Palm Oil Mill Effluent Biodegradation Potentials among Local Isolated Microorganisms

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1 Human Journals Research Article November 017 Vol.:8, Issue:1 All rights are reserved by D.R. Tiku et al. Palm Oil Mill Effluent Biodegradation Potentials among Local Isolated Microorganisms Keywords: Palm Oil, Biodegradation, Microorganisms ABSTRACT E.O. Eno, S.P. Antai, *D.R. Tiku Department of Microbiology, University of Calabar, Calabar Submission: 19 October 017 Accepted: 9 October 017 Published: 0 November The research study was aimed at investigating palm oil mill biodegradation potentials among locally isolated microorganisms. Palm Oil Mill Effluent (POME) polluted soil samples (0-1cm and 1-0cm depth) were collected into sterile polythene bags from Uruk Etta Ikot in Etim Ekpo L.G.A and Eyop industries at Akamkpa L.G.A, while POME wastewater was collected from Mfamosing at Akamkpa L.G.A, into glass containers with Teflon lined cap. The samples were placed in an ice-cool chest and transported to the laboratory for analysis. The study was completed within a duration of six months. Standard microbiological techniques were used to isolate, characterize and identify isolates from the collected samples. The biodegradation potentials of the isolates were adjudged based on their ability to reduce Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Oil and grease and Total Suspended Solids (TSS) of the palm oil mill effluents. The results obtained from the study showed that the BOD of the raw POME samples were reduced from ±.mg/l to 7000±.7mg/l (Bacillus spp), 6100±.77mg/l (Micrococcus spp),,100mg/l (Pseudomonas spp),,00±.08mg/l (Aspergillus spp),,00±.mg/l (Fusarium spp) and,600±1.81mg/l (Mucor spp). However the COD, Oil and grease, and TSS reduction potentials of the isolates followed similar pattern, with Pseudomonas spp (9,000±1.87mg/l), showing the highest COD reduction potential and Mucor spp (1,800±0.7mg/l, and 11,11±1.1mg/L) showing the highest oil and grease and TSS reduction potential respectively after a duration of days. However, from this study, it is a clear indicator that Pseudomonas spp (with.% BOD and 7.7% COD reduction efficiency), Aeromonas spp, (7.% oil and grease reduction potential). Fusarium spp and Mucor spp (with 6% oil and grease reduction potential) and Mucor spp (.0% TSS reduction potential) could be useful in enhancing the bioremediation and treatment of POME polluted environments.

2 INTRODUCTION Palm oil industry has become one of the main agro-industries in Nigeria. Palm oil mills release POME in colossal amounts with its attendant polluting impending (Mohammadreza et al., 01). POME has unfavorable environmental ramifications effects including land and aquatic ecosystem contamination, loss of biodiversity and increase in COD and BOD in environment (Wu et al., 010). Today, the penetration of palm oil has been considered due to the entry of its effluents into the waterways and ecosystems, making it a meticulous concern towards the food chain interference and water consumption (Foo and Hameed et al., 010). This can cause considerable environmental problems such as land pollution and effectively suffocating aquatic life if discharged without effective treatment (Cheng et al., 010). In the process of palm oil milling, POME is mainly generated from sterilization and clarification of palm oil, in which a large amount of steam and hot water are used (Ohimain et al., 010). POME is a thick brownish liquid that is compiled with high concentrations of total solids, oil and grease, chemical oxygen demand (COD) and biological oxygen demand (BOD) (Neoh et al., 01). The biological treatment of POME depends enormously on consortium of microbial activities, which operate on the organic substrates present in the POME as supplements and eventually degrade these organic matters into simple by product such as methane, carbon dioxide hydrogen and water (Mohammed et al., 01). A variety of microorganisms have been investigated to be capable of biodegrading oil wastewater with high profits. Although, the anaerobic and aerobic treatment are one of the most sort biological methods for POME treatment. However the suspended and colloidal components are neither effectively decomposed biologically nor by other conventional means because they float the surface of wastewater and this the major problem to cause failure of treatment system (Ahmad et al., 011). However, with due consideration of the aforementioned, this study intends to investigate POME biodegradation potentials among locally isolated microorganisms. MATERIALS AND METHODS POME polluted soil samples: POME polluted soil samples for this study was obtained from Uruk Etta Ikot in Etim Ekpo L.G.A and Eyop industries at Akamkpa L.G.A. The site was chosen with the intent of using soil samples that probably had in the past been exposed to POME.

