This EAGER research is to (1) identify proteins statistically relevant to the feast-famine process for microbial synthesis of polyhydroxyalkanoates (PHA) and (2) integrate proteomics and statistics to develop a preliminary understanding of feast-famine PHA synthesis. These are first steps in a vision to create microbial processes for producing plastics through fermentation from wastes such as manure and wastewater.
The purpose of this research was to develop advanced molecular-level information on a bacterial process for producing biological, biodegradable plastics (known as polyhydroxyalkanoates, or PHA) from fermented dairy manure. Bacteria can store different forms of organic carbon (in this case they were fed principally acetic acid, which is the primary ingredient in vinegar) inside their cell. The so-called PHA represents a carbon and energy storage reserve for bacteria. However, when recovered from the bacterial cell the intracellular granules are a form of thermoplastic (not unlike polypropylene or polyethylene). Our interest lies in ultimately commercially producing PHA using wild bacteria fed acetic acid produced from fermenting organic rich waste. In this project we completed the following: - We isolated and cultured bacteria capable of PHA synthesis from a mixed microbial consortium. To the best of our knowledge, this is the first time anyone has accomplished this task. - We identified the isolated bacterium as Acinetobacter spp., which are known PHA producers. - We confirmed the presence of intracellular PHA granules visually using fluorescent microscopy. - Analysis of the Acinetobacter isolate confirmed PHA synthesis, with a maximum intracellular PHA concentration of 43% (w/w). Distinct differences in protein abundance were observed over the course of PHA synthesis compared to the PHA degradation. Higher molecular weight proteins were observed in the later stages of the feast period. Multiple protein signals were observed in the non-cytoplasmic fraction of the lysates, which may represent important membrane-bound proteins and possibly a class of proteins known as phasins, which are key regulators in PHA synthesis. - Proteins were recovered from PHA granules for analysis. Proteins were detected with molecular weights ranging from 11 kDa to 45 kDa (identification of these proteins is pending). The research funding was used to support a PhD student in biochemistry as well as a number of undergraduate students in civil engineering. The graduate student and undergraduate students, along with the PI, gained valuable experience in performing proteomic studies on mixed microbial consortia cultured in complex wastewater environments. Very little research has been conducted to apply advanced molecular techniques on undefined microbial consortia being utilized for biotechnological purposes. Furthermore, minimal research has been conducted on the PHA synthesis meta-metabolism (to the best of our knowledge we are the only research team in the U.S. conducting such research - we have networked with researchers in Portugal, Netherlands, and Italy who have conducted similar work), and the protocols were originally developed for use in pure cultures. Thus, we have had to overcome many challenges in successfully applying these advanced molecular techniques on such complex matrices. Our preliminary results are very encouraging. Historically environmental engineers have studied microbiome systems principally as a "black box," extracting gross process performance parameters based on extrapolations of bulk solution process indicators. While such an approach has been very successful, for all intents and purposes we have fully mined that approach. The research conducted on this project takes the next big leap to apply advanced molecular techniques on complex bacterial microbiomes for the purposes of enhancing a fundamental understanding on structure/function related to a specific biotechnological process (PHA production). The research is broadly contributing to both the molecular/microbiology and environmental engineering disciplines. To date such interdisciplinary approaches have only been very minimally applied, largely due to the intrinsic complexities and challenges associated with applying pure-culture based techniques to highly complex systems. However, the interdisciplinary approach has great potential to yield significant advances in sustainable technologies that leverage the myriad organic wastes generated by developed societies. We are advancing a blueprint for such integrated approaches while concurrently advancing a high value biotechnology process.
