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ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 Current Science Perspectives 2(4) (2016) 105-115 iscientic.org. Comparison of degradation of benzene, toluene and phenol in both fresh and salt water media C. P. Ukpaka Department of Chemical/Petrochemical Engineering Rivers State University of Science and Technology Nkpolu PMB 5080, Port Harcourt, Nigeria *Corresponding author’s E-mail: [email protected] A R T I C L E I N F O Article type: Research article Article history: Received December 2015 Accepted June 2016 October 2016 Issue Keywords: Comparison Degradation Benzene Toluene Phenol Fresh water Salt water A B S T R A C T Research work was conducted to demonstrate the relationship between some aromatic hydrocarbon in fresh and salt water media. The comparison of the degradation rate of toluene in fresh and salt water media revealed a good match in the substrate degradation per unit time. The rate of degradation of benzene, toluene and phenol decreases with increase in period of exposure. The reaction mechanisms were inhibited by various factors such as salinity, P + , temperature etc. The medium of contamination play an activity role in effective remediation of contaminated environment. The nature of the functional parameters of environment may cause inhibition in the process in which the substance found in the area may activate the programme. Degradation of Benzene, Toluene and Phenol is faster in fresh water them salt water medium; this can be attributed to the variation in the physiochemical parameters of the media considered during the investigation. © 2016 International Scientific Organization: All rights reserved. Capsule Summary: The rate of degradation of benzene toluene and phenol was monitored and predicted in fresh and salt water media in Niger Delta of Nigeria. The characteristics of the aquatic environment was used in examining the degradation of the component investigated and results obtained reveals the significance of the physiochemical parameters (functional parameters) on the system. Cite This Article As: C. P. Ukpaka. Comparison of degradation of benzene, toluene and phenol in both fresh and salt water media. Current Science Perspectives 2(4) (2016) 105-115 INTRODUCTION The publication of Spies et al., (1996) cited in Okoh, (2006), claims that Marine oil spills; especially large-scale spill accidents can be hazardous to its coastal environment. They cited the case of the oil spill of the North Slope crude oil into the Prince William Sound, from Exxon Valdez, in 1989. This case is still being referred to today, because of the death of thousands of Sea bird and other marine mammals that the caused pollution. www.bosaljournals/csp/ 105 In a more recent report, Knightes and Peters, (2006) carried out a study on Polycyclic Aromatic Hydrocarbons (PAHs), and described them as complex mixtures occur as distinct organic phases, often called ‘non-aqueous-phase liquids’. PAHs are called Colliod-associated solutes in groundwater, soils and sediments. As for their hazardous effects of the contaminants in the environment today, knightes and Peters said that there is pressing need to develop and improve on previous biochemical methods of treating these pollutants (PAHs), in a controlled and effective manner. Polycyclic Aromatic Hydrocarbons are so dangerous, as environmental contaminants, that over 16 PAHs have been [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 named as ‘priority contaminants’, by the US Environmental Protection Agency (EPA). They are said to be extremely carcinogenic. They are carcinogenic in that, they can induce cancer or cancerous growth in humans. Bioremediation is the process of employing the biodegradative potentials of micro-organisms or their attributes (Caplan, 1993, cited in Okoh, 2006). This definition could not have been coined better! Bioremediation is an effective technology in that it also treats the physical and chemical aspects of the contaminants alongside the biochemical aspect. Simply put, bioremediation encourages volume reduction and detoxification. According to Zhu et al., (2001), a major setback in bioremediation is the lack of guidelines regarding which and how to use the technology. This problem would be solved as the technology advances. At the moment, a lot of research is being conducted on bioremediation. Bio-augmentation is a potential strategy for Oil bioremediation since the 1970s Zhu et al., (2001). Their rationale behind the augmentation of microbial population is that indigenous microbial population may not be able to degrade a wide range of potential substrates, present in