3 Collection: Surface soil (0 to 1cm depth) and subsurface soil (1cm-0cm depth) were collected using a Dutch auger. Collection was made from to random point, then bulked to form a composite sample and deposited in sterile labeled polyethylene bag and transported to the laboratory for further analysis. POME wastewater POME wastewater was collected into glass containers with Teflon lined cap from Eyop industries and Mfamosing, both located at Akamkpa L.G.A. The samples were placed in an ice-cool chest and transported to laboratory for analysis. Media: The media used in the study were; nutrient agar (Oxoid Ltd.), Sabouraud Dextrose Agar (Oxoid Ltd.) and Tributyrin agar (Oxoid Ltd.). The media were prepared in accordance to the manufacture instructions. METHODS Isolation of isolates The method used was that of (Antai et al., 016). In this method, surface- spreading technique was used. Ten fold serial dilution of the POME polluted soil samples and wastewater samples were prepared. 0.1ml of 10-6 dilution was plated into nutrient agar and sabouraud dextrose agar plates. The nutrient agar plates were supplemented with 0µgml -1 of nystatin so as to inhibit fungal growth whole the sabouraud dextrose agar plates were supplemented with100µml -1 of streptomycin, so as to inhibit bacterial growth. The plates were prepared in duplicates and incubated at 7ºC for hours (nutrient agar plate) and 8ºC for 7 hours (sabouraud dextrose agar plate). Purification and maintenance of microbial isolates The bacterial and fungal isolates obtained from the nutrient agar and sabouraud dextrose agar plates were purified by repeated sub-culturing. The isolates were subjected to series of transfer into newly prepared medium. The bacteria isolates were transferred into newly prepared nutrient agar medium and incubated at 7ºC for hours. The fungal isolates,

4 however, were transferred into newly prepared sabouraud dextrose agar and incubated at 8ºC for days. Pure colonies of the bacteria and fungi were maintained on slopes of nutrient agar and sabouraud dextrose agar slant and stored in a refrigerator at 8ºC. Characterization and identification of the isolates Standard inocula were prepared from the preserved stock culture by taking a loopful of the bacterial and fungal isolates and aseptically inoculating onto sterile nutrient agar and sabouraud dextrose agar respectively. The plates were incubated at 8ºC for hours. The characterization of the isolates was then performed by employing gram staining reaction, biochemical characterization and microscopic examination (Holt et al., 199; Cheebrough 000; Hunter and Bennet, 199). Biodegradation test 0ml of raw palm oil mill effluent was introduced into conical flash and sterilized at 11ºC for 0 minutes. The sterilized raw palm oil mill effluent was allowed to cool before inoculation. 0mls of broth sample containing the inoculums was used to inoculate 0mls of the palm oil mil effluent samples and was shaken and incubated at room temperature for hours (bacteria) and - days (fungi). The samples were aseptically drawn for days and analyzed for BOD, COD, oil and grease and TSS, with the control flasks not inoculated. The percentage reduction was measured by using the equation as described by (Jeremiah et al., 01). Reduction (%) = Where; Craw POME is the concentration of COD, BOD, TSS, OIL and grease of raw POME and cf is the concentration of these parameters after treatment. RESULTS Palm oil mill effluent biodegradation potential by isolates from collection sample. Table 1 presents result of POME biodegradation potential of selected isolates from collected samples. It showed that Bacillus spp (PCE 1 ) was able to reduce the BOD of the raw POME 6

5 from.87mg/l to 7,000±.87mg/l after a duration of days reduced while BOD value of 6,100±.77mg/l,,100±1.7mg/l, 9,700±.18mg/l,,600±1.81mg/l were obtained from Micrococcus spp (PSC 1 ), Pseudomonas spp (PSC ), Aeromonas spp (PSC 6 ), Geotricum spp (PSU 18 ), Aspergillus spp (PSC ), Fusarium spp (PSC 18 ) and Mucor spp (PUE 6 ) respectively (fig 1). The COD of the POME was reduced from ±.89 to 0,01±.1mg/l (Bacillus spp), 6,1±.1mg/l (Micrococcus spp), 9,000±1.87mg/l (Pseudomonas spp), 0,100±.1mg/l (Aeromonas spp),,00±.6mg/l (Geotricum spp),00 ±.98 (Aspergillus spp),,100±.89mg/l (Fusarium spp) and 8,00±.0mg/l (Mucor spp) (fig ). However, the oil and grease and TSS of the palm oil mill effluent substrate followed similar pattern (fig and ). Table 1: Palm oil mill effluent biodegradation potential of isolated isolates from collected samples Isolate code PCE 1 1 Numbe r of days BOD(mg/L) COD(mg/L) Oil and Grease(mg/L) 0,00 8,700 1,00 7,00 7,70 8,000 6,700 0,01,60,100,000,800 TSS(mg/L) 1,00 1,800 1,00 1,900 Probable organism Bacillus spp PSC 1 1 8,10 6,00 9,01 6,100 0,00 1,10 8,000 6,1,900,700,0,00 1,100 1,900 1,0 1,000 Micrococcus spp PSC 1,00 8,800,0 1,000,190,000 0,100 9,000,00,180,190 1,900 1,101 1,068 1,780 1,660 Pseudomonas spp PSC 6 1 8,800,00 0,00 9,700,00 9,0 7,60 0,100,00,100,080,18 1,098 1,0 1,90 1,00 Aeromonas spp 7

