THE USE OF CHLORELLA SP FOR BIOREMEDIATION OF DIESEL OIL SPILLAGE

THE USE OF

CHLORELLA SP

FOR BIOREMEDIATION OF DIESEL

OIL SPILLAGE

Chioma Nwakanma*1 and Eneh Pamella I.2

1

*Department of Environmental Management and Toxicology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

2

Department of Biological Sciences, Godfrey Okoye University, Enugu State, Nigeria.

ABSTRACT

The use of Chlorella specie for bioremediation of diesel oil spillage was carried out in the study using gravimetric analysis. From the study, experimental setup C Group 4 showed more growth and degradation than the others. The Chlorella sp. degraded a maximum of 33.3% diesel oil after 30days of incubation. This was followed by 32% in experimental setup B Group 2 after 25days and 28.7% in experimental setup A Group 2. These results demonstrated that with conditions that enhanced degradation capacity, Chlorella sp. could be effective in biodegradation of diesel oil spill. It was conducted to support or reject the predictions of the literature study. It was discovered that the physical, chemical and biological composition of the oil contaminated water play an important role in influencing oil spill bioremediation.

KEYWORDS: Oil pollution, Bioremediation, Algae, Gravimetric analysis, Biodegradation.

INTRODUCTION

Bioremediation is defined as any process that uses microorganisms or their enzymes to destroy or reduce the concentrations of hazardous wastes from contaminated sites without further disruption to the local environment. It is a relatively slow process, requiring weeks to months to effect cleanup. If done properly, it can be very cost-effective. It uses naturally occurring bacteria and fungi or plants to degrade or detoxify substances hazardous to human health and the environment. This is an attractive process due to its cost effectiveness and the benefit of pollutant mineralization to CO2 and H2O (Mills et al., 2004). The key component

in bioremediation is the microorganisms, which produce the enzymes involved in the

Volume 5, Issue 9, 248-256. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. Chioma Nwakanma

Department of

Environmental Management

and Toxicology, Michael

Okpara University of

Agriculture, Umudike, Abia

State, Nigeria. Article Received on 13 July 2016,

Revised on 03 August 2016, Accepted on 23 August 2016

degradative reactions leading to the elimination or detoxification of the chemical pollutant (Rahman et al., 2002). Cyanobacterial species such as Oscillatoria salina, Plectonema terebrans, Aphanocapsa sp. and Synechococcus sp., developed as mats in aquatic environments, have been successfully used in bioremediation of oil spills in different parts of the world (Raghukumar et al., 2001; Radwan and Al-Hasan, 2001; Cohen, 2002). Most of the biological treatment technologies involve the use of bacteria, but microalgae have already been applied for effluent treatment, either as single species, as is the case of Chlorella, Scenedesmus or Arthrospira (Lee, 2001; Lima, 2004; Mulbry, 2001 and Voltolina, 2005) or as mixed cultures/consortia (Mulbry et al., 2001; Ogbonna et al., 2000 and Tarlan, 2002) to treat and remove nitrogen, phosphorus and chemical oxygen demand, from different types of effluents. Investigations conducted by several researchers demonstrated that Spirogyra sp. is capable of accumulating heavy metals like Copper, Chromium, Zinc and Fluoride (Bisnhnoi

et al., 2005). In relatively recent times in Nigeria, there has been remarkable increase in population, urbanization and industrial activities (Eze and Okpowasili, 2010). Pollution caused by petroleum and its derivatives is the most prevalent in the industrial environment. The low solubility and adsorption of high molecular weight hydrocarbons limit their availability to microorganisms. Oils are hydrophobic in nature, their availability to bacteria are limited which leads to the slow degradation because petroleum hydrocarbon compounds bind to soil components and it's difficult to be removed or degraded. Bioremediation can be done on site, is often less expensive and site disruption is minimal, it eliminates waste permanently, eliminates long-term liability and has greater public acceptance, with regulatory encouragement, and it can be coupled with other physical or chemical treatment methods. The release of crude oil into the environment by oil spill is receiving worldwide attention (Millioli et al., 2009) The aim of this research is to study the physio-chemical properties of the oil contaminated water, to collect, identify, isolate and culture specific alga (Chlorella specie) and to assess the specific algal isolate (Chlorella specie) at different concentration in the bioremediation of oil contaminated water samples and screening the most efficient concentration.