complex mixtures such as petroleum. It is for this reason that oil-degrading micro-organisms are added to the indigenous microbial population. This practice may come in handy when conducting experiments in this project. Biostimulation involves the addition of rate-limiting nutrients to speed up the biodegradation process. In most shoreline ecosystems that have been heavily contaminated with petroleum hydrocarbons, nutrients are likely the limiting factors in the oil biodegradation. He stated that laboratory studies have shown that addition of growth limiting nutrients, namely Nitrogen and Phosphorous, have enhanced the rate of oil biodegradation Venosa, (1998) cited in Zhu et al., (2001). Okoh, (2006) stated that is important to expose the limiting factors of biodegradation of petroleum hydrocarbons in both Fresh and Sea water (Salt water). These limiting factors should definitely affect the kinetic modeling of the biodegradation of petroleum hydrocarbons, in subsequent chapters. The limiting factors are: petroleum hydrocarbon composition (PHC), physical state of the petroleum hydrocarbons, mineral nutrients availability, salinity, pH, temperature, oxygen, water and micro-organisms. Enumerations and subsequent explanation of the roles played by these mentioned factors, while not lengthy in content, used as few words as possible in order to simplify the factors. The highpoint of Bossert and Bartha’s work, (1984) cited in Okoh, (2006) is the approximation of the temperature ranges for biodegradability of petroleum to be: 20-30o in fresh environment, and 15oC – 20oC , in water (sea water) environments. Temperature affects the biodegradability of petroleum hydrocarbons in two ways: the properties of spilled petroleum hydrocarbons, and the activity or population of micro-organisms. It was also stated in their work that the biodegradability of petroleum www.bosaljournals/csp/ 106 iscientic.org. hydrocarbons decreases more viscous, the micro-organisms become less Okoh, (2006). In terms of the physical state of the petroleum hydrocarbons, Morrrison and Boyd, (1974), noted that oxidation increases the biodegradability of petroleum hydrocarbon by increasing its availability. This enhances microbial activities. Oxygen’s role in biodegrability of petroleum hydrocarbons is very important one, depending on the type of micro-organism. The Microbial utilization of hydrocarbons require an exogenous “electron sink”. This electron sink, according to them in the absence of molecular oxygen, further biodegradation of partially oxygenated intermediates may be supported by nitrate and Sulphate reduction. What they didn’t explain clearly was the concept of the “electronsink” Reardon etal., 2003; Prasad, 2000; Okoh, 2002; Egberongbe et al., 2006; Keyzig, 199). Oxygen is most important to this project because a lot of micro-organisms are aerobic, especially bacteria, while some are facultative by nature. That is, they depend on oxygen from aeration in order to survive in their substrates. Oxygen is crucial for rapid bioremediation. Dibble and Bartha, (1976) cited in Okoh, (2006). Nutrients affect the biodegradability of petroleum hydrocarbons. Inadequate mineral nutrients especially Nitrogen and Phosphorus, often limits the growth of hycarbon utilizing microbes in water. This case can be especially found in fresh water plants for nutrients. On the other hand, the nutrients level could increase as a result of the deposal of industrial waters, which may and may not contain nutrients Zhu, et al; (2001). Nitrogen is at a low level in Sea (salt) water. This publication contrasts the findings of Okoh in 2006. It is also evident that the case of ‘bends’ which occurs when a sea diver swims directly upwards especially from a very deep sea level, is as result of the Nitrogen struggling to burst through the skin pores of the Sea Diver who is a now suffering from the ‘bends’ condition (Ukpaka, 2013, 2013a; 2013b; Ukpaka, 2015, 2015a; 2015b; 2015c; 2015d; Belcher et al., 1970). The increase of salinity leads to a sharp drop in the growth rate of micro-organism. This is only temporary as the culture recovers and adapts to its harsh environment. The effect, however, is difficult to quantify. It is transient, and applies to mixed culture Richardson and Peacok (1991); Ukpaka (2014; 2014a; 2014b; 2014c; 2014d) Inhibition can be illustrated through simple reaction equation, where a substance ‘B’, when introduced to a reactant ‘A’, causes the slowdown, pauses, or completely stops further enzyme-substrate reaction of A to R. Levenspiel (1999), as shown in equation (1a) below: A+I R (1) Equation (1a): Single reversible reaction. The aim and objectives