Bioremediation of Petroleum Refinery Wastewater Effluent via Augmented Native Microbes

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1 Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1): 1-6 Scholarlink Research Institute Journals, 2015 (ISSN: ) jeteas.scholarlinkresearch.com Journal of Emerging Trends Engineering and Applied Sciences (JETEAS) 6(1):1-6 (ISSN: ) Bioremediation of Petroleum Refinery Wastewater Effluent via Augmented Native Microbes 1 N. M. Musa, S. Abdulsalam, A.D.I. Suleiman and 2 Sale, Abdullahi 1 Department of Chemical Engineering, Abubakar Tafawa Balewa University (ATBU), PMB 0248, Bauchi, Bauchi State, Nigeria. 2 Microbiology Laboratory, Department of Biological Science, Abubakar Tafawa Balewa University (ATBU), PMB 0248, Bauchi, Bauchi State, Nigeria. Corresponding Author: N. M. Musa Abstract In Nigeria, like other developing countries, industries discharge their wastewater without effective treatment due to the high cost of existing treatment technologies. Wastewater containing petroleum hydrocarbon is highly toxic and pose a great danger to the nearby communities. Therefore, there is a need for a more robust and cost effective treatment technology. In this research, a study was carried out on bioremediation of petroleum refinery wastewater via augmented native microbes. Four dominant microbial strains isolated from the petroleum refinery wastewater were identified to be Bacillus subtilis (S1), Micrococcus luteus (S2), Staphylococcus aureus (S3) and Staphylococcus epidermidis (S4) base on their gram reaction, morphology, microscopic appearance, and biochemical characteristics. The four strains showed their capability to degrade hydrocarbon as indicated by their growth in Bushnell-Haas medium. The four native microbes were used to boost the performance of the indigenous microbes (note that only two out of these four strains were used to boost bioremediation). Five different treatments (T1-T5) were investigated for 63 days. T1 (Natural attenuation) showed O&G degradation of %. T5 (Aeration + Bioaugmentation with S1 & S2) showed O&G degradation of 66.7 %. From the result of this study, it has been established that bioaugmentation with native microbes using treatment option five can be used to developed a robust, cost effective and environmentally friendly process for the treatment of hydrocarbon contaminated wastewater. Keywords: bioremediation, bioaugmentation, effluent, hydrocarbons, native-microbes, wastewater. INTRODUCTION Wastewater released from petroleum refineries is characterized by the presence of large quantity of petroleum products, polycyclic and aromatic hydrocarbons, phenols, metal derivatives, surface active substances, sulfides, naphthylenic acids and other chemicals. Due to ineffective purification systems, the pollutants finds its way into nearby water bodies and soil with potentially serious consequences on the ecosystem (Otunkunefor and Obiukwu, 2005; Bay et al., 2003; Beg et al., 2001). According to World health organization (WHO), the mortality rate of water associated diseases exceeds five million people annually with microbial intestinal infections accounting for more than 50 % (Cabral, 2010). Wastewater may be treated by physiochemical or biological methods, biological treatment is preferred over physicochemical as the former is cost effective, efficient and environmentally friendly (Suleimanov, 1995, Hamza, 2012). Bioremediation strategies involved the use of microbes to transform the harmful pollutants into harmless products. Bacteria and fungi are often used in the biochemical decomposition of wastewaters to stable end products. More microorganisms, or sludge are formed and the portion of the waste is converted to carbon dioxide, water and other end products. This is achieved by enhancing the conditions (ph, nutrients and aeration) of the indigenous microbes to carry out the bioremediation process (biostimulation). Alternative to this is bioaugmentation, where exogenous microbes are introduced to carry out the process (Hamza et al., 2012; Long, 2007). In this study, bioaugmentation using native microbes was used to treat petroleum refinery wastewater using various treatment options. The aim of this study is to develop a robust cost effective process for the treatment of refinery wastewater using bioremediation technique via augmented native microbes. The research objectives are: i. To develop a bench scale bioreactor for the treatment of refinery wastewater. 1