MATERIALS AND METHODS

collected will be subjected to the bioremediation of the diesel oil contaminated water. It will be isolated from the freshwater sample and identified on the basis of colony and microscopic morphological characteristics. The algal isolate will be sub-cultured in Central Food Technological Research Institute (CFTRI) medium composition (Jainab et al., 2014). Sample serial dilution was prepared by 9ml of distilled water which was aseptically pipette into five (5) test tubes labelled as thus, 101, 102, 103, 104 and 105. 1ml of the water sample was transferred into 101 test tube. 1ml of 101 sample was pipette into 102 test tube. this was repeated accordingly for the remaining 103, 104, 105 test tubes. The pour plate method was used for the inoculation of nutrient media agar. 0.1ml of 101 test tube sample was pipette into the petri-dish labelled 101. Pour the nutrient medium at 45oC into the plate and carefully swirl to mix. Same process was carried out for 103 and 105 test tube sample. Then the plates was incubated in an incubator at 37oC for 48 hours. The algal isolate on each sub-cultured plates were inoculated into various conical flasks and labelled accordingly for easy identification. The mouth of the conical flasks were covered with cotton wool held firm by the help of a masking tape to avoid it falling into the medium. The inoculums were incubated on a laboratory bench for 5 days with occasional shaking. Fifteen (15) conical flasks and three (3) experimental setup namely A, B and C were used for the empirical study. Each of the experimental setup had Group 0-4. Group 0 serving as the control. Group 0 contained, 150ml of freshwater with 30ml of diesel oil. Group 1 contained, 150ml of freshwater, 30ml of diesel oil with 10ml of Chlorella solution. Group 2 contained, 150ml of freshwater, 30ml of diesel oil with 20ml of Chlorella solution. Group 3 contained, 150ml of freshwater, 30ml of diesel oil with 30ml of Chlorella solution. Group 4 contained, 150ml of freshwater, 30ml of diesel oil with 40ml of Chlorella solution. Experimental setup A were incubated for 20days, B were incubated for 25days and C for 30days with occasional shaking. 2ml of n-hexane was added to the flasks containing the sample and shook. The content was transferred to a separating funnel with filter paper and extracted. The extract was treated with 0.4g of anhydrous sodium sulphate to remove the moisture and decanted into an empty conical flasks leaving behind the sodium sulphate. It was then evaporated in a hot air oven under low temperature of 135oC to remove every other solute except the diesel oil.

Calculations where made using gravimetric analysis as thus:

% degradation = Amount of oil degraded x 100 Amount of oil added in the media

RESULT

Table 1: Physio-chemical Analysis of Fresh Water Sample and Oil Contaminated

Sample

Parameters (ppm) Freshwater Sample Oil Contaminated Sample

Nitrate 30 0.0

Nitrite 0.0 3

Ammonium 0.0 1

Total Hardness 250 150 Total Alkalinity 125 100

pH 9.0 8.0

Colour Transparent Brown

From the study, Nitrate in freshwater was 30ppm while in oil contaminated water it was 0.0ppm. In freshwater, nitrite was 0.0ppm and 3ppm in oil contaminated water. Ammonium was 0.0ppm in freshwater and 1ppm in oil contaminated water. Total hardness was observed to be 250ppm for freshwater and 150ppm for oil contaminated water. Total alkalinity of 125ppm and 100ppm for freshwater and oil contaminated water respectively was observed. The pH range was 9.0 for freshwater and 8.0 for oil contaminated water. The colour changed from being transparent in freshwater to brown in oil contaminated water. This results showed that when oil spill occurs in freshwater there tend to be a change in the physio-chemical properties of the water leading to the loss of flora and fauna in freshwater. Hence, there is a change in the ecosystem of the aquatic environment.

Table 2: Viable Counts of the Microbes

Water Sample

101 (Number of Colonies)

103(Number of Colonies)

105(Number of Colonies)

Fresh Water 4 Flooded Growth 16

The viable counts for the microbes was obtained by counting the numbers of colonies on the petri dish for each dilution factor. Four (4) colonies was observed in petri dish of dilution factor 101 and sixteen (16) colonies in petri dish of dilution factor 105. There was flooded growth in petri dish with dilution factor 103.

served as the control. During the treatment in Group 1, 10ml of Chlorella solution and 30ml of diesel oil were added to 150ml of fresh water, in Group 2, 20ml of Chlorella solution and 30ml of diesel oil were added to 150ml of freshwater, in Group 3, 30ml of Chlorella solution and 30ml of diesel oil were added to 150ml of freshwater, in Group 4, 40ml of Chlorella

solution and 30ml of diesel oil were added to 150ml of freshwater, in Group 0,0ml of

Chlorella solution and 30ml of diesel oil were added to 150ml of freshwater.

Table 3: Gravimetric Analysis of Experimental Setup A Group 0-4

Experimental Setup A Group 0 A Group 1 A Group 2 A Group 3 A Group 4

Amount of Diesel Oil Added (ml) 30ml 30ml 30ml 30ml 30ml

Chlorella sp. Solution Added (ml) 0ml 10ml 20ml 30ml 40ml

Weight of Residual Diesel Oil (ml) 24.4ml 24.2ml 21.4ml 22.3ml 26.7ml Amount of Diesel Oil Degraded (ml) 5.6ml 5.8ml 8.6ml 7.7ml 3.3ml Percentage Degradation 18.7% 19.3% 28.7% 25.7% 11%

Gravimetric analysis conducted on Experimental Setup A for Group 0 -4 indicated a decrease in percentage degradation from 28.7% (Group 2) to 11% (Group 4). While Group O had a percentage degradation of 18.7%, Group 1 increased in percentage degradation of 19.3%.