of the research work is to determine the rate of degradation of each component investigated such as benzene, toluene, and phenol as well as the significance of the water medium environment in enhancing biodegradation. Another important aim and objective is to [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 iscientic.org. Fig. 1: Comparison of Degradation Rate of Toluene in Both Media determine the specific rate of substrate, maximum specific rate of substrate and determination of the equilibrium constant. The substance ‘I’ is known as an inhibitor. Inhibitor reacts with enzyme ‘E’ to make the inert Y. The Equation (1) illustrates competitive inhibition: MATERIALS AND METHODS Inhibition can be illustrated through simple reaction equation, where a substance ‘B’, when introduced to a reactant ‘A’, causes the slowdown, pauses, or completely stops further enzyme-substrate reaction of A to R. Levenspiel, (1999), as shown in equation (1a) below: A+I R (1) (1) Equation (1a): Single reversible reaction. Equation (1): Cooperate Inhibition reaction Enzyme Inhibitors can be described as molecules that reduce or abolish enzyme activity. There are several types of inhibitation namely: Competitive, Non-competitive and Mixed Inhibition. Competitive Inhibition occurs when a substrate ‘A’ and a substrate ‘I’ competes for the same site on the enzyme ‘E’. The competition usually results in either the slowing down or stops the reaction completely. Levenspiel (1999). The following equation rates are derived from the reaction equations above (equation 1): TR K 3CEOC A K 3CEOC A CM C A NC EOCM CM 1 NC EO C A (2) Equation (2): Competitive Inhibition equation. www.bosaljournals/csp/ 107 [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 K 2 K3 , Michealis' cons tan t , mol / m3 K1 N And Ch t 1 Pt e8r t t Where, CM iscientic.org. K4 K5 In non-competitive Inhibition, a Substrate ‘A’ attacks the Enzyme ‘E’ on one site, while, Inhibitor ‘I’ attacks the enzyme on another site. ‘I’ would still be an inhibitor because, its action of attacking the enzyme on a different site results in the slowdown or stopping of the reaction. This form of Inhibition is illustrated below. Substrate ‘A’ attacks the enzyme to form ‘intermediate ‘X’. Intermediate ‘X’ is completed into product ‘R’. On a different site but, on the same enzyme, ‘I’ attacks the enzyme, either stopping it, or slowing it down, as seen in equation (3) below. (5) Where, Ch(t) = time-varying hopane-normalised concentration, P = polar fraction of the Oil, r = ratio of the average residual nitrogen concentration to Oil loading, ε = assumed multiplicative error term, and α, δ, γ, ε = fitting parameters determined from the multiple regression analysis. They also claimed that the equation (5) model matched the experiment results, when parameters were chosen to fit the data. The Model has a limitation though, and that is because; the data set used was limited to only a small area in the field. Venos, et al. (1996) cited in Zhu, et al. (2001) developed a model from field data first-order biodegradation rate constants for Alkanes and several PAHs presents in Light Crude oil. This model is shown below in equation (6): e kt A A A A 0 (6) Where, A A time-varying hopane-normalised concentration of an analyte, A A 0 Equation (3) is a non-competitive Inhibition reaction. From Equation (3) above, equation (4) is derived: TR time-varying hopane-normalised concentration of an analyte K 3C EO C A C M C A NC BO C M LC AC B K3 CE C 1 LC BO 0 A 1 NC BO C A C M 1 LC BO First-order biodegradation rate constant for an analyte and is was stated that actual first-order biodegradation rates are not constant, but are a function of the residual nutrient concentration. Equation (7) shows this relationship between nutrient concentration and 1st –order biodegradation rates: N K obs K max Kn N (4) Biodegradation Kinetics Oil biodegradation rates are difficult to prevent due to the complexity of the environment Zhu et al. (2001). According to Zhu, the Kinetic model development by the Exxon Valdez monitoring computer programme is based on field studies conducted by researchers from Exxon. This model is depicted below in equation 5: www.bosaljournals/csp/ 108 (7) Where, Kobs = Observed first-oder hydrocarbon biodegradation rates. Kmax =Maximum first-order hydrocarbon biodegradation rates. Kn = Half-saturation concentration for a specific nutrient (MnL-3), N = Interstitial pore water residual nutrient conc. (MnL-3). According to Reardon et al. (2002), the rate of biodegradation of Phenol is shown below in equation (8) we have dS X dt Yx / s (8) [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 