The Comparative Study of the Invitro Production of Amylase by Species of Bacillus and Pseudomonas(Bacillus Subtilis and Pseudomonas Aeruginosa) During the Biodegradation of Palm Oil Mill Effluent.

The Comparative Study of the Invitro Production of Amylase by Species of Bacillus and Pseudomonas(Bacillus Subtilis and Pseudomonas Aeruginosa) During the Biodegradation of Palm Oil Mill Effluent.

CHAPTER FIVE

Discussion, Conclusion and Recommendation

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).

The highest turbidity was recorded on the 16^th day of the biodegradation process with Bacillus subtilis (0.806±0.011) and Pseudomonas aeruginosa (0.782±0.009).the increase observed  in turbidity withincrease in days is as a result of enzyme released in the medium as well as the rapid growth of the bacterial isolates during the  degradation of the POME in the medium.

Further identification procedure was carried out on the bacterial isolates, Bacillus subtilis showed characteristic Cream colour on Nutrient Agar plates, round colonies with entire margins and a raised elevation which are all characteristics of Bacillus species. Pseudomonas aeruginosa also showed a characteristic cream colour on the Nutrient plate, the elevation of the colony is flat and has wrinkled colony,

The isolates were subjected to biochemical tests, Bacillus subtilis  was catalase positive and reactedpositively to gram staining through its ability to retain the primary stain (crystal violet).where as Pseudomonas aeruginosa  was negative to Gram stain.

The total viable count of the Bacterial isolates was also recorded at intervals of 4 days starting with initial Bacterial count of 1.79×10¹⁰±1.50cfu/ml and 1.27×10¹⁰±1.50cfu/ml. for both Bacillus subtilis and Pseudomonas aeruginosa respectively. The result of the viable count from days 4,8,12, and 16 included 1.49×¹⁰±1.50cfu/ml,2.01×¹⁰±1.00cfu/ml,1.84×10¹⁰±3.50cfu/ml,1.15×10¹⁰±1.00cfu/ml,and1.18×10¹⁰±2.00cfu/ml,1.32×10¹⁰±1.50cfu/ml,1.37×10¹⁰±2.00cfu/ml and 2.75×10¹⁰±2.50cfu/ml for both Bacillus subtilis and Pseudomonas aeruginosa respectively. The Result of the plate count showed that there was increase in the number of organisms with increase in days. day 16 (2.75×10¹⁰±2.50cfu/ml) been the highest for Pseudomonas aeruginosa and day 8(2.01×¹⁰±1.00cfu/ml) been the highest for Bacillus subtilis. Analysis of the plate count shows that there is a significant difference in the viable count of the two isolates at 5% level of significance.

The test consist of the Mineral salt medium (Zajic and Supplison 1972), The organisms with 1% POME,while the control consist of the Mineral salt medium, and the organism only without  the POME which should serve as essential as essential carbon source, hence at the end of the biodegradation process,the growth reduced because the organisms rapidly utilized the inorganic nutrient for growth and since there is no supplementary source of carbon they began to move into the death phase.

The Amylase activity was observed to increase from day 4, 8, 12, and 16 .For Bacillus subtilis. it included 46.50±1.50U/ml,43.50±1.00U/ml,41.20±1.20U/ml and    61.50±0.50U/ml respectively. and the values observed for Pseudomonas aeruginosa  for     the same interval included 19.50±0.50U/ml ,23.5±0.50U/ml, 26.5±0.40U/ml and 60.0±3.00U/ml respectively. The highest amylase activity was produced by Bacillus subtilis on the 16^th day as 61.50+0.50U/ml while the least amylase activity was recorded on the 4^th day by Pseudomonas aeruginosa as 19.50+0.50u/ml as seen in (table 4 andfigure 4).This agreed with Kretscher et al.,(2002) who reported a high amylase activity during the biodegradation of  POME for Bacillus species than Pseudomonas species.

This study showed that Bacillus subtilis showed maximum Amylase activity than Pseudomonas aeruginosa. But however both species of Bacteria can be optimized for improved production of the enzyme (Amylase) industrially.

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

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