The Comparative Study of the Production of Amylase by Species of Bacillus Subtilis and Pseudomonas Aeruginosa During the Biodegradation of Palm Oil Mill Effluent.
CHAPTER ONE
OBJECTIVES OF THE STUDY
The Objectives of the Study includes:
- To collect POME sample from a local processing plant
- To obtain pure isolates of Bacillus subtilis and Pseudomonas aeruginosa
- To screen the pure isolates for amylase production
- To investigate the invitro biodegradability of Palm Oil Mill Effluent by Pseudomonas aeruginosaand Bacillus subtilis.
CHAPTER TWO
LITERATURE REVIEW
Palm Oil Mill Industry History
As of early 1900, Nigeria was producing all palm oil sold in the world market and it was considered a dominant source of foreign exchange. Up until the 1960’s, Nigeria was the world’s largest producer of palm oil. accounting for 43% of global palm oil production. Over reliance on traditional production methods. Excessive tapping of palm trees for palm wine and the civil war between 1967 – 1970 are the factors that contributed to Nigeria’s inability to meet up with the global rise in demand for palm oil (Eshalomi, 2009).
The Nigerian oil palm belt covers twenty four state including all nine state of the Niger Delta (Akwa – Ibom, Abia, Rivers, Edo, Imo, Ondo, Bayelsa, Cross river and Delta). Within the oil palm bet in Nigeria, 80% of production comes from dispersed small holders who harvest semi – wild plants and use manual processing techniques. Several million small holders are spread over an estimated area ranging from 1.65 million hectares to 2.4 million hectares and to a maximum of 3 million hectares. The estimates for oil palm plantations in Nigeria ranges from 169,000 hectares (72,000 ha of estate plantations) and 97,000 ha of small holder plantations) to 360,000 hectares of plantations (Eshalomi, 2009).
PALM OIL MILL PRODUCTION PROCESSES
The fresh fruit bunches (FFB) of palm trees are transported to the mills for processing after harvest. Each FFB consists of hundred of fruit. Each of which contains the palm oil these FFB are sterilized with steam at a pressure of 3 bar and a temperature of 140oc for 75 – 90 min. the objective of this process are to prevent further formation of free fatty acid due to enzyme action, facilitate shipping and prepare the fruit mesocarp for subsequent processing the steam condensate coming out of the sterilizer constitutes one of the major sources of liquid effluent (Thani et al., 1999). Figure 1 present typical process flow diagram for the extraction of crude palm oil.
After sterilization the FFBs are fed to a rotary drum – stripper where the fruits are stripped from the bunches, the detached fruit are passed through he bar screen of the stripper and are collected below by a bucket conveyor anddischarged into a digester, the fruits are mashed by the rotating arms in this stage the mashingof the fruit under heating breaks the oil from the digested mash of fruit. Under high pressure hot water is added to enhance the flow of the oils. The crude oil slurry is then fed to a clarification system for oil separation and purification. The fibre and nut (Press Cake) are conveyed to a depericarper. For separation (Thani, et al., 1999). The crude palm oil (CPO from the screw presses consists of a mixture of palm oil (35- 45%), water (45 – 55%) and fibrous materials in varying proportion. It is then pumped to a horizontal or vertical clarification tank for oil separation. In this unit, he clarified oil is continuously skimmed off from the top of the clarification tank. It is then passed through a high speed centrifuge and a vacuum dryer before sending it to the storage tanks. The press cakes discharged from the screw press consist of moisture, oily fibre and nuts, and the cakes are conveyed to a depericarper for nuts and fibres separation. The fibre and nuts are separated by strong air current induced sent to boiler house and is used as boiler fuel.
CHAPTER THREE
MATERIALS AND METHODS
STUDY AREA
This study was carried out at the Microbiology laboratory of the Department of Microbiology, University of Abuja, Abuja,Nigeria.
MATERIALS USED
The materials used for the collection of sample include; sterile hand gloves, sterile containers and sterile spoons. Equipment used in the laboratory include; LDZX-50 FBS Autoclave,Galaxy R(Co2) Incubator, Samsung Refrigerator, Weighing balance, Microscope, Jenway 66 UV/Vi Spectrophotometer, Water bath, Esco Laminar flow cabinet.Colony counter,NYC-101 Oven. Other Materials include; Sterile Petri dishes, Wattman filter paper, Inoculating wire loop, Bunsen burner, Stoppers, Sterile hand gloves, Aluminium foil, Spatula, Cotton wool and syringes, Test tube rack. Glass wares used include; Conical flask, measuring cylinder, Bijoux bottles, Pipettes, Beakers, Test tubes, Microscopic glass slides and cover slips. Reagents used include; Inorganic salts, distilled water, sterile water, Ethanol, Crystal violet, Gram’s iodine, Acetone, Safranin, immersion oil, Peptone water, Maltose standard solution, DNSA(dinitrosalicylic acid).e.t.c
CHAPTER FOUR
Results
Determination of Amylase Potential.