iscientic.org. Fig. 2: Comparison of the degradation rates of benzene in fresh and salt water Where, S = Substrate concentration, T= time, = specific growth rate,YX/S= Biomass yield, andX= Biomass concentration. Author attributed the emergence of this model to the non-volatile nature of phenol. As for the model for Toluene and Benzene (being volatile hydrocarbons), the equation was modified. This, according to them is because Toluene is present in both gas and liquid phases in the bioreactor. Since the microbial growth rate depends on the liquid phase substrate concentration only, and biomass yield depends on the change in total mass of substrate, the masses of Toluene (liquid and gas) are shown in equations (9) and (10) below: M TOT M L M G H VG M TOT M L 1 M L RT VL (9) dSL S L X dt YX /S Where, (10) 109 (11) SL = Liquid-phase Their first setoff experience involved the use of P. Putida F1 microorganism to biodegrade benzene, toluene and phenol, alongside their binary and tertiary mixtures. Their results proved that their single-substrate experiment’s growth kinetics fit the Monod model (toluene and benzene, respectively). Equation (12) below show the Monod model: Where, ML= mass of Hydrocarbon in liquid phase, MG = mass of Hydrocarbon in Gas phase, MTOT = mass of Hydrocarbon in the entire system, H = Henry’s constant, R = Gas constant. T = temperature, VG = Gas-pahse volume,VI = Liquid - Phase Volume,VG = Gas-phase volume, and VL = Liquid- Phase volume. From the equations above, during their experiments, they discovered that the temperature and volume of liquid www.bosaljournals/csp/ remained essentially constant. Therefore, the rate of substrate consumption was written below in equation (11) below, as: m SL K S S L (12) Bearing in mind that Were 1 dX X dt max max imum specific growth rate, KS = Monod half-saturation constant. [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 However, they chose to adopt the Monod model over Andrew’s because the differences between the model fits were small, also, Andrew’s model did not improve the prediction of mixture experiments. They also adopted a model for Cell growth on homologous substrate mixtures. In this model, the specific growth rate is the sum of the specific growth rate of each substrate. Equation (14) depicts this model: max 1 S1 K S ,1 S1 μ = μ0 = specific growth rate at temperature T0, R = gas constant, 3.2 103 Ea = activation energy = KJ Kmol Richardson and Peacock (1991). Effect of salinity on microbial growth rate max 2 S 2 K S ,2 S 2 iscientic.org. Increase in salinity leads to sharp drop in growth rate of microbes. This is only temporary, as the culture recovers after adaptation. The effect is however, difficult to quantify. It is transient, and applies to mixed culture. (14) μ1 + μ2 + ……… μi it should be noted though that this model is strictly a no interaction model. This study is laudable. The never-say-die attitude of the researchers to the various setbacks and challenges they were in gigantic contributions to field of Bioremediation. This piece will be directly relevant to this project, as their findings would be improved on, and innovated. The model below was developed by Park and Marchaland, (2005) to explain the effect of salinity on the maximum specific growth rate of the biomass: Effect of pH on microbial growth rate Where, IS = Salinity inhibition constant (h-1), K1 = Substrate inhibition constant (mg I-1). Micro-organisms have an optimum pH for growth. This is usually close to neutrality (7.0), the effect of hostile environments, caused by unusually on pH specific growth rate is similar to the product inhibition. That is, it is considered as a modification of μm. Note that a number of species of micro-organisms can actually cope and even grow in spite of their harsh environments. The equation (15) below shows the modified version of μm: m m pH 1 H K H K a2 a1 Where, Ka1 and Ka2 are constants. [H+] = Hydrogen ion concentration. The specific growth rate is not necessarily at maximum at the optimum Ph. The formula below is used to calculate the effect of temperature on the microbial growth rate: K S S S 2 / K1 (17) I S % NaCl 0.01 % NaCL (18) Where, I*S = Constant depending on culture. The PARK and MARCHLAND’S model was modified, with simpler parameters introduced to show the relationship between the maximum specific growth rate and salinity. A basis of 1% salinity level was used. A specific growth rate of 0.01 (h-1), and the estimated density of salt water of 1.027 g/cm3 were also used as a basis. However, this is a Hypothesis that would be tested in the experimental chapter of this project: Salinity is directly proportional to the