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Assessing and improving sustainability of Camelina sativa through rhizobacterial inoculants and soil enzymatic activity
(Montana State University - Bozeman, College of Agriculture, 2023) Stowell, Henry Douglas; Co-chairs, Graduate Committee: Catherine A. Zabinski and Jed O. Eberly
Camelina sativa is an oilseed crop with potential to be used in biofuel production as an alternative to contemporary fossil fuels. To ensure biofuels are a more sustainable alternative , considerations and improvements must be made regarding the inputs and land-use needs of producing biofuel feedstocks. This research assessed the beneficial effects of inoculating C. sativa plant growth-promoting rhizobacteria candidates in greenhouse trials. Additionally, we explored agronomic responses of C. sativa and bulk soil enzymes in field trials across Montana to nitrogen and sulfur fertilizer treatments applied as pelleted urea and gypsum respectively. Co- inoculations of Pseudomonas putida ATCC 12633 and Bacillus thuringiensis ATCC 33679 were associated with seed yield increases of 60% relative to the uninoculated control. Co-inoculations of Pseudomonas brassicacearum 36D4 and B. thuringiensis ATCC 33679 were associated with significantly shortened root lengths of early seedlings but did not reduce total biomass. Field trials found a strong seed yield response to nitrogen treatments, with yields increasing with each treatment up to 168 kg N/ha. No significant yield response to sulfur treatments was observed. Additionally, fertilizer treatments did not have any significant effects on the activities of arylsulfatase, beta-glucosaminidase, beta-glucosidase, or urease in soils sampled at crop flowering. Rhizobacterial inoculants have potential to improve crop yields without additional inputs and should be tested on C. sativa in field settings. Urea applications can be used to improve C. sativa yields without any short term effects on soil enzymatic activity, but longer-term studies are needed to accurately determine the effects of the crop and its inputs on soil properties.
Phycosomal dynamics in xenic cultures of the alkalitolerant green Microalga chlorella sp. SLA-04
(Montana State University - Bozeman, College of Agriculture, 2023) Miller, Isaac Robert; Chairperson, Graduate Committee: Matthew Fields; This is a manuscript style paper that includes co-authored chapters.
The production of microalgal biomass and biofuel is an important component of the transition away from a petroleum-based economy. Industrial scale microalgal cultures are often xenic, meaning they are comprised of microalgae as well as a phycosome (i.e., microbiome). The microalgal field has begun to appreciate the ubiquity and potential influence of the phycosome, but there remains a critical need for comprehensive research to unravel the intricate metabolic and ecological relationships between microalgae and the respective phycosome that can be comprised of mainly bacteria but also other microorganisms (i.e., archaea, fungi, protists, viruses). Phycosome research is essential for potentially using these interactions to enhance the stability, productivity, and cost-efficiency of industrial microalgal cultivation. Chlorella sp. SLA-04 is an oleaginous, alkalitolerant microalga isolated from the alkaline Soap Lake (Washington, USA). Under alkaline conditions, SLA-04 can be grown to high biomass levels without reliance on the delivery of concentrated CO 2, an improvement in producing competitively priced biomass and biofuel. The high pH, high alkalinity systems are able to capture CO 2 directly from the air in open systems (e.g., raceway ponds) but the open systems can be dynamic in terms of stability and productivity. Despite growing knowledge of the importance of phycosomes in open production systems, little is known about how alterations to cultivation conditions can be used to maintain a xenic system with controllable outputs, especially under high pH, high alkalinity conditions. The work outlined in this dissertation employed long term temporal community studies, open outdoor raceway experiments, diel-cycle-resolved temporal sampling coupled with activity-based probing (bioorthogonal non-canonical amino acid tagging (BONCAT)), and quantitative measures of algal physiology to better understand the relationship between microalgal phenotype and the respective phycosomes. SLA-04 phycosome composition and culture physiology were consistent over time when maintained in xenic cultures under low and high alkalinity. When xenic cultures were used in successive open, outdoor raceway experiments, compositional community changes coincided with seasonal temperature and light shifts, providing evidence that abiotic and biological environmental stresses impact directly and indirectly SLA-04 productivity and phycosome composition. By employing temporally resolved sampling and probing the relationship between diel-cycle-dependent metabolism and the phycosome, we identified active bacterial populations that may play a role in culture productivity. Expanding beyond augmenting SLA-04 productivity, aggregation of xenic cultures was assessed as a quantifiable phenotype, uncovering a relationship between aggregation, taxonomic composition and algal growth conditions (i.e., alkalinity level). All together, these results represent an initial description of the ecology (e.g., composition, succession, activity) of alkaline microalgae cultures and provide methodology and perspective for future phycosome studies.
