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Document Type

Dissertation - University Access Only

Award Date

2015

Degree Name

Doctor of Philosophy (PhD)

Department

Biology and Microbiology

First Advisor

Bruce H. Bleakley

Abstract

Enrichments cultures were performed using soil as inocula and water samples which were collected from different locations within South Dakota including, creeks, compost piles, and the Homestake Mine in Lead, SD, the location of the Sanford Underground Research Facility (SURF), formerly prospective Deep Underground Science and Engineering laboratory (DUSEL). A total of 290 culturable isolates were screened for the presence of carboxymethylcellulase (CMCase) on agar containing carboxymethylcellulose (CMC) as the sole carbon source. Of the 105 CMCaseproducing isolates [89 positive for CMCase and 25 thermophiles (all CMCase negative)] screened for extracellular cellulase production on RBBR-cellulose, eight fungi and three bacteria produced measurable cellulase. Ten of these microorganisms were then characterized and identified through a series of biochemical and genetic analyses. Fungal strains were identified as: Scytalidium lignicola, Clonostachys rosea rosea, Phoma glomerata M8757, Gliocladium virens, Acrodontium salmoneum, Penicillium solitum, and Fusarium spp. Three Gram positive bacteria, two-thermophilic strains of Geobacillus toebii and a strain of Bacillus amyloliquefaciens were identified. The endo-β-1,4- glucanase (CMCase) produced by the strain of Bacillus amyloliquefaciens was partially purified and characterized. Ammonium sulfate precipitation along with gel filtration chromatography were used to fractionate and partially purify the enzyme. The partially purified enzyme was characterized through enzymatic assays allowing for the calculation of kinetics, optimal temperature, and optimal pH. The molecular weight of the protein was determined using an SDS-PAGE gel with a 4% stacking and 10% resolving gel. The optimal temperature for this enzyme was 60 to 70°C. Enzyme activity occured between a pH of 5 to 10 with 8.0 being optimal. Two separate bands were identified using SDSPAGE. The molecular weight of band A was 21.3 ± 0.4 kDa and band B 11.5 ± 0.2 kDa. After elution of bands from the SDS-PAGE gel, both bands retained enzymatic activity independent of one another. The DGGE analysis of sample sites indicated that the depth within the mine or the level of the sampling in the fountain had an effect on bacterial and fungal community compositions. Both bacterial and fungal community compositions varied from one another with the bacterial communities appearing much more diverse than the fungi.

Library of Congress Subject Headings

Cellulase -- South Dakota
Microorganisms -- South Dakota
Lignocellulose

Description

Includes bibliographical references (pages 209-223)

Format

application/pdf

Number of Pages

239

Publisher

South Dakota State University

Rights

In Copyright - Educational Use Permitted
http://rightsstatements.org/vocab/InC-EDU/1.0/

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