density of sea water, but inversely proportional to the maximum specific growth rate, as shown below in equation (19). S 1 / m (19) S = salinity level (% g/cm3), = density of sea water. S Da / m (20) constant for the salinity inhibition of Where, D maximum specific growth rate. (16) It affects the Monod kinetic parameters Ks and μm. Fixing the values m T Specific growth rate at temperature T , www.bosaljournals/csp/ IS m 1s S Where, Effect of temperature on microbial growth rate E 1 1 m T 0 exp a R T T0 110 [email protected] ISSN: 2410-8790 0.01 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 Da 1.027 0.01 Headquarters, Diobu. The water runs around Eagle Island, to Iwofe –Siapem Company and up to the Aka Naval Base and beyond. The water is used for bathing around the Eagle Island, and fishing around the Aka (Naval base) region. The samples were gotten from the following sites: (21) Da 0.00009737 h 1 Site I: Siapem Company, which is an upstream operation company. The water serves as a passage way for vessels and local fishermen. Substituting the constant above into equation (17) we have m Da S K S S S 2 / K1 Site II: The Siapem Company water front. This water is a few kilometers from the Aka Naval base. It is used as a way for boats to go to sea. (22) Henry’s Equation Henry’s equation will be used to calculate the masses and volumes of volatile hydrocarbons, which are present in both gas and liquid phases in the bioreactor. Since the microbial growth rate depends on the liquid-phase substrate concentration only, and biomass yield depends. Description of Sampling Site/Station (Fresh Water Source) The location of sampling site is the new Calabar River, which passes through Choba Community. It flows from Aluu through to Ibefaway at Emohua Local Government area and then to Kalabari area and lined up with the Niger River. The river is used for bathing, drinking and washing. Samples were obtained from the following station / site: Site I: Aluu Community which is the upstream with little contamination from the inhabitants through activities like washing of cloths and plates and dumping of used water from domestic works, bathing and dredging activities. Site II: Spot close to the Wilbros Company just directly under the bridge. This station is polluted with a lot of diesel oil from the barges. Also some meters away are the local dredges and standard dredgers, carrying out their dredging activities. Site III: is the Choba Community, which is located a few kilometers from the Wilbros Company. Here the diesel-oil also pollutes the water and a lot of feacal pollution (human wastes) is seen here because the inhabitants defecate, bathe, wash and dump refuse on this site. Other activities like fishing and swimming are also carried out. Sample collection Sub-surface water sample was collected at three (3) different locations or stations from the New Calabar River, in sterile sample bottles. This was taken from the sub-surface water and the sample collected was not up to the brim of the sample bottles. The sample were immediately kept in an ice bag and taken to the laboratory within two (2) hours of collection. Samples were also collected for dissolved oxygen, biochemical oxygen demand, salinity pH and conductivity. Description of sampling site/ station (salt water) The salt water samples were collected from the Eagle Island waters. Eagle Island is situated behind AGIP Port Harcourt www.bosaljournals/csp/ iscientic.org. 111 Site III: This is the Aka Naval base. It is more saline than the two previous sites. The Nigerian Naval base runs her activities on these waters. Sample collection Sub-surface water sample was collected at three (3) different locations or sites from the Aka Naval Base, Saipem Company water front, and the Eagle Island waters, respectively. This was taken from sub surface water. The samples were not up to the brim of the sample bottles. The samples were immediately kept in ice bags. They were promptly taken to the laboratory within two hours of collection. The Benzene, Toluene and Phenol samples were collected in sterile containers from Oil Test Group of Companies laboratory, Trans-Amadi, Port Harcourt. Dissolved oxygen, Biochemical oxygen demand, salinity pH and conductivity tests were conducted on the water samples. Temperature Mercury in Glass thermometer was dipped into water for 3 minutes, and then the result gotten was read and recorded. The unit for temperature is Celsius/ Centigrade. Biochemical oxygen demand Water samples collected in the same way as the DO were incubated at 20oC for five days. At the end of the incubation period, the samples were treated in the same manner as the DO