Algal biofilms and lipids: bicarbonate amendment and nitrate stress to stimulate lipid accumulation in algal biofilms
(Montana State University - Bozeman, College of Engineering, 2022) Rathore, Muneeb Soban; Chairperson, Graduate Committee: Brent M. Peyton; This is a manuscript style paper that includes co-authored chapters.
Algal biofuels are compounds obtained by transesterification of algal lipids to fatty acid methyl esters (FAMEs) which can be used as biodiesel. Algal biofilms have a potential for commercial applications of algal biomass for biofuel production and provide concentrated biomass requiring less water removal to reduce biofuel production costs. Lipid production in algal biofilms is low as compared to planktonic algal growth systems and strategies for enhancing lipid content in algal biofilms need to be developed. The overarching goal of the studies presented herein was to develop lipid accumulation strategies in algal biofilms using nutrient stresses to increase triacylglycerides (TAGs) and FAMEs. First, a reactor was designed for photoautotrophic biofilm growth incorporating a novel algal biomass harvesting mechanism. Chlorella vulgaris biofilm growth was demonstrated to establish the reactor characteristics under three different inorganic carbon regimes and the presence of excess calcium to facilitate biofilm attachment and accumulation. Excess calcium resulted in precipitate formation and increasing ash content in biomass and caused difficulty in biofilm detachment. However, the highest biomass accumulation was observed in the bicarbonate and the bicarbonate with calcium treatments. Second, two different algal strains were tested for lipid accumulation under two nutrient conditions: nitrate limitation and bicarbonate addition. Algal strains included, an extremophilic freshwater diatom RGd-1, a Yellowstone National Park (YNP) isolate, and oleaginous chlorophyte C. vulgaris. High bicarbonate content at low nitrate concentration in the bulk medium provided the highest lipid accumulation as determined by Nile Red fluorescence and Gas Chromatography Mass Spectrometry (GCMS) analysis of extracted FAMEs (7-22 % wt/wt). For prevention of biomass loss and quick response to nutrient stresses to stimulate lipid accumulation, the growth medium was exchanged after initial biofilm accumulation and operated in batch mode. This was implemented to quickly introduce nutrient stresses using fresh medium to vary bicarbonate and nitrate concentrations as needed. Thus, the work presented here demonstrated enhanced lipid production in algal biofilms with nitrate stress and bicarbonate amendment is a viable strategy to increase lipid accumulation. Increased lipid content may help offset the cost for biodiesel production with more lipid product and lower processing requirements for water removal.
Alkaline microalgae from Yellowstone National Park: physiological and genomic characterization for biofuel production
(Montana State University - Bozeman, College of Agriculture, 2021) Moll, Karen Margaret; Chairperson, Graduate Committee: Brent M. Peyton; This is a manuscript style paper that includes co-authored chapters.