2 ii. To determine the hydrocarbon degrading potential of refinery wastewater in the bench scale bioreactor developed. iii. To determine the effect of boosting the microbial population by adding enriched native microbes (bioaugmentation) on the rate of hydrocarbon degradation. STATEMENT OF THE PROBLEM In Nigeria, like in other developing countries, the conventional cleaning technologies (physicochemical) are being used for the remediation of refinery wastewater, these methods are expensive and does not lead to complete degradation of pollutants, therefore, the need for a robust, cost effective and environmentally friendly cleaning technology (Hamza et al., 2009; Ojo, 2006). LIMITATION OF THE STUDIES This study is limited to the development of an effective bench scale bioremediation technology for the treatment of refinery wastewater, characterization of the physicochemical and microbiological properties of the refinery wastewater, isolation and identification of native microbes in the wastewater and bioremediation of refinery wastewater using augmented native microbes in the bench scale bioreactor developed. MATERIALS AND METHODS Sample Collection The wastewater sample for this research was collected using sterile containers at the outlet of the bio-filter of a typical petroleum refining company in Nigeria. The wastewater sample was then preserved at low temperature in a deep freezer before commencement of the experiment. Wastewater Characterization The wastewater was analyzed for physicochemical, organic and heavy metal constituents. Turbidity was determined with DR\2000 spectrophotometer using absorphotometric method, COD was determined using calorimetric method with DR\2000 spectrophotometer (Long, 2007). BOD was determined using HACK BOD track instrument (Anonymous, 2007). O&G was determined using reflux method with DR\2000 spectrophotometer. Conductivity was determined using a Wagtech WEDIST6 conductivity/ TDS meter. Total solids and suspended solids were determined following the procedures given in (Anonymous, 1997). Isolation and Identification of Bacteria Following the aseptic technique, the standard dilution methods for recovery of bacteria from the wastewater was used. The nutrient agar plates were inoculated with 0.1 ml of diluted sample and then incubated at 37 o C. Growth was observed after 48 hours and the samples were left for up to 96 hours (Four days) after which distinct colonies were formed. Single colonies were removed from these plates and then subcultured into fresh nutrient agar plates. They were then incubated at 37 o C for 48 hours. The pure isolated organisms were identified by microscopic appearance, morphological features, gram reaction, and biochemical characteristics. The isolates were identified according to description given in Bergeys manual of systematic bacteriology to be Bacillus subtilis (S1), Micrococcus luteus (S2), Staphylococcus aureus (S3) and Staphylococcus epidermidis (S4). Determination of Hydrocarbon Degrading Potentials The isolates were inoculated in Bushnell-haas medium and incubated at 37 o C, microbial growth was observed in all the four isolates after three days indicating their ability to degrade hydrocarbon compounds (Hamza et al., 2012). Preparation of Enriched Native Microbial Cultures for Bioremediation Culture media was prepared using manufacturers standard methods and procedures. For example in a typical run, 1000 cm 3 of distilled water was measured and 28 g of agar was added to it in a conical flask after weighing. The mixture was shaken until the agar was totally dissolved in the distilled water. The solution was heated on