Investigating the Molecular Mechanisms Associated With Feast-Famine PHA Synthesis by Mixed Microbial Consortia View Homepage


Ontology type: schema:MonetaryGrant     


Grant Info

YEARS

2012-2015

FUNDING AMOUNT

126000 USD

ABSTRACT

More than 300 bacterial species synthesize PHAs as intracellular carbon and energy storage granules in response to starvation. The study conveniently can exploit this feast-famine PHA synthesis response to produce commercial quantities of PHAs while simultaneously treating dairy manure in an environmentally benign manner. To advance this technology, the study must first expand the fundamental knowledge of the metabolic steps involved with feast-famine PHA production by bacterial consortia. The primary objective of this research is to identify the key proteins responsible for feast-famine PHA synthesis in a bacterial consortium when using fermented dairy manure as a substrate. With this molecular information, the critical metabolisms associated with feast-famine PHA synthesis will be further defined and translated into engineering models that will help steer the large-scale production of this biodegradable plastic. Mass spectrometry-based proteomic techniques will be utilized to determine the proteins responsible for the observed biochemical transformations occurring during feast-famine PHA synthesis. Because the bacterial consortium exhibits two distinct physiological states during feast-famine PHA synthesis, two-dimensional gel electrophoresis will be employed to separate proteins and visualize differences in protein abundance. Proteins of interest will be identified using nano-liquid chromatography coupled to tandem mass spectrometry. Through a statistical analysis of differential protein abundance, the critical metabolisms associated with feast-famine PHA synthesis will be characterized. This research also will investigate the proteins that are bound to the surface of PHA granules, as these proteins have been shown to play key roles in PHA synthesis. Finally, this research will begin to more carefully characterize how the global dynamic of a bacterial consortium changes during feast-famine PHA synthesis by integrating the proteomic data with other molecular investigations. Knowing the identity and relative expression of relevant proteins in a bacterial consortium performing feast-famine PHA synthesis will allow the reconstruction of the metabolic pathways that predominate the process. Detailed metabolic information will further aid in refining the stoichiometry of feast-famine PHA synthesis and thus metabolic flux analysis, thereby improving engineering models for large-scale production. Furthermore, information on metabolic intermediates can be leveraged to develop process monitoring techniques and aid model calibration. Lastly, understanding the expression of proteins relative to bulk solution parameters and consortium history could allow for the expansion of current engineering models from simple substrate/product-inhibition kinetics to more global forms of metabolic regulation.Potential to Further Environmental/Human Health ProtectionAdvancing this technology would provide not only a sustainable strategy for the effective management of dairy manure, but also introduce more biodegradable alternatives to conventional petroleum-based plastics. Treating dairy manure in this manner would capture the nutrients from this resource to make valuable products while minimizing the human health and environmental hazards associated with inadequate manure disposal practices. Considering the abundance of organic-rich waste streams generated nationally, results from this research will help facilitate expanding and applying PHA production technologies at a larger scale. This, in return, would help decrease the amount of plastic waste accumulation in the environment, as well as lessen the dependence on fossil fuels for plastic manufacturing. Mass spectrometry-based proteomic techniques will be utilized to determine the proteins responsible for the observed biochemical transformations occurring during feast-famine PHA synthesis. Because the bacterial consortium exhibits two distinct physiological states during feast-famine PHA synthesis, two-dimensional gel electrophoresis will be employed to separate proteins and visualize differences in protein abundance. Proteins of interest will be identified using nano-liquid chromatography coupled to tandem mass spectrometry. Through a statistical analysis of differential protein abundance, the critical metabolisms associated with feast-famine PHA synthesis will be characterized. This research also will investigate the proteins that are bound to the surface of PHA granules, as these proteins have been shown to play key roles in PHA synthesis. Finally, this research will begin to more carefully characterize how the global dynamic of a bacterial consortium changes during feast-famine PHA synthesis by integrating the proteomic data with other molecular investigations. Knowing the identity and relative expression of relevant proteins in a bacterial consortium performing feast-famine PHA synthesis will allow the reconstruction of the metabolic pathways that predominate the process. Detailed metabolic information will further aid in refining the stoichiometry of feast-famine PHA synthesis and thus metabolic flux analysis, thereby improving engineering models for large-scale production. Furthermore, information on metabolic intermediates can be leveraged to develop process monitoring techniques and aid model calibration. Lastly, understanding the expression of proteins relative to bulk solution parameters and consortium history could allow for the expansion of current engineering models from simple substrate/product-inhibition kinetics to more global forms of metabolic regulation.Potential to Further Environmental/Human Health ProtectionAdvancing this technology would provide not only a sustainable strategy for the effective management of dairy manure, but also introduce more biodegradable alternatives to conventional petroleum-based plastics. Treating dairy manure in this manner would capture the nutrients from this resource to make valuable products while minimizing the human health and environmental hazards associated with inadequate manure disposal practices. Considering the abundance of organic-rich waste streams generated nationally, results from this research will help facilitate expanding and applying PHA production technologies at a larger scale. This, in return, would help decrease the amount of plastic waste accumulation in the environment, as well as lessen the dependence on fossil fuels for plastic manufacturing. Knowing the identity and relative expression of relevant proteins in a bacterial consortium performing feast-famine PHA synthesis will allow the reconstruction of the metabolic pathways that predominate the process. Detailed metabolic information will further aid in refining the stoichiometry of feast-famine PHA synthesis and thus metabolic flux analysis, thereby improving engineering models for large-scale production. Furthermore, information on metabolic intermediates can be leveraged to develop process monitoring techniques and aid model calibration. Lastly, understanding the expression of proteins relative to bulk solution parameters and consortium history could allow for the expansion of current engineering models from simple substrate/product-inhibition kinetics to more global forms of metabolic regulation.Potential to Further Environmental/Human Health ProtectionAdvancing this technology would provide not only a sustainable strategy for the effective management of dairy manure, but also introduce more biodegradable alternatives to conventional petroleum-based plastics. Treating dairy manure in this manner would capture the nutrients from this resource to make valuable products while minimizing the human health and environmental hazards associated with inadequate manure disposal practices. Considering the abundance of organic-rich waste streams generated nationally, results from this research will help facilitate expanding and applying PHA production technologies at a larger scale. This, in return, would help decrease the amount of plastic waste accumulation in the environment, as well as lessen the dependence on fossil fuels for plastic manufacturing. More... »