Alternatives are needed to avoid future economic and environmental impacts from continued exploration, harvesting transport, and combustion of conventional hydrocarbons resulting in a rise of atmospheric CO 2. Microalgae, including diatoms, are eukaryotic photoautotrophs that can utilize inorganic carbon (e.g., CO 2) as a carbon source and sunlight as an energy source, and many microalgae can store carbon and energy in the form of neutral lipids. In addition to accumulating useful precursors for biofuels and chemical feed-stocks, the use of autotrophic microorganisms can further contribute to reduced CO 2 emissions through utilization of atmospheric CO 2. Most microalgal biofuel research has focused on green algae. However, there are good reasons to consider diatoms for biofuel research. Diatoms are responsible for approximately 40% of marine primary productivity, are important in freshwater systems, and are known to assimilate 20% of global CO 2. Identification and implementation of factors that can contribute to rapid growth will minimize inputs and production costs, thus improving algal biofuel viability. Nine green algae strains that were isolated from Witch Creek, Yellowstone National Park, were compared to two culture collection strains (PC-3 and UTEX395) for growth rates, dry cell weights and lipid accumulation. The strains exhibiting the fastest growth rates were WC-5, WC-1 and WC-2b. The culture collection strain was the best biomass producer and WC-5 and UTEX395 were the most productive for lipid. Based on the growth rates and lipid content, the best strains for biodiesel production were WC-1 and WC-5. In addition to the green algae strains, diatom strain, RGd-1 has previously been found to accumulate 30-40% (w/w) triacylglycerol and 70-80% (w/w) fatty acid methyl esters that can be transesterified into biodiesel. The RGd-1 was sequenced via Illumina 2x50 and PacBio RSII reads and genome comparisons revealed that the RGd-1 genome is significantly divergent from other publicly available genome sequences. RGd-1 was found to have nearly complete metabolic pathways for fatty acid elongation using acetyl-CoA in the mitochondrion or malonyl-CoA in the cytoplasm. The ability to switch between two different starting substrates may confer an advantage for fatty acid and neutral lipid biosynthesis. Further, RGd-1 was found to use the glyoxylate shunt as part of its central carbon metabolism. This carbon conservation pathway may potentially explain why RGd-1 is able to produce high concentrations of lipids. Using Illumina R MiSeq sequencing it was possible to obtain thorough community analysis of bacteria associated with RGd-1 in culture. Nine primary taxa were identified and further research will elucidate their roles as potential phycosphere bacteria that may have specific functional roles that contribute to RGd-1 health. With long-range PacBio reads, RGd-1 was found to have a potential bacterial symbiont, Brevundimonas sp.
Evaluation of nitorgen and carbon supplementation strategies for optimizing biomass generation during cultivation of Chlorella sorokiniana, strain SLA-04
(Montana State University - Bozeman, College of Engineering, 2021) Jackson, Matthew Clifford; Chairperson, Graduate Committee: Catherine Kirkland
Algal cultivation requires significant nitrogen and carbon inputs, which are expensive and can offset benefits associated with biofuel production. This research investigates growth of an alkali-tolerant Chlorella sorokiniana, strain SLA-04, using different nitrogen and carbon regimes to improve physiological knowledge of this novel organism, and improve biomass production and resource demand. Nitrate, ammonium, and urea were used efficiently by SLA-04, however pH changes during utilization of nitrate and ammonium impacted inorganic carbon availability (species and concentration). Generation of OH- during use of nitrate increased pH, increasing mass transfer of CO 2 into solution and increasing the ratio of HCO 3-/CO 2. Ammonium utilization resulted in proton generation, lowering pH and inhibiting growth. When bicarbonate, rather than CO 2, was provided, productivity improved for the urea and mixed nitrogen conditions. This likely resulted from upregulation of genes related to nitrogen and carbon assimilation in the presence of bicarbonate, however Na + cotransport with urea and nitrate is required in some organisms. It is possible that Na + was insufficient when CO 2 was provided, but not in conditions with bicarbonate since it was added as NaHCO 3-. The impact of Na +, as well as other ions, on nitrogen and carbon utilization is not well understood, but it may alter gene regulation. Bicarbonate and CO 2 both promoted increased growth relative to cultures without inorganic carbon supplementation. The highest productivities were observed when carbon supplementation, either as continued CO 2 augmentation to the air sparge or as a 50mM bicarbonate amendment, was provided during nitrogen deplete growth. Glucose availability improved productivity for conditions without CO 2 supplementation. The use of urea or a combination of nitrogen sources with bicarbonate, instead of CO 2, was promising due to (a) the low cost of urea, relative to the other nitrogen sources; (b) the potential for using wastewater containing a mix of nitrogen sources; and (c) the low cost and easy transport of bicarbonate. Future research should evaluate changes in SLA-04 gene expression resulting from the supply of different nutrients, including nitrogen and carbon sources, as well as other ions essential for growth.