a hot plate then sterilized in an autoclave at 121 o C for 15 minutes; the sterile media was then placed in a water bath to cool the media to 47 o C before pouring into plates. Same procedure was done for peptone water but with different amount of agar (i.e. 15 g in 1000 cm 3 ) and for macconkey agar (38.5 g in 1000 cm 3 ). Pure isolates S1 & S2 were then enriched by inoculating them in all the three sterile media before incubating it for at 37 o C. Procedure for Serial Dilution and Pour Plate Counting Method Pour plate method was employed as the technique for recovery of microorganisms from wastewater on solid media. After autoclaving, the agar was cooled to 45 o C before pouring to plates. 0.1 ml of 10-4, 10-6 and 10-8 dilutions were inoculated using pour plate method. The plates were incubated at room temperature for 24 h before counting all the colonies using colony counters. The procedure was repeated for the same dilution in bushnell-hass media and for pure isolates S1 & S2 in peptone water. Bioremediation Studies Five plastic buckets (20 L) each were used for this experiment. Four of these plastic buckets, each containing 13.3 L of wastewater were aerated while the fifth bucket containing the same quantity of wastewater (13.3 L) was left un-aerated (figure 1); 2

3 Treatment 1 (T1), the first plastic bucket (un-aerated) was left as collected to monitor natural degradation (control). Treatment 2 (T2), the second bucket contains the same sample as the first but was aerated at 1.2 L air /min. Treatment 3 & 4 (T3 & T4), 50 ml each of enriched pure isolate S1 & S2 were added to plastic buckets 3 & 4 respectively and the reactors were aerated at 1.2 L air /min. Treatment 5 (T5), combination of 25 ml each of enriched pure isolates S1 & S2 were added to plastic bucket 5. Effect of aeration, potentials of adding pure isolates S1 & S2 separately and the use of consortium of bacteria were compared with natural attenuation. RESULTS AND DISCUSSION Wastewater Characterization The results of physicochemical characterization (Table 1), showed that the refinery discharge its wastewater without treating as required by the international standards for discharge of environmental pollution. Therefore the need for a more robust treatment process. Substrate Reduction COD and BOD tests are commonly used to determine the amount of organic pollutants found in water, making COD and BOD a useful measure of water quality (Okerentugba and Ezeronye, 2003, Hamza et al., 2009). Biodegradation potentials of five different treatments (T1 T5) were measured in a bench scale bioreactor by monitoring O & G contents. Fig 2 4 show results of bioremediation of a typical Petroleum refinery wastewater. This result (fig. 2-4) showed Treatment 1 (natural attenuation), which is a very slow process for biodegradation can be boosted by augmentation with strain of bacteria or consortium of bacteria. In T1 (Treatment 1), only %, COD reduction of % and BOD reduction of % compared to T5 (Treatment 5) boosted with a consortium of bacillus subtilis and micrococcus luteus which showed O&G decrease of 67 %, COD decrease of % and BOD reduction of %. Micrococcus and Bacillus species were reported to be hydrocarbon degraders (Long, 2007; Anonymous, 2007, 1997). T2 (Treatment 2) showed that injecting air can boost biodegradation process compared to natural attenuation. T2 showed % O&G decrease, COD decrease of % and BOD decrease of %. Table 2 & 3 showed the summary of results obtained after 63 days and parameters used for the bioremediation studies. Biomass Growth and Conductivity Increase In figure 5, the biomass growth was measured by turbidity/optical density increase (Leah and Colwell, 1990). In figure 6, the rate of conductivity increased with decrease in O&G (Adekunle, 2011; Adekunle et al., 2012). CONCLUSION The following conclusions were drawn from the results obtained: i. Bioremediation of petroleum refinery wastewater was effective to an appreciable extent via augmented native microbes (67 % O&G removal in 63 days) in the bench scale bioreactor rig