URL

http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/9936/report/0

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Mass spectrometry-based proteomic techniques will be utilized to determine the proteins responsible for the observed biochemical transformations occurring during feast-famine PHA synthesis. Because the bacterial consortium exhibits two distinct physiological states during feast-famine PHA synthesis, two-dimensional gel electrophoresis will be employed to separate proteins and visualize differences in protein abundance. Proteins of interest will be identified using nano-liquid chromatography coupled to tandem mass spectrometry. Through a statistical analysis of differential protein abundance, the critical metabolisms associated with feast-famine PHA synthesis will be characterized. This research also will investigate the proteins that are bound to the surface of PHA granules, as these proteins have been shown to play key roles in PHA synthesis. Finally, this research will begin to more carefully characterize how the global dynamic of a bacterial consortium changes during feast-famine PHA synthesis by integrating the proteomic data with other molecular investigations. Knowing the identity and relative expression of relevant proteins in a bacterial consortium performing feast-famine PHA synthesis will allow the reconstruction of the metabolic pathways that predominate the process. Detailed metabolic information will further aid in refining the stoichiometry of feast-famine PHA synthesis and thus metabolic flux analysis, thereby improving engineering models for large-scale production. Furthermore, information on metabolic intermediates can be leveraged to develop process monitoring techniques and aid model calibration. Lastly, understanding the expression of proteins relative to bulk solution parameters and consortium history could allow for the expansion of current engineering models from simple substrate/product-inhibition kinetics to more global forms of metabolic regulation.Potential to Further Environmental/Human Health ProtectionAdvancing this technology would provide not only a sustainable strategy for the effective management of dairy manure, but also introduce more biodegradable alternatives to conventional petroleum-based plastics. Treating dairy manure in this manner would capture the nutrients from this resource to make valuable products while minimizing the human health and environmental hazards associated with inadequate manure disposal practices. Considering the abundance of organic-rich waste streams generated nationally, results from this research will help facilitate expanding and applying PHA production technologies at a larger scale. This, in return, would help decrease the amount of plastic waste accumulation in the environment, as well as lessen the dependence on fossil fuels for plastic manufacturing. Mass spectrometry-based proteomic techniques will be utilized to determine the proteins responsible for the observed biochemical transformations occurring during feast-famine PHA synthesis. Because the bacterial consortium exhibits two distinct physiological states during feast-famine PHA synthesis, two-dimensional gel electrophoresis will be employed to separate proteins and visualize differences in protein abundance. Proteins of interest will be identified using nano-liquid chromatography coupled to tandem mass spectrometry. Through a statistical analysis of differential protein abundance, the critical metabolisms associated with feast-famine PHA synthesis will be characterized. This research also will investigate the proteins that are bound to the surface of PHA granules, as these proteins have been shown to play key roles in PHA synthesis. Finally, this research will begin to more carefully characterize how the global dynamic of a bacterial consortium changes during feast-famine PHA synthesis by integrating the proteomic data with other molecular investigations. Knowing the identity and relative expression of relevant proteins in a bacterial consortium performing feast-famine PHA synthesis will allow the reconstruction of the metabolic pathways that predominate the process. Detailed metabolic information will further aid in refining the stoichiometry of feast-famine PHA synthesis and thus metabolic flux analysis, thereby improving engineering models for large-scale production. Furthermore, information on metabolic intermediates can be leveraged to develop process monitoring techniques and aid model calibration. Lastly, understanding the expression of proteins relative to bulk solution parameters and consortium history could allow for the expansion of current engineering models from simple substrate/product-inhibition kinetics to more global forms of metabolic regulation.Potential to Further Environmental/Human Health ProtectionAdvancing this technology would provide not only a sustainable strategy for the effective management of dairy manure, but also introduce more biodegradable alternatives to conventional petroleum-based plastics. Treating dairy manure in this manner would capture the nutrients from this resource to make valuable products while minimizing the human health and environmental hazards associated with inadequate manure disposal practices. Considering the abundance of organic-rich waste streams generated nationally, results from this research will help facilitate expanding and applying PHA production technologies at a larger scale. 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This, in return, would help decrease the amount of plastic waste accumulation in the environment, as well as lessen the dependence on fossil fuels for plastic manufacturing.
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