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.
Solid acid catalysts for biomass and sugar upgrading to furans
(Montana State University - Bozeman, College of Engineering, 2019) Romo, Joelle Elise; Chairperson, Graduate Committee: Stephanie Wettstein; Nathan V. Bollar, Coy J. Zimmermann and Stephanie G. Wettstein were co-authors of the article, 'Conversion of sugars and biomass to furans using heterogeneous catalysts in biphasic solvent systems' in the journal 'ChemCatChem' which is contained within this thesis.; Ting Wu, Xinlei Huang, Jolie Lucero, Jennifer L. Irwin, Jesse Q. Bond, Moises A. Carreon and Stephanie G. Wettstein were co-authors of the article, 'SAPO-34/5A zeolite bead catalysts for furan production from xylose and glucose' in the journal 'ACS omega' which is contained within this thesis.
Platform chemicals derived from biomass provide a viable alternative to petroleum-based fuels, chemicals, and materials. The efficient production of chemical building blocks, such as 5-hydroxymethylfurufral (HMF) and furfural, requires an optimized catalyst and reaction system, as well as an efficient system in which catalysts and products can be easily recovered. While homogeneous acid catalysts have historically been a popular choice for furan production, additional safety, material, and corrosion considerations motivates the exploration of heterogeneous solid acid catalysts. Furthermore, biphasic reaction systems, which use an organic solvent to continuously extract products, have shown increased furan yields over aqueous and monophasic systems and can allow for easy product recovery if the boiling point is selected carefully. One class of heterogeneous catalysts known as zeolites, has unique potential for furfural and HMF production with its controlled acidic and structural properties. A novel SAPO- 34/5A zeolite bead is presented in this thesis, showing promise in catalyst design for activity, product selectivity, and stability. The combination of optimized solvent systems with carefully designed solid acid catalysts lays a framework for the progression of platform chemical production from biomass. Additionally, a comprehensive review of heterogeneous catalysts for furan production in biphasic systems is presented here, which informs decisions on optimized solvent selection.
Fungal production of biofuel and flavor compounds in liquid and solid state
(Montana State University - Bozeman, College of Engineering, 2017) Schoen, Heidi Renee; Chairperson, Graduate Committee: Brent M. Peyton; Kristopher A. Hunt, Gary A. Strobel, Brent M. Peyton and Ross A. Carlson were co-authors of the article, 'Carbon chain length of biofuel- and flavor-relevant volatile organic compounds produced by lignocellulolytic fungal endophytes changes with culture temperature' submitted to the journal 'Mycoscience' which is contained within this thesis.; Brent M. Peyton and W. Berk Knighton were co-authors of the article, 'Rapid total volatile organic carbon quantification from microbial fermentation using a platinum catalyst and proton transfer reaction-mass spectrometry' in the journal 'AMB express' which is contained within this thesis.; W. Berk Knighton and Brent M. Peyton were co-authors of the article, 'Volatile organic compound production at varying oxygen conditions in a solid state fungal reactor' submitted to the journal 'Bioresource Technology' which is contained within this thesis.; W. Berk Knighton and Brent M. Peyton were co-authors of the article, 'Production of volatile organic compounds under varying nitrogen conditions by ascocoryne sarcoides' submitted to the journal 'Biotechnology and Bioengineering' which is contained within this thesis.