developed. ii. A full scale treatment technology can be sized using treatment option 5 (T5). iii. Four microorganisms namely bacillus subtilis, micrococcus luteus, staphylococcus aureus and staphylococcus epidermidis were identified in the petroleum refinery wastewater and can be enriched to boost the rate of biodegradation. (remember only two were used for boosting) REFERENCE Adekunle A. A., Adekunle, I. M. and Igba U. T Assessing and Forecasting the Impact of Bioremediation Product Derived From Nigeria Local Raw Materials on Electrical Conductivity of Soils Contaminated with Petroleum Products. Journal of applied hytotechnology 2 (1): Adekunle, I.M Bioremediation of soils contaminated with Nigerian petroleum products using composted municipal wastes. Bioremediation Journal. 15(4): Bay S., Jones B.H., Schiff K. and Washburg L Water quality impacts of storm-water discharges to santa monica Bay. Marine environmental research 56: Beg M. U., Al-Muzaini S., Saeed T., Job P.G., Beg K.R., Al-Bahloul M., Al-Matrouk K., Al-Obaid T. and Kurian A Chemical contamination and toxicity of sediment from a costal area receiving industrial effluents in kuwait. Archives of environmental contamination and toxicology 41: Cabral J.P., Water Microbiology. Bacteria Pathogens and water. Int. J. Environ Res. Pub. Health, 7: Hach Company, DR/2000 Procedure manual. 8 th edition, U.S.A Hamza, U. D., I.A Mohammed, I. M., Ibrahim, S Kinetics of Biological Reduction of Chemical Oxygen Demand from Petroleum Refinery Wastewater. Researcher, 1(2). Hamza, U.D., Mohammed, I. M., Sale, A Potentials of bacterial Isolates in bioremediation of petroleum refinery wastewater. Journal of applied hytotechnology 1(3):

4 Long, H. (2007); Wastewater treatment methods and disposal, 3441, Empire road U.S.A., htm (accessed 12th December, 2013) Noweco Laboratory, Norwalk Wastewater Equipment Company, Inc. 220 republic street Norwalk, Ohio U.S.A (accessed January 2014) Leahy J.G., Colwell, R Microbial degradation of hydrocarbons in the environment. American Society of Microbiology. Microbiological Reviews, 54(3): Ojo, O.A., Petroleum Hydrocarbon Utilization by Native Bacterial Population from a Wastewater Canal Southwest Nigeria. African Journal of biotechnology, 5(4): Okerentugba P.O. and Ezeronye, O. U Petroleum Degrading Potentials of Single and Mixed Microbial Cultures Isolated from Rivers and Refinery Effluent in Nigeria. African Journal of Biotechnology, 2 (9): Otunkunefor T. V., Obiukwu, C Impact of refinery effluent on the physicochemical properties of a water body in Niger Delta. Applied ecology and environmental research 3: Table 2: Summary of results O & G (mg/l) COD (mg/l) BOD (mg/l) Turbidity (NTU) Initial Value Treatment Treatment Treatment Treatment Treatment Standard Values Table 3: Summary of Results and Isolates Identified S/N Parameter Value obtained 1 Volume of bioreactor 20 L 2 Working volume or Effective volume 2/3 of Total volume (13.3 L) 3 Aeration flow rate 1.2 L/h (0.02L/min) 4 Geometry of bioreactor Cylindrical 5 Continuous monitoring 63 days 6 Isolates (indigenous microbes) Bacillus subtilis Micrococcus luteus Staphylococcus aureus Staphylococcus epidermidis 7 The best treatment option T5 (67 % O&G removal) Suleimanov, A.Y Conditions of waste fluid accumulation at petrochemical and processing enterprise prevention of their harm to water bodies, Meditsina Truda Promyswe Nnaia Ekologila. 12:31-36 APPENDIX Table 1: Physicochemical Characteristics of KRPC Wastewater before Treatment Parameter Value ph 6.55 Conductivity (µs.cm -1 ) 450 Turbidity (NTU) 47 TDS (mgl -1 ) 224 TSS (mgl -1 ) 47 Organics BOD (mgl -1 ) 155 COD (mgl -1 ) 285 O&G (mgl -1 ) 21 Inorganic Phosphate 7.36 Total-N (mgl -1 ) Total-C (mgl -1 ) Heavy Metals Fe (mgl -1 ) 0.93 Cu (mgl -1 ) 0.5 Zn (mgl -1 ) 0.08 Cr (mgl -1 )

5 Figure 1: Schematic representation of a bench-scale bioreactor rig. Figure 2: Rate of COD Reduction Figure 3: Oil and Grease Removal 5

6 Figure 4: Rate of BOD Reduction Fig. 5: Rate of Turbidity increase Fig. 6: Rate of Conductivity increase 6