When the two bacterial isolates were inoculated into a modified nutrient agar plate containing 1% soluble starch and incubated for 28hrs and then flooded with solution of gram’s iodine, allowed to stand for a minute. A clear zone of hydrolysis was observed on each plate (Plate1&2).which indicates amylase potentials of the isolates .The plate containing Bacillus subtilis showed the highest potentiality than that of Pseudomonas aeruginosa
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION
Discussion
The Bacterial isolates showed a clear zone of hydrolysis when inoculated on a nutrient agar plate supplemented with 1% soluble starch and was flooded with gram iodine after 24 hours of incubation. the clear zone of hydrolysis demonstrates the ability of the bacterial isolates to degrade complex carbon substrate “starch” in the culture plate .this clearing indicates hydrolysis of starch as a result of amylase production (Plates1&2). this phenomenon has been reported by Senthilkumar et al., 2012.Benson (2002) also reported that species of Bacillus are possessing higher amylolytic potential than that of the Pseudomonas species
During the process of degradation of POME invitro using both Bacillus subtilis and Pseudomonas aeruginosa, the turbidity of the substrate was determined at 4 days interval and the result from 4,8,12,and16 included 0.558±0.015,0.507±0.004,0.497±0.005 and 0.806±0.011 for Bacillus subtilis and 0.142±0.009, 0.199±0.002, 0.244±0.007, 0.782±0.009 for Pseudomonas aeruginosa. The turbidity of the medium was observed to increase with increase in days of biodegradation,therefore day 16 having the highest turbidity for Bacillus subtilis and Pseudomonas aeruginosa has 0.806±0.011 and 0.782±0.009 respectively (table2 and figure3) the turbidity was higher in the inoculated sample containing 1% POME than their respective controls that is lacking the Carbon Source.this indicates that the inoculated sample were able to grow and utilize the POME as a carbon source thus degrading it into smaller molecule by the elaboration of certain extracellular enzyme [amylase]. The mineral salt medium presence denotes the reason for the increase in turbidity and it is also important in overcoming theproblem of nutrient limitation during the biodegradation process (Adesodun and Mbagu,2008).
Conclusion
The present study revealed that Pseudomonas aeruginosa and Bacillus subtilis are good producers of amylase enzyme. in which the result of the amylase activity indicated a higher value for Bacillus subtilis .Research on palm oil mill effluent had progressed very rapidly over the last decades and potential applications of the enzymes in the industry especially in waste management and food production. The degradative enzyme (Amylase) produced by Pseudomonas aeruginosa and Bacillus subtilis were capable of breaking down complex substrates in nature, and thus may be implored for the degradation of POME in polluted habitat
Recommendation
The Results of the study showed that Pseudomonas aeruginosa and Bacillus subtilis were able to produce the essential enzyme (Amylase) that is of economic importance both in the Field of biotechnology and environmental protection and conservation.it is therefore recommended that:
- The Bacterial isolates could be used for the biodegradation of amylolytic compounds such as the POME which constitute high level of pollutant in the environment thus reducing the pollutants to simpler compounds that can be readily disposed in the environment without causing pollution.
- The Amylolytic isolates could be used the production fuel alcohol where starch is converted to sugar
- The Amylase produced could be extensively employed in processed-food industry such as baking and the preparation of starch syrups
- The Amylase produced could be extracted and used in the pulp and paper industries for production purposes.
- Most of the amylases used for the production of detergents can be produced by species of Bacillus and Pseudomonas
REFERENCES
- Okwute, O. L., E. Stephen, and P. I. Anyanwu. 2015. “Biodegradation of Palm Oil Mill Effluent (POME) and Lipase Activity by Pseudomonas Aeruginosa, Bacillus Subtilis and Candida Albicans”. Microbiology Research Journal International 9 (5):1-10.
- O. L. Okwute , E. Stephen and P. I. Anyanwu
- Department of Microbiology, University of Abuja, P.M.B. 117, Gwagwalada- Abuja, Nigeria.
- Department of Microbiology, Kogi State University, P.M.B. 1008, Anyigba-Kogi State, Nigeria.