samples stated previously to determine the dissolved oxygen. To ensure the presence of oxygen the BOD samples were dissolved and diluted before incubation and the DO of the dilution water determined. DO at day 5 was determined as in dissolved oxygen above and the BODs calculated using the following (A__ B) X Df. Where A is initial DO of dilution water, Bin DO after 5days incubation and DF in dilution water. Conductivity Measurement for pH, salinity, and conductivity were done using Horiba water Clucker (model U-10) after calibrating the instrument with the standard Horiba solution. The units of measurement, for salinity and conductivity are in percentage (%) and μs/cm respectively. [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 Table 1: Test Results from Samples Sites (Fresh and Salt Water) Locations iscientic.org. BOD (mg/L) Salinity % EC Fresh Salt fresh Salt Fresh salt Fresh Salt Fresh Salt Fresh Salt Location 1 Location 2 Location 3 19 19 19 17 17 15 6.1 6.3 6.2 7.2 7.3 7.3 4.5 4.1 4.1 5.9 6.3 6.4 18.3 18.3 18.3 19.6 19.5 19.4 0 0 0 1 1.1 1.4 17 15 15 19 19.6 19.9 Location 1 Location 2 Location 3 Location 1 Location 2 Location 3 Total 20 20 20 19.5 19.5 20 176.2 20 20 17 15 15 16 152.5 8.2 7.9 8.9 7.7 7.5 6 63.7 8.5 7.9 8.9 8.8 8.9 8.8 73.6 6.9 7.3 8.9 6.8 6.z5 5.9 51.4 WEEK II 6.9 7.9 7.9 7.8 7.7 7.8 64.6 12.2 9.1 12.2 15.4 10.1 15.3 129.2 19.5 19.3 19.5 19.3 19.4 19.6 175.1 0 0 0 0 0 0 0 1.1 1.1 1.4 1 1 1.6 10.7 21 16 15 18 14 16 147 22.8 19.6 19.5 19.3 19 119.2 177.9 19.6 16.9 21.2 8.2 5.7 7.2 14.4 19.5 0 1.19 16.3 19.8 0.3 1.9 0.5 0.7 0.6 0.8 1.7 0.1 0 0.21 0.6 1.1 Experiment to determine total heterotrophic bacteria Media: The media used for isolation of the organisms are: nutrient agar, manifold salt agar, and petroleum hydrocarbon agar. The solution for the nutrient agar contained 28g of powdered nutrient agar and 1 litre of distilled water Manitol contained NaCl (0.55mg) and 1 litre of distilled water, 0.5g of NH4Cl, 5g of Na2HPO4, 0.5g of KHPO4, 5ml of Toluene. The media were filtered with a filter paper of hole-size 20μm. Chemicals: NaCl powdered nutrient agar (Biology Laboratory, RSUST, Port Harcourt), Toluene (Oil test Group of Companies, Port Harcourt), distilled water (Biology Laboratory, RSUST, Port Harcourt), glycerol (Biology Laboratory, RSUST, Port Harcourt). All chemicals used for media preparation were reagent grade. of the test organism. This is in order to identify the total heterotrophic bacteria that are present in the colony. Experiment to determine the degradation rate of aromatic hydrocarbon content Aim: The aim of this experiment is to determine the rate of biodegradation of aromatics hydrocarbons, using aromatic hydrocarbon-utilizing bacteria. The bacteria isolated are Pseudomonas Putida (from pseudomonas sp). Micro-organism used: Pseudomonas Putida was gotten from the isolation of total petroleum hydrocarbon utilizing bacteria from samples of fresh water. The isolation and enumeration of the micro-organisms was carried out in the Biology laboratory, Port Harcourt. Apparatus: Hot oven, Conical flask, Test tubes, Cotton wool, Aluminum tool, Thermometer, Petri dishes, Filter paper (20μm hole size), Pipette (sterile), Glass spreader (sterile), Bijor storage bottle, Microscope, Wire loop and Wooden spatula. Media: The media used was an aqueous nutrient agar. It contained 1 litre of distilled water, in order to form a solution. It also contained about 0.5g of NH4Cl, 5g of Na2HPO4, 5.0g/I of KH2PO4. The Benzene, Toluene and phenol were added. Experimental Procedure: All glass waves were sterilized in a hot air oven for 1 hour at 160oC and left to cool. Culture was extracted and re-grown in another Petri dish. Plate count method was used to count the number of colonies of bacteria formed. The diluents were then prepared using normal saline. Chemicals: Benzene (Oil Test Group of Companies, Port Harcourt, HPLC Grade), Toulene (Oil Test Group of Companies, Port Harcourt, HPLC grade), Phenol (Oil Test Group of Companies, Port Harcourt, H2S04 acid (Biology Laboratory, RSUST, Port Harcourt),(CH3CN), (Biology Laboratory, RSUST, Port Harcourt over 99.5% purity), chlorofoam and p-xylene (Baxter GC Grade, Institute of Pollution Studies, RSUST, Port Harcourt), De-ionized water (RSUST Biology laboratory). Analytical Methods: One ml of the original was transferred in triplicate into test tubes containing 9ml of normal saline. The sample was then inoculated. The sample was isolated, using Gram’s stain method to prepare the micro-organism for observation under the microscope. The following tests were conducted: Biochemical test, Oxides test, Voges-Pros Kaner test, Catalase test, Methyl Red test, Sugar Fermentation test and Coagulase test. The tests were conducted on the colony www.bosaljournals/csp/ 112 Apparatus: GC Spectrophotometer, correlated to biomass concentration, Filter paper (0.33μm), Gas chromatograph, Thermometer, 2 ml screw cap vials, 25 μ! Gas-tight syringe and10ml Test tubes [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 iscientic.org. Fig. 3: Comparison of Degradation Rate of Benzene in Both Media Method used: Cell concentration was measured as optical density of 600 mM (oD600) using GC spectrophotometer. It was correlated to bio mass concentration. 