the National Institute of Standards and Technology (NIST)(Benson et al., 2011). Following this method, a stock culture was made by dissolving 13g of nutrient broth in 1liter of distilled water. The stock was divided into 10 parts of 100ml volumes containing 1.3g of nutrient broth each and were autoclaved at 121 °C. The broth (1.3g) (100 ml) solutions were allowed to cool. The resultant broth solutions were inoculated with a sterile micropipette tip full of Pseudomonas aeruginosa from the culture plate. The bacteria were allowed to grow at 37 °C in the incubator shaker for 10 hours to a turbidity recording an absorbance of 0.04 at 600nm measured from spectrophotometer serial number 122445 in the biochemistry laboratory. A sample of the resulting bacteria (1ml) was then added to 1ml of 50% glycerol in a vial for cryopreservation. The vials were kept in a deep freezer at -20°C. Glycerol was prepared as follows: a 20ml Glycerol was added to 20 ml of distilled water to make a 50% glycerol solution. The remaining Pseudomonas aeruginosa cultures formed the Working culture and the starter culture for the bioremediation experiments.

3.4 Petroleum Hydrocarbons (PHs) Extraction A UNEP/IOC/IAEA 1992 method for PHs extraction was adopted and used as follows: a 10 ml of n-hexane was introduced to each extract for 30 minutes to improve phase separation and then separated decantation. The mixtures were eluted through glass chromatographic columns having silanized glass wool in the effluent side and packed with approximately 5.0 cm length of silica gel followed by 5.0 cm length Alumina and 1.5 cm length of anhydrous sodium sulphate. Two 10ml preparations of hexane were used to rinse the flasks (and the sodium sulphate remaining in the glass) and the cleanup columns. All of the mixtures and rinsate were brought together. The resulting extracts were intensified to 1 ml (in a Turbo Vaporizer) for the low concentration level and diluted to 100 mL for the greater concentration level previous to analysis.

3.5 Instrumental analysis An Agilent 6890N gas chromatograph (GC), joined with Mass Selection detector (5975) on a fused silica capillary column coated with HP-5 MS 5% Phenyl methyl siloxane (30 m length and 0.25 mm ID 0.25 μm film thickness) was set. Injection of a 1.0μL aliquot of the extract as the injector port is held at150°C and operated in split mode and Helium carrier used to detect PHs at a split ratio of 1: 20. Temperature-programme was as follows: Initial temperature at 95 °C for 1 min, 95−190 °C at 20 °C/min, 190−250 °C at 15 °C/min, 250−300°C at 25 °C/min for 3.0 min, 18

giving a total run time of 18.5 min. The detector temperature was set at 150 °C. Alternative Temperature programme was from 100 °-300°C at 2.5°C/min at a column pressure of 2.0 atm while detector temperature was kept at 150°C.Agilent Chemstation software was used to obtain the chromatogram and for data calculations. Integration of the entire peaks area was used to determine the total area counts for each sample chromatogram before and after bioremediation. The difference in total area counts was used to calculate the biodegration efficiency (Reddy, 1999). The performance parameters used for the valuation of bioremediation were sum corrected area of chromatogram peaks and amount removed (RE). Amount removed (RE) was determined using the following equation, 𝑟𝑒𝑚𝑜𝑣𝑎𝑙 𝑒𝑓𝑓 =

𝑆𝑐𝑎1 −𝑆𝑐𝑎2 𝑆𝑐𝑎1

× 100% Where: 𝑆𝑐𝑎1 = sum corrected area of

chromatogram peaks before bioremediation, 𝑆𝑐𝑎2 = sum corrected area of chromatogram peaks after bioremediation. (𝑆𝑐𝑎1 − 𝑆𝑐𝑎2 ) = the difference between sum corrected areas before and after bioremediation,

𝑆𝑐𝑎1 −𝑆𝑐𝑎2 𝑆𝑐𝑎1

×concentration of the total petroleum hydrocarbon consumed during

bioremediation activity in grams per cubic centimeter, 𝐶𝑜̅ = average PHs Contamination spread on L. Albert water in grams per cubic centimeter. The data sets were amount of TPH before inoculation, 2) amount of TPH consumed after bioremediation for every 24 hours for 7 days.

3.6 Bioremediation procedure Microorganisms of the species Pseudomonas aeruginosa were used to degrade Lake Albert petroleum hydrocarbon contaminants. Growth of Pseudomonas aeruginosa was done in a sterile nutrient broth(100ml) incubated at 37°C for a period of 1.5 hours that was expected for the log phase of these species of bacteria(Goldman & Green, 2015). A sample of petroleum hydrocarbon contaminated water (10 ml), was quantitatively placed in a 250 ml flask containing a nutrient broth (100 ml). An aliquot of Pseudomonas aeruginosa starter culture (100µl) whose turbidity absorbance was 0.04 at 600nm (3.0x107 colony- forming unit (CFU)/mL)was introduced as it was reported by (Goldman & Green, 2015) that an absorbance at 600 nm (using a LB blank) of 1.0 is approximately equivalent to 7.5x108 colony- forming unit (CFU)/mL of Pseudomonas aeruginosa . Another set of petroleum hydrocarbon contaminated autoclaved Lake Albert water (10 ml) at a temptation of 121°C, was measurably placed in a 250 19

ml flask containing a nutrient broth (100 ml) but with no Pseudomonas aeruginosa starter culture to acted as the control experiment. The resulting mixture sets were shaken at a speed of 180 r/min for 24 hours at room temperature using a shaker model THZ-82. The bacterial activity was temporary stopped by reducing the temperature of the mixture to about 2°C to 8°C every after 24 hours]. Petroleum hydrocarbons were extracted from water with n-hexane following a method described by A UNEP/IOC/IAEA 1992 method for PHs. Gas Chromatography-Mass Spectroscopy following unresolved complex mixtures approach was used quantification of the amount removed.

3.7 Determination of the remediation Graphical method was used to determine initial rate basing on the fact that, bioremediation rate can be related to the time take to degrade PHs as (Sheth et al., 2009) reported a removal rate of 136 mgl-1 h-1 using Pseudomonas aeruginosa

though using a dye as an indicator. Typical

graphical analysis parameters were as follows: amount removed (g/L) versus time; and bioremediation rate in grams on a litre per day versus time in days.

3.8 Quality assurance and data analysis To guarantee data quality, duplicate analyses for accuracy control and the control reference were used. The results from this study were blank corrected. One procedural blank was made for every 4 samples. The limit of detection (LOD) were defined as 3 times the standard deviation divided by the slope of the regression line (𝐿𝑂𝐷 = 3 ×

𝑆𝑇𝐷𝐸𝑣 𝑏

). The limit of quantification (LOQ) was

defined as 10 times the standard deviation divided by the slope of the line relating sum corrected area and mass of PHs (𝐿𝑂𝐷 = 10 ×

𝑆𝑇𝐷𝐸𝑣 𝑏

). This method was described by (Ripp, 1996) in the

detection limit guidance. The limits of detection and quantification was reported as a function of mass of the PHs extracted. The statistical significance of the study results were defined by a p