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ADVANCING THE CULTIVABILITY OF SOIL BACTERIA USING A DYNAMIC SOIL ENVIRONMENT AND SOIL EXTRACT METHOD

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Date Issued:
2019
Abstract/Description:
Bacteria are inarguably the most ubiquitous and adaptive organisms on the planet. The vast, diverse community of microbes residing in soil are mostly studied using sequencing technologies because over 99% of them are currently uncultivable in the laboratory. This lack of diverse bacterial cultivation presents a serious challenge for modern microbiological and medical science where the discovery of novel antibiotic producers and microbial products has been outpaced by the rise in drug resistance. This study designed and tested two new cost-effective culture systems called the “Dynamic Soil Environment” and Soil Extract Systems with the goal of increasing the cultivable communities of diverse bacteria in a soil sample over standard methods. Illumina MiSeq sequencing and DADA2 pipeline protocols were used to analyze community DNA from cultivated samples and source soil metagenomes. Autoclaved soil extract media in the Soil Extract Experiment yielded a statistically significantly greater Shannon’s (p = 0.008) and Simpson’s diversity (p = 0.007) of bacteria over pH modified (6.4) nutrient agar media over 30 days of incubation. Autoclaved soil extract media was also able to cultivate, on average, 33% of species in bulk soil sequences compared to 27% from standard nutrient agar however these differences weren’t statistically significant. The length of incubation had a lesser effect than media type on yield of bacteria over 30 days in batch culture conditions. Species richness and diversity generally decreased over time except in soil extract samples. In the Dynamic Soil Environment experiment, membrane plates placed on a live soil environment produced a slightly higher diversity than autoclaved membrane plates and control plates without soil, however, these differences were not statistically significant except when analyzed with Chao1 diversity (0.041). Cultivated bacterial diversity and communities differed more according to media type than soil environment with statistically significant differences between standard and pH modified nutrient agar. Media with a 5.8 pH buffer produced a significantly higher relative abundance of the well-known antibiotic-producers, Actinobacteria (t(10) = -5.715, p < .000) and also Proteobacteria (t(10) = -10.127, p < .000). This study establishes cost-effective methods of cultivating more diverse bacterial communities for low-funded laboratories. Culture conditions for the reliable cultivation of higher relative abundances of bacterial groups belonging to Actinobacteria and Proteobacteria are also established with the Dynamic Soil Environment Experiment.
Title: ADVANCING THE CULTIVABILITY OF SOIL BACTERIA USING A DYNAMIC SOIL ENVIRONMENT AND SOIL EXTRACT METHOD.
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Name(s): España, Erick, author
Brooks, Randy W., Thesis advisor
Esiobu, Nwadiuto, Thesis advisor
Florida Atlantic University, Degree grantor
Charles E. Schmidt College of Science
Department of Biological Sciences
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Date Created: 2019
Date Issued: 2019
Publisher: Florida Atlantic University
Place of Publication: Boca Raton, Fla.
Physical Form: application/pdf
Extent: 91 p.
Language(s): English
Abstract/Description: Bacteria are inarguably the most ubiquitous and adaptive organisms on the planet. The vast, diverse community of microbes residing in soil are mostly studied using sequencing technologies because over 99% of them are currently uncultivable in the laboratory. This lack of diverse bacterial cultivation presents a serious challenge for modern microbiological and medical science where the discovery of novel antibiotic producers and microbial products has been outpaced by the rise in drug resistance. This study designed and tested two new cost-effective culture systems called the “Dynamic Soil Environment” and Soil Extract Systems with the goal of increasing the cultivable communities of diverse bacteria in a soil sample over standard methods. Illumina MiSeq sequencing and DADA2 pipeline protocols were used to analyze community DNA from cultivated samples and source soil metagenomes. Autoclaved soil extract media in the Soil Extract Experiment yielded a statistically significantly greater Shannon’s (p = 0.008) and Simpson’s diversity (p = 0.007) of bacteria over pH modified (6.4) nutrient agar media over 30 days of incubation. Autoclaved soil extract media was also able to cultivate, on average, 33% of species in bulk soil sequences compared to 27% from standard nutrient agar however these differences weren’t statistically significant. The length of incubation had a lesser effect than media type on yield of bacteria over 30 days in batch culture conditions. Species richness and diversity generally decreased over time except in soil extract samples. In the Dynamic Soil Environment experiment, membrane plates placed on a live soil environment produced a slightly higher diversity than autoclaved membrane plates and control plates without soil, however, these differences were not statistically significant except when analyzed with Chao1 diversity (0.041). Cultivated bacterial diversity and communities differed more according to media type than soil environment with statistically significant differences between standard and pH modified nutrient agar. Media with a 5.8 pH buffer produced a significantly higher relative abundance of the well-known antibiotic-producers, Actinobacteria (t(10) = -5.715, p < .000) and also Proteobacteria (t(10) = -10.127, p < .000). This study establishes cost-effective methods of cultivating more diverse bacterial communities for low-funded laboratories. Culture conditions for the reliable cultivation of higher relative abundances of bacterial groups belonging to Actinobacteria and Proteobacteria are also established with the Dynamic Soil Environment Experiment.
Identifier: FA00013304 (IID)
Degree granted: Thesis (M.S.)--Florida Atlantic University, 2019.
Collection: FAU Electronic Theses and Dissertations Collection
Note(s): Includes bibliography.
Subject(s): Bacteriological Techniques--methods
Bacteriology--Cultures and culture media
Soil
Held by: Florida Atlantic University Libraries
Sublocation: Digital Library
Persistent Link to This Record: http://purl.flvc.org/fau/fd/FA00013304
Use and Reproduction: Copyright © is held by the author with permission granted to Florida Atlantic University to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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Host Institution: FAU
Is Part of Series: Florida Atlantic University Digital Library Collections.