Theses and Dissertations at Montana State University (MSU)
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Item Understanding physiological adaptations, metabolic potential and ecology in a novel photoautotrophic alga for biofuel production(Montana State University - Bozeman, College of Letters & Science, 2019) Corredor Arias, Luisa Fernanda; Chairperson, Graduate Committee: Matthew Fields; Elliot B. Barnhart, Al Parker, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Impact of temperature, nitrate concentration, PH and bicarbonate addition on biomass and lipid accumulation of a sporulating green alga' which is contained within this dissertation.; Thiru Ramaraj, Huyen Bui, Mensur Dlakic, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Genomic insights into a sporulating, non-motile, oligotrophic green microalga (PW95)' which is contained within this dissertation.; Huyen Bui, Thiru Ramaraj, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Transcriptomic profiling of Chlamydomonas-like PW95 cultivated in coal bed methane production water with the native microbial community' which is contained within this dissertation.; Anna J. Zelaya, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Associations between sympatric bacterial groups and a novel green alga cultivated in coal bed methane production water' which is contained within this dissertation.Commercial implementation of microalgal biomass as bio-oil/chemical feedstocks has been difficult to achieve, and challenges include water/nutrient sources, CO 2 delivery, and community dynamics of mixed cultures. We employed an integrated approach to the study of microalgal production systems to advance towards sustainable implementation of industrial microalgal biofuel production using a native alga (Chlamydomonas-like alga, PW95) isolated from Coal Bed Methane (CBM) production water. Our approach was based on the evaluation of PW95 physiological responses to combinations of growth constraints, the determination of its genomic and functional potential, phylogenetic relations and the implementation of an ecosystem view to algal biomass production. PW95 growth and lipid accumulation (biofuel potential) were ascertained in standardized media and CBM water through the evaluation of mixed effects of temperatures, nitrate levels, pH, and bicarbonate to elucidate interactions between multiple environmental variables and nutritional levels. The biofuel potential of PW95 ranges between 20-32% depending on culture conditions and our results suggest an important interaction between low nitrate levels, high temperature, and elevated pH for trade-offs between biomass and lipid production in the alga. Whole genome sequence was employed to predict biological and metabolic capacity in PW95, and the expression of these capabilities during growth in CBM water with the native microbial consortia was evaluated using RNA sequencing. genome determination and assembly resulted in a draft genome size of 92 Mbp with 14,000 genes predicted and 402 pathways mapped in the KEGG database. The gene complement of PW95 provided a glance into life in an oligotrophic environment (CBM water) and evidence of essential metabolic pathways for cell growth, survival and maintenance, also relevant for cultivation and value-added products generation. Microbial composition and shifts during growth were identified, as well as the algal phycosome. During growth in CBM water, PW95 appeared to be supported by a native microbial consortium and differential expression analysis showed basic metabolic functions and adaptive physiological responses. Our findings build on previous knowledge for improved algal culturing for biomass and industry-valued products while exploring the biology of an organism with relevant impact in energy and water resource management.Item The role of ribosome hibernation factor and the stringent response in the survival of Pseudomonas aeruginosa during dormancy(Montana State University - Bozeman, College of Letters & Science, 2019) Theng, Sokuntheary; Chairperson, Graduate Committee: Michael FranklinPseudomonas aeruginosa survives in a dormant state in low nutrient environments and is able to resuscitate when favorable conditions are available. In response to stressful environmental conditions including antibiotic stress, osmotic stress, and starvation, P. aeruginosa undergoes the (p)ppGpp-mediated stringent response to induce a variety of genes for entry into the dormant state. One critical mechanism for P. aeruginosa dormancy involves in inactivating translation machinery by converting