Oil reserves are limited, so new sources of fuels and petroleum byproducts must be found. Some endophytic, filamentous fungi produce fuel and flavor relevant compounds from minimally pretreated cellulosic materials. Additionally, fungal volatile organic compounds can act synergistically as mycofumigants to inhibit bacteria, insects, and fungi. This dissertation identifies and quantifies fungal volatile organic compounds. A new method was created to quantify the total volatile organic carbon in the gas phase. A platinum catalyst was used to completely oxidize organic compounds to carbon dioxide, which was then measured with a carbon dioxide detector. This method agreed to within 94% of volatile organic carbon measurements taken with proton transfer reaction-mass spectrometry. Additionally, fungal production of fuel and flavor relevant volatile organic compounds was measured with varying pH and temperature in liquid cultures from Nodulisporium isolates EC, CO and TI. Production was also measured for TI when grown in solid state on the agricultural byproduct beet pulp at varying oxygen conditions. Finally, the model volatile organic compound producing organism Ascocoryne sarcoides was grown in liquid state with varying nitrogen sources, including amino acids. The three Nodulisporium isolates produced longer carbon number compounds at lower temperatures, which are better biofuels and are more likely to be bioactive. This trend was especially strong among volatile organic compounds associated with fatty acid metabolism. The fungi produced fewer compounds at lower pH. In solid state, TI had the highest total production of ethanol and carbon number four and higher compounds under anoxic conditions, but the highest production rates under microaerophilic conditions. Additionally, ethanol appeared to be the only major anoxic fermentation byproduct. Finally, A. sarcoides produced the most ethanol and carbon number four and higher compounds in the gas phase with ammonium chloride as the nitrogen source. Nitrogen sources that are reactants for volatile organic compounds, like leucine and phenylalanine, had lower gas phase concentrations of volatile organic compounds.
The stoichiometry of nutrient and energy transfer: from organelles to organisms
(Montana State University - Bozeman, College of Engineering, 2016) Hunt, Kristopher Allen; Chairperson, Graduate Committee: Ross Carlson; James P. Folsom, Reed L. Taffs and Ross P. Carlson were co-authors of the article, 'Complete enumeration of elementary flux modes through scalable, demand-based subnetwork definition' in the journal 'Bioinformatics' which is contained within this thesis.; Ashley E. Beck was an author and Hans C. Bernstein and Ross P. Carlson were co-authors of the article, 'Interpreting and designing microbial communities for bioprocess applications, from components to interactions to emergent properties' in the journal 'Biotechnology for biofuel production and optimization' which is contained within this thesis.; Ryan deM. Jennings, William P. Inskeep and Ross P. Carlson were co-authors of the article, 'Stoichiometric modeling of assimilatory and dissimilatory biomass utilization in a microbial community' in the journal 'Environmental microbiology' which is contained within this thesis.; Ryan deM. Jennings, William P. Inskeep and Ross P. Carlson were co-authors of the article, 'Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community' submitted to the journal 'The International Society for Microbial Ecology journal' which is contained within this thesis.; Natasha D. Mallette, Brent M. Peyton and Ross P. Carlson were co-authors of the article, 'Theoretical and practical limitations of hydrocarbon production for a cellulolytic, endophytic filamentous fungus' submitted to the journal 'Metabolic engineering' which is contained within this thesis.