Table 4: Gravimetric Analysis of Experimental Setup B Group 0-4

Experimental Setup B Group 0 B Group 1 B Group 2 B Group 3 B Group 4

Amount of Diesel Oil Added (ml) 30ml 30ml 30ml 30ml 30ml

Chlorella sp. Solution Added (ml) 0ml 10ml 20ml 30ml 40ml

Weight of Residual Diesel Oil (ml) 26.2ml 28.7ml 20.4ml 23.4ml 21ml Amount of Diesel Oil Degraded (ml) 3.8ml 1.3ml 9.6ml 6.6ml 9ml Percentage Degradation 12.7% 4.3% 32% 22% 30%

For Experimental Setup B, Group 2 and 4 had the maximum percentage degradation of 22% and 30%. While the lowest percentage degradation was recorded with Group 1 having 4.3%.

Table 5: Gravimetric Analysis of Experimental Setup C Group 0-4

Experimental Setup C Group 0 C Group 1 C Group 2 C Group 3 C Group 4

Amount of Diesel Oil Added (ml) 30ml 30ml 30ml 30ml 30ml

Chlorella sp. Solution Added (ml) 0ml 10ml 20ml 30ml 40ml

Weight of Residual Diesel oil (ml) 28.8ml 24.3ml 24ml 24.1ml 20ml Amount of Diesel Oil Degraded (ml) 1.2ml 5.7ml 6ml 5.9ml 10ml Percentage Degradation 4% 19% 20% 19.7% 33.3%

However, the experimental setup C Group 4 had the highest percentage degradation of 33.3% after 30days of incubation. This was followed by B Group 2 with percentage degradation of 32% after 25days and 28.7% degradation by A Group 2 after 20days of incubation. The lowest percentage degradation was observed in C Group O which had 4% degradation, followed by B Group 1 with 4.3% and A Group 4 with 11% degradation.

DISCUSSION

The physio-chemical data collected from this study as presented in table 1, indicated that there were changes in the physio-chemical properties of the freshwater samples to that of the oil contaminated water sample. It showed that nitrate in freshwater was 30ppm while in oil contaminated water it was 0.0ppm. In freshwater, nitrite was 0.0ppm and 3ppm in oil contaminated water. Ammonium was 0.0ppm in freshwater and 1ppm in oil contaminated water. Total hardness was observed to be 250ppm for freshwater and 150ppm for oil contaminated water. Total alkalinity of 125ppm and 100ppm for freshwater and oil contaminated water respectively was observed. The pH range was 9.0 for freshwater and 8.0 for oil contaminated water. The colour changed from being transparent in freshwater to brown in oil contaminated water. This is in line with reports by scientist that the aquatic ecosystem is depended on the physio-chemical and biological characteristic (Venkatesharaju

findings of (Adenipekun et al., 2005, Margesin et al., 2001 and Singh et al., 2006). In Table 3, 4 and 5, Gravimetric analysis of the experimental setup showed that the experimental setup C Group 4 had the highest percentage degradation of 33.3% after 30 days of incubation. This was followed by B Group 2 with percentage degradation of 32% for 25days and 28.7% degradation by A Group 2 after 20 days. The increase in percentage degradation after 30 days was as a result of the Chlorella sp. being able to recover from the naphthalene damage and effect its optimal degradation potential on the diesel oil contaminated water. Naphthalene which is a component of diesel oil is known to affect the cell components of microorganisms. Chandra et al. (2011), observed that Chlorella sp. can potentially recover from naphthalene damage within 7 days of incubation with nitrogen and phosphorous present in the medium. The nitrogen and phosphorous present in the CFTRI medium was utilized by the Chlorella sp. as its nutrient source during the remediation process. This shows that nutrient is vital to

Chlorella sp. in the bioremediation of diesel oil. According to Edward et al., 1993, nutrients are likely the limiting factors in biodegradation of oil. Munoz et al., (2003), found out that

Chlorella sp. has the quality of releasing biosurfactants that further enhances bioremediation. Biosurfactants are surface-active biomolecules produced by microorganisms with wide range of applications (Vijayakumar et al., 2015). Biosurfactants are suitable for environmental applications such as bioremediation (Mulligan et al., 2001). This quality was also observed in this study as the Chlorella sp. survived in the diesel oil contaminated water without any external supply of oxygen, under photosynthetic conditions. Chlorella sp. is known to degrade oil pollution but its degradation potentials are not as high as that of Scenedesmus sp.

which is another photosynthetic micralgae (Petkov et al., 1992). The necessary enzymes needed for complete biodgradation cannot be found in a single organism. This was demonstrated in the experimental setup as none of them were able to degrade 100% of the diesel oil present in the freshwater. This result agrees with Friello et al., (2001), who reported that a wide variety of metabolic and physiological factors are required for the degradation of different compounds in diesel oil.

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