Title: Carbonaceous Resource Recovery Through Flexible Engineered Biological Systems and Platforms - Process Engineering and Systems Biology
Description: This dissertation research represents efforts towards development of organic waste fueled bio-refineries, capable of achieving circular economy and resource recovery through conversion of the embedded chemical energy and nutrients present in the organic waste, into biochemical products of commercial value. The specific objectives were: 1. Evaluation of anaerobic fermentation derived volatile fatty acid as a recovery platform: Anaerobic fermentation of organic wastes is a cost and energy efficient way of recovering the inherent chemical energy potential associated with such waste streams. Additionally, since the carbon is recovered in the form of short chain volatile fatty acids (VFA), the high solubility in water results in fairly easy recovery and handling than the gas phase endpoints (methane, hydrogen). It also opens up the possibility to biologically or chemically redirect those VFA to high-value endpoints. Anaerobic fermentation of such streams to recover VFA also results in reducing the organic strength of the waste streams, thereby achieving waste treatment and sanitation in a far more sustainable manner than the existing practices of the organic waste management and wastewater treatment, which were inherently designed with the goal of removal of contaminants and are focused upon sequential removal of those undesirable ‘contaminants’ including carbon (energy), nitrogen and phosphorus.

Therefore, the focus of this review was to evaluate the feasibility of anaerobic fermentation derived volatile fatty acids as a substrate for potential bio-conversion into products of high commercial value. The key advantage of such a pipeline would be to intrinsically couple applications such as sanitation or wastewater treatment with resource recovery in an energetically and economically neutral or positive manner. The pipeline consists of two major steps, namely (i) Production and recovery of carbon in the form of volatile fatty acids through anaerobic fermentation and, (ii) Biological or chemical conversion of VFA to other endpoints including but not limited to biofuels, bio-plastics, butanol, organic acids and solvents and dihydrogen. 2. Microbial conversion of synthetic and food waste-derived volatile fatty acids to lipids: Lipid accumulation in the oleaginous yeast Cryptococcus albidus was evaluated using mixtures of volatile fatty acids (VFA) as substrates. In general, batch growth under nitrogen limitation led to higher lipid accumulation using synthetic VFA.

During batch growth, an initial COD:N ratio of 25:1 mg COD:mg-N led to maximum intracellular lipid accumulation (28.33±0.74% g/g dry cell weight), which is the maximum reported for C. albidus using VFA as the carbon source, without compromising growth kinetics. At this feed COD:N ratio, chemostat cultures fed with synthetic VFA yielded statistically similar intracellular lipid content as batch cultures (29.88±1.92%, g/g). However, batch cultures fed with VFA produced from the fermentation of food waste, yielded a lower lipid content (14.99±0.06%, g/g). The lipid composition obtained with synthetic and food-waste-derived VFA was similar to commercial biodiesel feedstock. We therefore demonstrate the feasibility of linking biochemical waste treatment and biofuel production using VFA as key intermediates. 3. Genome sequencing of oleaginous yeast Cryptococcus albidus and evaluation of its genetic and biotechnological potential: We reported the complete draft genome sequence of Cryptococcus albidus var. albidus, an oleaginous yeast, which can utilize various organic carbon sources for lipid synthesis.

The basidiomycetous oleaginous yeast has been gaining popularity as a non-conventional yeast with the ability to metabolize and transform diverse organic substrates. The 24.8 Mb genome of C. albidus was sequenced and the metabolic reconstruction revealed that C. albidus contains several essential pathways for metabolism of various carbon sources (including glucose, sucrose, glycerol, acetic, propionic and butyric acids), accumulation of carbon compounds (tri-acyl glycerol (TAGs) and glycogen) and for assimilation of various nitrogen (ammonia, nitrate, nitrite, and urea) and sulfur sources (sulfate, sulfite, thiosulfate). It is also capable of secreting enzymes of industrial significance. Here, we presented a comprehensive overview of the biology and biotechnology of C. albidus, specifically focusing on its microbial physiology, metabolic pathways and its potential for production of commercially and industrially important chemicals. 4. Evaluation of the global transcriptomic and proteomic responses of the Cryptococcus albidus to nitrogen limitation: ‘Non-ideal’ carbon sources could be an ideal substrate for economically feasible lipid production by oleaginous yeast Cryptococcus albidus; however, there have been no studies thus far, on biochemical pathways governing its oleaginity and metabolism.

Here, we report for the first time, a comprehensive account of the transcriptome and proteome level changes in continuous cultures of Cryptococcus albidus in response to nitrogen limitation. Proteome and differential gene expression data revealed a tight co-regulation of nitrogen and carbon metabolism, wherein nitrogen limitation resulted in a complete redistribution of carbon flux throughout the cellular processes, including nitrogenous compound recycling, autophagy and cessation of nucleic acid and ribosome biosynthesis. Lipid accumulation by C. albidus does not seem to involve transcriptional regulation but is a passive consequence of carbon flux redistribution during nitrogen limitation. This study therefore, provides a valuable resource to understand oleaginity and metabolism of alternate carbon sources by C. albidus and provides opportunities for metabolic re-engineering of its lipid production pathways. 5. Organic waste fueled biorefineries: future perspectives on production of chemicals of industrial significance from volatile fatty acids: The focus of this review was to propose alternate bio-based pipelines for recovery and conversion of organic waste streams into high value commercial products, using VFA as central precursors for further aerobic/anaerobic carbon cycling.

Herein, we present various pathways, microorganisms, culture conditions and current status of bio-based production of certain building-block chemicals such as adipic acid, butanol, organic acids such as citric, malic and succinic acids. These chemicals have the highest potential to be economically produced from VFA since the pathways for their bioconversion either exist natively or have been metabolically engineered. Nevertheless further research would be needed to reduce the costs and enhance productivity. In conclusion, this dissertation represents the first attempt at a holistic evaluation of a VFA based resource recovery platform. In first phase, microbial conversion of volatile fatty acids into lipids by the yeast C. albidus was evaluated and nitrogen limitation was identified as the inducer of lipogenesis in C. albidus through operation of batch and chemostat cultures. Next, entire genome was sequenced and transcriptome and proteome level changes were evaluated in conjunction to understand the metabolic basis of nitrogen-mediated oleaginity in C. albidus.

This genomic and the comparative transcriptome and proteome data is expected to help further elucidate factors driving lipid accumulation in C. albidus and contribute toward bioprocess development and optimization for engineered lipid production from ‘waste’ streams. Finally, the feasibility of microbial conversion of VFA into several other bio-based chemicals of commercial value was also evaluated.