6 PSU 18 1,00 8,700 6,100,00,100,00,600 8,100,00,000,800,0,00 1,11 1,011 1,81 1,11 Geotricum spp PCE 1,00,100 1,00 8,00,00,00 9,00 9,100,00,600,00,00,100 1,11 1,001 1,11 1,010 Aspergillus spp PSC ,700 6,800 7,600,00 9,100 7,600,00,100,600,00,0,800 1,1 1,11 1,611 1,0 Fusarium spp PUE 6 1 6,00,00 8,00,600,00,000 1,600 8,00,000, ,800 1,781 11,81 11,1 11,11 Mucor spp Keys; BOD= Biological oxygen demand, COD= Chemical oxygen demand, TSS= Total suspended solids. Days Fig 1: BOD reduction by isolates from samples 8

7 Key: PCE 1 : Bacillus spp PSC 1 : Micrococcus spp PSC : Pseudomonas spp PSC 6 : Aeromonas spp PSU 18 : Geotricum spp PCE : Aspergillus spp PSC 18 : Fusarium spp PUE 6 : Mucor spp Fig : COD reduction by isolates from samples Key: PCE 1 : Bacillus spp PSC 1 : Micrococcus spp PSC : Pseudomonas spp PSC 6 : Aeromonas spp PSU 18 : Geotricum spp PCE : Aspergillus spp PSC 18 : Fusarium spp PUE 6 : Mucor spp Fig : Oil and grease reduction by isolates from samples 9

8 Key: PCE 1 : Bacillus spp PSC 1 : Micrococcus spp PSC : Pseudomonas spp PSC 6 : Aeromonas spp PSU 18 : Geotricum spp PCE : Aspergillus spp PSC 18 : Fusarium spp PUE 6 : Mucor spp Fig : TSS reduction by isolates from samples Key: PCE 1 : Bacillus spp PSC 1 : Micrococcus spp PSC : Pseudomonas spp PSC 6 : Aeromonas spp PSU 18 : Geotricum spp PCE : Aspergillus spp PSC 18 : Fusarium spp PUE 6 : Mucor spp Palm oil mill effluent percentage reduction potential of selected isolates from samples Table presents the result of palm oil mill effluent percentage reduction potentials of selected isolates from samples. It showed that Pseudomonas spp (PSC ) had the highest BOP percentage reduction (.%) (fig ), and COD percentage reduction (7.7%) (fig 6). Mucor spp (PUE 6 ) and Fusarium spp (PSC 18 ) had the highest oil and grease percentage reduction (6%) (fig 7) while Mucor spp (PUE 6 ) had the highest TSS percentage reduction (.0%) compared to other isolates (fig 8) 60

9 Table : Palm oil mil effluent percentage reduction potential of selected isolates from samples Isolate code BOD (%) COD (%) Oil and Grease (%) TSS (%) Probable organism PCE Bacillus spp PSC Micrococcus spp PSC *** Pseudomonas spp PSC 6 *** Aeromonas spp PSU Geotricum spp PCE Aspergillus spp PSC 18 *** Fusarium spp PUE 6 *** Mucor sspp Keys: ***= Potential microorganisms for biodegradation of palm oil mill effluents, TSS= Total suspended solid, BOD= Biological oxygen demand. Fig : BOD percentage reduction potential of selected isolates from samples Fig 6: COD percentage reduction potential of selected isolates from samples 61

10 Oil and grease (%) Fig 7: Oil and grease percentage reduction potential of selected isolates from samples Fig 8: TSS percentage reduction potential of selected isolates from samples DISCUSSION Palm mill effluent microorganisms have been known to survive in only wastewater or soils by producing the enzyme lipase and or spores (as the case may be with fungi) which enables them in surviving the anaerobic nature of POME (Ohimain et al., 01). Orji et al., (006) stated that oily substrate provides a good environment for lipolytic microorganisms to flourish. The biodegradation of oil in the environment is a complex process, whose 6

