Identification and Characterization of Endophytic Bacteria Isolated from Oil Palm Using Enzyme-Based Biochemical Assays

Chiar, Kelvin Kai Boon (2018) Identification and Characterization of Endophytic Bacteria Isolated from Oil Palm Using Enzyme-Based Biochemical Assays. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Two endophytic bacteria, sample A and B which possess potentially suppressive ability towards G.boninense, a white rot fungi that causes Basal Stem Root disease in oil palm. The purpose of this study was to identify and characterise of the isolated strains from sample A and B, by establishing a pattern of activities using enzyme-based biochemical assays. The microscopic analysis indicates both sample A and B as gram-negative bacteria with rod shape. Under aseptic conditions, the pure and unidentified cultures of bacteria were cultured on different media to study the biochemical characteristic which will help in the identification. The bacteria were cultured on both skim milk agar and tributyltin agar to study the presence of extracellular enzymes caspase and lipase and it was shown that both strains gave negative result which indicates the absence of the above enzymes. The utilizations of carbohydrate of sample A and B were studied using methyl red test, citrate utilization test, and fermentation of carbohydrate (sucrose) using Durham tubes. Both strains were able to demonstrate the ability to utilize citrate. As for the fermentation of sugars, sample A showed negative response while sample B were shown to ferment sucrose and glucose. Degradation of amino acids were studies in the form of Hydrogen Sulphide Production Test. Sample B was able to produce hydrogen sulphide as the product from the degradation of sulphur-containing cysteine amino acid. In the same SIM agar for Hydrogen Sulphide test, the motility of the bacteria were also studied, only sample B showed an outward growth from stab in the agar tube, indicating the strain is motile. Both strains produced bubbles of oxygen in the catalase test, proving the presence of catalase. The stains were cultured in urea broth for urea hydrolysis test and only sample B showed positive result showing the presence of urease. The strains were also cultured on selective and differential media, Mannitol Salt agar ,MacConkey agar and EMB agar were used in this study. On MacConkey agar, both stains were selected and differentiated as gram negative, non-lactose fermenter. On Mannitol salt ager, sample A is both non-tolerant to salt and non-mannitol fermenter while sample B is shown to be tolerant to salt but cannot ferment mannitol. On EMB agar both samples were selected and shown to be gram negative bacteria with abilities to ferment lactose and/or sucrose.By compiling the responses above and using the identification matrix to compare the responses of the bacteria, the suggested organism for sample A is Pseudomonas aeruginosa and sample B is Proteus vulgaris. By using online unknown bacterial identification tool (ABIS), sample A is identified as Obesumbacterium proteus with 80.4% similarity, while sample B is identified as Citrobacter wekmanii with 98.2% similarity.