All life requires the acquisition and transformation of nutrients and energy, driving processes from cellular nutrient flow to planetary biogeochemical cycling. However, the organisms and communities responsible for these processes are often uncultivable and too complex to observe directly and understand. Stoichiometric modeling, a systems biology approach, analyzes the reactions in an organism and incorporates data from multiple sources to extract biologically meaningful parameters, such as theoretical limits of conversion and yields of a metabolism. These limits and yields quantify relationships between organisms to establish governing principles, from resource requirements to community productivity as a function of population composition. The presented work expanded the stoichiometric modeling algorithm and identified fundamental principles that govern nutrient and energy transfer associated with heterotrophy, community composition, and intracellular compartmentalization. A scalable routine capable of analyzing complex metabolic networks by dividing them into tractable subnetworks was demonstrated for a eukaryotic diatom. The metabolic model contained approximately two billion routes through the network and established an international benchmark for elementary flux mode analysis. Additionally, a heterotrophic archaeon was examined for the resource requirements while consuming 29 different forms of biomass derived dissolved organic carbon. These resource requirements and limitations establish a basis to analyze heterotrophy with regard to the limiting nutrient in a variety of systems. The resulting resource requirements of heterotrophy were incorporated into a community where an iron oxidizing autotroph was hypothesized to be the primary source of carbon and energy. Analysis of the community model and in situ measurements of iron and oxygen utilization indicated additional electron donors were required to account for the observed acquisition of nutrients in some communities. Finally, limits and resource requirements for fungal production of hydrocarbons were identified as a function of carbon and energy partitioning using simulated genetic modifications, providing context regarding endophytic production of bioactive molecules for host resistance as well as endophyte capacity as a petroleum producing alternative.
The microbial community ecology of various systems for the cultivation of algal biodiesel
(Montana State University - Bozeman, College of Letters & Science, 2017) Bell, Tisza Ann Szeremy; Chairperson, Graduate Committee: Matthew Fields; Bharath Prithiviraj, Brad D. Wahlen, Matthew W. Fields and Brent M. Peyton were co-authors of the article, 'A lipid-accumulating alga maintains growth in outdoor, alkaliphilic raceway pond with mixed microbial communities' submitted to the journal 'Frontiers in microbiology' which is contained within this thesis.; Emel Sen-Kilic, Tamas Felfoldi, Gabor Vasas, Matthew W. Fields and Brent M. Peyton were co-authors of the article, 'Bacteria and eukarya community during eutrophication and toxic cyanobacterial blooms in the alkaline Lake Velence, Hungary' which is contained within this thesis.; Lakotah Doig, Brent M. Peyton, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Characterizing the microbial community and its intrinsic ability to produce algal biodiesel in wastewater treatment lagoons' which is contained within this thesis.
Algal based biofuel has the potential to aid in offsetting future fossil fuel consumption and demand, and lowering CO 2 emissions. Cultivation strategies are a pivotal component of achieving high biomass yield. Open outdoor pond systems are currently the most economically viable method for large-scale algae cultivation due to less energy for maintenance than closed systems. However, open pond cultivation is subject to microbial colonization, sometimes negatively impacting the algal crop. Thus, large-scale production is hindered by gaps in our fundamental understanding of microbial interactions and ecology. The following research aims to explore the interplay between cultivation methods, nutrient availability, community composition, lipid metabolism, and system ecology and identify cost effective concepts for algal lipid production. Using alkalinity to limit microbial colonization of an open system is investigated in Chapter 2 in which a monoculture of Chlorella vulgaris was successfully cultivated. A putative relationship with a Pseudomonas sp. was identified in which the exchange of key metabolites could have enhanced algal growth and limited contamination. Such interactions may minimize the need for pesticides and fertilizer subsequently reducing pollution and operating costs. Findings suggested that potentially beneficial algal-bacterial relationships occurring in alkaline conditions supported a productive and stable monoculture. Alkalinity, in addition to nutrient abundance, is further explored in a natural freshwater terminal lake system, presented in Chapter 3. Lake eutrophication coupled with temperature increases led to a toxic cyanobacterial bloom that reduced overall eukaryotic diversity. Insight gained on the interplay between alkalinity, nutrients, and community dynamics from this natural system was then applied to a series of artificial wastewater lagoons Chapter 4. Elevated lipid (g/L) was observed in this system partially facilitated by increased water residence time in the lagoons and elevated nitrogen availability. Differing alga community composition were observed during periods of elevated lipid in addition to higher biomass (cells/mL) suggesting that higher lipid volumes were the result of high biomass concentration and not necessarily the lipid productivity of specific alga taxa. The research presented utilizes traditional ecologic concepts like diversity and contributes to a more comprehensive understanding of community interactions helping to minimize cost, reduce pollution, and ultimately contribute to the realization of viable biodiesel.

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