11 quantitative and qualitative aspects depend on nature and amount of the oil present, the ambient and the seasonal environmental condition, and the constitution of the indigenous microbial community (leachy and Colwell, 1990; Hinchee and Olfenbuttel, et al., 1991). This is in compliance with this study, whereby constitution of in POME (Izah and Ohimain. 01). (Orji et al., 006) stated that oily substrate provides a good environment for lipolytic microorganisms to flourish. This is in compliance with this study, whereby constitution of indigenous bacteria and fungi isolated from POME and its polluted soil samples were used for degradation of palm oil mill effluent samples. The ability of the bacterial and fungi isolates to biodegrade palm oil mill effluent was demonstrated in terms of reduction in BOD (mg/l), COD (mg/l), TSS (mg/l), Oil and Grease(mg/L) and percentage degradation determined from the equation earlier mentioned. Amongst, the bacteria isolates screened for palm oil mill effluent biodegradability for days, Pseudomonas spp (PSC ) showed the highest BOD (.%) and COD (7.7%) while Fusarium spp (PSC 18 ) and Mucor spp (PUE 6 ) showed the highest Oil and grease (6% each) and Mucor spp (PUE 6 ) had the highest TSS reduction potential (.0%). In compliance with the observation from this study, other researchers have reported biodegradation of oily wastewaters with significant reduction in TSS, Oil and Grease and similar parameters (Elmasery et al., 00; Raj and Murthy, 1999). Generally, the BOD, COD, TSS and Oil and grease reduction observed is as a result of microbial oil degradation which is considered to occur as a result of hydrolysis of oil by secretion of lipase (oil degradation enzyme), which degrades the oil to organic acids and Volatile Fatty Acids (VFA) or reduces it to a low molecule via beta oxidation (Fatty acid degradation pathway) and finally the oil is decomposed to CO and H 0 (Koshimizu et al., 007). Since Palm oil mill effluent contains a very high organic matter (Organic carbon) (as evident in the result of Physicochemical analysis obtained in this study) which is generally biodegradable, this facilitates the application of biological treatment based on aerobic process (Chin and Wong et al., 00). This biological treatment depends greatly on active microorganisms, which utilizes the organic substances present in the POME as nutrients and eventually degrades these organic matters into simple by-products such as methane, carbondioxide, hydrogen sulphide and water (Jameel and Olanrewaju et al., 011). 6

12 Thus, the exploitation of these microorganisms isolated from palm oil mill effluent for biodegradation and bioremediation purposes will offer a very efficient tool for purifying contaminated effluents water. CONCLUSION From this study, it is evident that POME degrading microorganisms can be isolated from POME polluted area and the degrading ability of these microorganisms; Pseudomonas spp (PSC ), Aeromonas spp (PSC 6 ), Fusarium spp (PSC 18 ), Mucor spp (PUE 6 ) is a clear indicator that they can be applied in bioremediation techniques for biodegradation of POME to enhance treatment. REFERENCES 1. Ohimain, E., Oyedeji, A & Izah, S. (01). Empolyment effects of small holder oil palm processing plants in Elele Rivers State, Nigeria. International Journal of Applied Research and Technology, 1(6), Orji, M., Nwokolo, S & Okoli, I. (006). Effects of palm oil mill effluent on soil microflora. Nigerian Journal of Microbiology, 0(), Leachy, J & Colwell, R. (1990). Microbial degradation of hydrocarbons in the environment. Microbiology Review,, Hinchee, R & Olfenbuttel, R. (1991). Enzyme production from palm oil mill effluent. Journal of Engineering and Life Science, 1(1), EL-masery, M., EL-Bestawy, H & EL- Adi, N. (00). The potentiality of free Gram- negative bacteria for removing oil and grease from contaminated industrial effluents. World Journal of Microbiology and Biotechnology, 0, Raj, S & Murthy, O. (1999). Synthetic diary wastewater treatment using cross flow medium trickling filter. Journal of Environmental Science and Health, : Jeremiah, D., Japareng, L. & Norli, I. (01). Biodegradation of palm oil mill effluent (POME) by bacterial. International Journal of Scientific and Research Publications, (), Chin, K., & Wong, K.(00). Thermophilic anaerobic digestion of palm oil mill effluent. Water Resources, 17, Jameel, A., Muyibi, S & Olanrewaju, A. (011). Comparative study of bioreactors used for palm oil mill effluent treatment based on chemical and oxygen removal efficiencies. Current Research and Development in Biotechnology,, Mohammadreza, S., Soleimanin, A. & Soheila, M. (01). Enhancement of biodegradation of palm oil mill effluents by local isolated microorganisms. International Scholarly Research Notices, 1: Wu, T., Mohammad, A., Md-Jhaim, J., Anuar, N. (010). Pollution Control technologies for the treatment of palm oil mill effluent (POME) through end- pipe- processes. Journal of Environmental Management, 91, Foo, K., & Hameed, B. (010). Insight into the applications of Palm oil mill effluent: A renewable utilization of the industrial agricultural waste. Renewable Sustainable Energy Review. 1, Cheng, X., Zhu, J., & Borthwick, A. (010). Palm oil mill effluent treatment using a two-stage microbial fuel cells system integrated with immobilized biological aerated filters. Bioresource Technology, 101(8): Ohimain, E., Daokoru, C., Izah, S. Eke, A. & Okonkwo, C. (01). Microbiology of palm oil mill effluents. Journal of Microbiology and Biotechnology Research, (6):8. 6

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