active ribosomes into inactive 70S and 100S ribosome monomers and dimers. Hibernation promoting factor (HPF~11.6kDa) is a ribosome-associated protein that stabilizes inactive ribosomes. Here, I investigated the relationship between HPF and the stringent response in survival of P. aeruginosa during dormancy. I also investigated role of HPF preserving ribosomes in dormant cells by quantifying the abundance of two ribosomal proteins (L5 of the large ribosomal subunit and S13 of the small ribosomal subunit), during P. aeruginosa starvation. For quantitative analysis during nutrient-limited conditions, I used immunoblotting and image analysis to quantify L5 and S13 abundances in the wild-type strain, PAO1, and in a relA/spoT double mutant strain, which is incapable of producing (p)ppGpp. The results show that the relA/spoT mutant loses HPF proteins after four days of starvation. To explore the role of HPF in preserving ribosomal proteins, I quantified L5 and S13 in wild-type PAO1 and in the Delta hpf deletion mutant and in the relA/spoT mutant. Immunoblots showed that both L5 and S13 rapidly decrease by day 2 of starvation in the Delta hpf mutant strain, but that these proteins are maintained throughout eight days of starvation in the wild-type strain. Notably, L5 and S13 are maintained in the Delta relA/Delta spoT strain throughout starvation. Lastly, I determined if the amount of cellular HPF required for P. aeruginosa ribosome maintenance. Growth in minimal medium (MOPS medium) affects the amount of HPF produced, based on the carbon source. Therefore, I tested ribosome maintenance during starvation of cells first cultured in MOPS-fructose or MOPS-glucose. The results indicate that HPF production during growth in MOPS-fructose is higher than in MOPS-glucose. However, the increased amount of HPF did not affect the amount of L5 and S13 during starvation. Therefore, the amount of HPF is not critical for P. aeruginosa to maintain its ribosomes during starvation. These results demonstrate HPF is essential for maintenance of ribosomal proteins during starvation of P. aeruginosa, and that the ribosomal proteins are likely degraded in the absence of HPF. P. aeruginosa needs a minimum amount of HPF to preserve ribosomes during nutrient-limited condition.Item Characterization of the stability of Pseudomonas aeruginosa ribosomal proteins under stress conditions(Montana State University - Bozeman, College of Letters & Science, 2017) Yanardag, Sila; Chairperson, Graduate Committee: Michael FranklinIn this study, I aimed to standardize western blot methods for probing large and small ribosomal subunits of Pseudomonas aeruginsa grown under different environmetal conditions, and to characterize the stability of ribosomal proteins to bring light to the heterogeneous composition of the population, which is hypothesized as one mechanism for antibiotic tolerance. Long-term studies done with P.aeruginosa PAO1 showed that mRNA transcripts of two proteins, RMF and HPF, are highly abundant at the biofilm-nutrient interface of the thick P.aeruginosa biofilms. Also, it was previously shown by Perez et al. and Williamson et al. (Pérez-Osorio et al., 2010; Williamson et al., 2012) that the cells located at the oxygen limited interphase of the biofilm were metabolically inactive or slow-growing. Akiyama et al. (Akiyama et al., 2017) and Williamson et al. (Williamson et al., 2012) found that HPF is a critical protein for the maintenance of 23S rRNA and overall ribosomal RNA stability after prolonged stress exposure (Akiyama et al., 2017; Williamson et al., 2012). In light of this information, Akiyama et al. (Akiyama et al., 2017) showed that in the absence of the HPF protein, P.aeruginosa cannot protect its ribosome integrity and cannot resuscitate from dormancy after the environmental stressors are gone. Perez et al. (Pérez-Osorio et al., 2010) showed that P.aeruginosa biofilms are heterogeneous in physiology, and it is posited that persister cells of the biofilm are located at the bottom of the biofilm, unaffected by the antibiotic exposure and therefore can repopulate the biofilm (Williamson et al., 2012). Localization of ribosomal subunits and determination of the abundance of ribosomes within the heterogeneous biofilms will provide valuable insights on the mechanisms of persister cell formation, dormancy, and resusication from dormancy. In order to do so, I have isolated two ribosomal proteins, L5 and S13, and HPF. In this study, I generated polyclonal antibodies against those three proteins. I used the antibodies to determine the abundance of these