Document Type

Thesis - Open Access

Award Date

2021

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

Bruce Bleakley

Keywords

Bacillus amyloliquefaciens, bioremediation, petroleum, Surfactin

Abstract

Several Bacillus amyloliquefaciens isolates obtained from wheat residue have been studied as biological agents to control wheat diseases. Notable traits of isolates 1BA and 1D3 include growth with high salt (10% NaCl), at temperatures up to 50°C and over a wide pH range. BIOLOG Gen III plates were used to study and further characterize Bacillus amyloliquefaciens 1BA and 1D3. Both isolates showed tolerance to high salt concentration supporting previous studies. They also grew in the presence of lithium chloride, potassium tellurite, and sodium bromate. Both isolates grew at pH 5, with almost identical carbon source utilization fingerprints. However, D-serine, quinic acid, and N-acetylglucosamine were utilized by 1BA but not by 1D3. Potential for the bacteria to carry out dissimilatory nitrate reduction was studied using nutrient broth (NB) media and tryptic soy broth (TSB) amended with 5 mM potassium nitrate. The isolates were able to reduce nitrate better in richer (tryptic soy broth) culture medium compared to the less nutrient rich medium (nutrient broth), suggesting that the isolates may carry out dissimilatory nitrate reduction to ammonium. Utilization of phenolic acids that are known to occur in plant and soil habitats, including, cinnamic acid, ferulic acid, salicin, caffeic acid, vanillin, and benzoic acid, was assayed for both the bacterial isolates. Both the isolates were able to utilize caffeic acid and ferulic acid as sole carbon source, indicating the possibility of utilizing phenolic compounds that are found in soil and plant materials. The isolates were also assayed for their ability to metabolize of volatile aromatic hydrocarbons. The isolates were incubated in a sealed glass a desiccator on a carbon-free agar growth medium in the presence of toluene, phenol, or kerosene. After a 30-day incubation in the dark at 27°C, soluble proteins of bacterial growth were measured using a bicinchonic acid (BCA) assay. Bacillus amyloliquefaciens isolates 1BA and 1D3 showed greater growth with kerosene compared to the individual volatile aromatic hydrocarbons (VOCs) toluene and phenol. In broth studies, isolates 1BA and 1D3 were grown in TSB amended with sterile (v/v) 1.0 % kerosene, and in minimal broth media (MSM) amended with sterile (v/v) 1.0% kerosene. The petroleum degradation/utilization was tracked every two days Gas Chromatography (GC) connected with an Agilent Mass spectrometer. There was no significant reduction in total kerosene observed. Using the same broth media production of surfactin was tracked using a High-Pressure Liquid Chromatography (HPLC). Of the two isolates only 1D3 was found to produce the biosurfactant surfactin. In the TSB medium isolate 1D3 produced 1,021 ppm of surfactin after 5 days and 1,055.5 ppm at 10 days incubation in the presence of kerosene. In the absence of kerosene 3,310 ppm was produced after 5 days of incubation, and 3,271.3 ppm after 10 days incubation. The reduction in surfactin concentration in flasks with kerosene compared to media without kerosene could be due to surfactin-kerosene interaction. The concentration of surfactin was higher in the TSB broth compared to minimal broth medium. Surfactin was detected in the minimal medium, but could not be quantified. Bacillus amyloliquefaciens isolates 1BA and 1D3 were found to utilize some of the volatile compounds and phenolic acids that are commonly found in plant and soil environments. They also were found likely to carry out dissimilatory nitrate reduction, and to produce surfactin. Future studies could evaluate the impact of surfactin on degradation of longchain hydrocarbons; and examine their utilization of other volatile compounds which can be harmful to human and environmental health.

Library of Congress Subject Headings

Bacillus (Bacteria)
Biosurfactants.
Petroleum.
Hydrocarbons.
Phenolic acids.
Bioremediation.

Number of Pages

78

Publisher

South Dakota State University

Rights

Copyright © 2021 the Author

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