0.22 μm filtered samples were used as optical density blanks. Deionized water was used as the OD blank for toluene (1.00 OD600= 1000mg/L). Benzene, Toluene and Phenol concentrations were mentioned by gas chromatography. Aqueous samples were extracted (0.75ml of sample to 0.75ml of chlorofoam continuing 25mg/l p-xylene as an internal standard). The chlorofoam layer was removed and analyzed using an HP 5890 II gas chromatography equipped with a mass selective detector (HP 5971A). Samples were stored at 4oC in 2ml screw cap vials with Teflon-lived rubber septa until analysis. Chlorofoam was used to extract the Benzene, Toluene and Phenol standard solutions. The gas phase concentrations for the volatile hydrocarbons (Benzene and Toluene) were determined through gas chromatography. Samples were injected into the gas chromatography equipment using 25Μl gas tight syringe. Aqueous intermediated were formed in the biodegradation experiments. RESULTS AND DISCUSSIONS www.bosaljournals/csp/ 113 The results obtained from the investigation are presented in the paper, in terms of comparison of benzene, toluene and phenol degradation in both fresh and salt water media. Comparison of the degradation rates of toluene in fresh and salt water The degradation rate of Toluene was similar irrespective of the water present (fresh or salt water). Salinity only inhibited it slightly. This is depicted in figure (1) Comparison of degradation rate of phenol in fresh and salt water The degradation pattern of Phenol in both fresh and salt water followed the same trend at first. This was during the lag period. However, the patterns deviated with phenol, degrading better in fresh water than salt water. This is illustrated in Figure 2. Comparison of the degradation rates of benzene in fresh and salt water [email protected] ISSN: 2410-8790 Ukpaka / Current Science Perspectives 2(4) (2016) 105-115 The degradation rates of Benzene in both fresh and salt water were roughly the same. Slight occasional deviation was only briefly experienced. This illustrated in Figure 3. After a rather interesting study of the biodegradation of petroleum hydrocarbons, the following recommendations have been made. More work needs to be done on the role of salinity in the degradation of petroleum hydrocarbons in water. Judging from the Biodegradation of Phenol and Benzene, especially phenol, in salt water, there is no doubt that they were inhibited by their harsh saline environment. The assumptions made on the kinetic model equation for the degradation of petroleum hydrocarbons should be proven and compared to the experimental data. The success of this equation would make bioremediation much easier for Engineers and other professionals in the field of Biochemical engineering. An improved version of petroleum-utilizing bacteria should be genetically engineered. This will enable a broader range of petroleum hydrocarbons to be biodegraded. A ‘super-bacteria’ should be developed, which would be used to completely degrade Phenol at a very fast rate. In a situation where there is limited time to degrade Phenol, the microbe could become useful. CONCLUSIONS The following conclusion was drawn from the investigation such as: 1. Physicochemical properties of the fresh and salt water media influence the rate degradation that is to say that the active site of microbe will be inhibited. 2. The degradation of benzene is faster than toluene whereas toluene is faster than phenol in both fresh and salt water media. 3. The reaction pathway is the same as observed in the bioreactor set up 4. The microorganism in non diluted on the reactor was capable of degrading the substrate, thereby producing product that are environmental friendly. 5. The major inhibiting factors considered during the investigation includes, Ph, temperature and salinity for both salt and fresh water media. 6. In this investigation lag phase and other phase was also experienced in the bioreactor set up. REFERENCES Belcher, R., Nutten, A.J., Macdonald, A.M.G., 1970. 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