proteins during the normal course of growth of the wild type and Deltahpf mutant strains. Growth analysis in nutrient rich media gave us an understanding of the stability of 70S ribosomes when the bacterium was growing without any stress. Later, the wild type and Deltahpf strain were grown in a carbon and nitrogen-limited environment for seven days to examine the response of the cells to the starvation stress regarding ribosomal stability. Finally, I tested the hypothesis that cells located at the bottom of the biofilm are abundant in the HPF protein, and therefore contain more inactive ribosomes compared to the cells located at the top of biofilm.Item Microbial interactions and the role of environmental stress in natural and synthetic consortia(Montana State University - Bozeman, College of Letters & Science, 2018) Beck, Ashley Esther; Chairperson, Graduate Committee: Ross Carlson; Kristopher A. Hunt, Hans C. Bernstein and Ross P. Carlson were co-authors of the chapter, 'Interpreting and designing microbial communities for bioprocess applications, from components to interactions to emergent poperties' in the book 'Biotechnology for biofuel production and optimization' which is contained within this thesis.; Kristopher A. Hunt and Ross P. Carlson were co-authors of the article, 'Measuring cellular biomass composition for computational biology applications' submitted to the journal 'Processes, methods in computational biology special issue' which is contained within this thesis.; Hans C. Bernstein, and Ross P. Carlson were co-authors of the article, 'Stoichiometric network analysis of cyanobacterial acclimation to photosynthesis-associated stresses identifies heterotrophic niches' in the journal 'Processes, microbial community modeling: prediction of microbial interactions and community dynamics special issue' which is contained within this thesis.; Kathryn Pintar, Diana Schepens, Ashley Schrammeck, Tim Johnson, Alissa Bleem, Hans C. Bernstein, Tomas Gedeon, Jeffrey J. Heys and Ross P. Carlson were co-authors of the article, 'Escherichia coli co-metabolizes glucose and lactate for enhanced growth' submitted to the journal 'Applied and Environmental Microbiology' which is contained within this thesis.; Ross P. Carlson was a co-author of the article, 'Synthetic consortia engineered for push and pull dynamics show conditional optimality over metabolic generalist' which is contained within this thesis.Microbial communities are critical underpinnings of most natural processes, e.g. biogeochemical cycling, and can also be harnessed and engineered for a variety of industrial applications. Despite the abundance of detailed physiological characterization of many individual microorganisms, as well as large data sets describing microbial community composition, the area of interspecies interactions requires further research to truly appreciate and harness the potential of microbial capabilities. Using a combination of in silico metabolic modeling and in vitro laboratory approaches linked to guiding ecological theories, this dissertation investigates metabolite exchange as a mechanism of interspecies interactions and focuses on the role of environmental stress in mediating interactions. A stoichiometric metabolic network model was constructed for the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 and was analyzed with elementary flux mode analysis to predict metabolic acclimations to light and oxygen, two common environmental stressors in photoautotrophic habitats. High stress levels were predicted to activate organic byproduct secretion pathways, which opens a niche to support growth of heterotrophic partners. To further investigate metabolite exchange in the laboratory, synthetic consortia were designed through genetic engineering and pairing of Escherichia coli strains to form metabolically partitioned organic acid cross-feeding systems. These controlled systems were used to investigate the impact of division of labor as well as the effect of byproduct detoxification. Kinetic data from these systems were also applied to interpret ecological theories regarding microbial community structure. Altogether, these studies demonstrate an integrated approach to studying microbial community interactions by combining in silico metabolic modeling and in vitro laboratory experiments with ecological theory as a basis for interpretation. This dissertation provides insight into rationale for microbial community structure and highlights the role of environmental stress, particularly byproduct inhibition, in driving microbial consortia interactions.