Performing Department
Bacteriology
Non Technical Summary
Biobased and easily biodegradable bioplastics, such as polyhydroxyalkanoates (PHAs), are alternatives to petroleum-based plastics which are difficult to degrade in the environment. and are rapidly gaining plastic market share. PHAs hold promise for a sustainable future owing to their biological origin, biodegradability, biocompatibility, and tunable properties. PHAs biodegrade entirely into carbon dioxide and water, and the degradation rate is faster compared to other bioplastics. Despite the environmental benefits and significant interest, the commercial success of PHAs is limited, mainly due to the high production cost and scalable supply of carbon sources. Currently, most of the PHAs are produced using carbon sources derived from food-based feedstocks, which contribute significantly to the production cost (> 50%) and also compete with food production. Agro-food and forest-product industries generate several million tons of various wastes and low-value co-product streams during their operations. Traditionally, most of the solid waste streams are disposed of at landfills and liquid waste streams are directed to wastewater treatment plants. Disposal of these waste is costly to food industries, and at the same time causes potential environmental problems, with direct and indirect emissions of greenhouse gasses, and contamination of groundwater. Agro-food processing industries are increasingly looking toward diversifying their product stream through the valorization of waste materials and achieving zero waste.Our goal is to establish an economically and environmentally sustainable bioprocess that converts agriculture and forest product low-value and underutilized waste streams into polymers for the production of more elastic and biodegradable biobased bioplastics. This project would use an innovative approach by developing a mixing strategy for waste streams from the dairy industry (acid whey and whey permeate phospholipid concentrate) and paper mills (waste fines) to produce a polymer with desired properties for improved bioplastics. Acid whey is a byproduct of the production of cream cheese and Greek yogurt. Currently, more than 4 million tons of acid whey are produced in the United States. The acid whey waste is hard to dispose of and thus poses serious problems for the dairy industry and the environment. Waste fines are fragments of fibers that are produced during the processing of recycled pulps into linerboard and similar paper products. A large amount of these fibers is rejected as a solid waste stream to avoid these challenges in the paper production process. Acid whey and waste fines burden dairy and paper industries economically and environmentally but contain desirable substrates for microbial growth during the bio-production of bioplastics. Besides increasing bioplastic feedstock availability and decreasing cost, mixing these waste streams and use as co-substrates addresses individual bioconversion challenges through less neutralization chemicals, better carbon utilization efficiency, and pretreatment elimination, all while improving economic and environmental sustainability of the process. We will optimize the bioprocess by converting the mixture of acid whey and waste fines to maximize a common PHA, polyhydroxybutyrate, yield by controlling hydrolysis conditions. While there are many uses for poluhydroxybutyrate, it is brittle and not suitable for all applications. To make a more flexible bioplastic, we will incorporate lipids from underutilized dairy industry co-product Whey Permeate Phospholipid Concentrate into the bioplastic polymer by metabolically engineering our production microbe. Process simulations, techno-economic analysis, and life cycle assessments will determine economic and commercial feasibility and the potential impact of the PHA production on the environment. Accomplishing this goal will enable future work in scaling the bioplastic production process towards industrial levels and testing other unexplored agricultural wastes as innovative bioproduct substrates.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
Development of technologies to valorize agricultural and forest industrial waste streams through conversion to high value bioplastics can provide green and sustainable alternative to petroleum-based plastics and improve profitability to agricultural product and bioplastic producers. Our long-term goal aims to design a sustainable bioprocessing system utilizing agro-processing waste streams as feedstock for production of bio-based and biodegradable plastic precursors. This project is based on extensive preliminary data on the bioconversion of acid whey (AW), a major liquid waste (5-7% solids) from the dairy industry, and waste fines (WF), a solid waste from the recycled paper mills, into bioplastics which enables us to focus on engineering solutions to specific limitations we identified in the system. Our process is hindered by ratio of substrates and nutrients and in the type of biopolymer being produced. In Objective 1, we will optimize i) the mixing of AW and WF, and ii) the performance of a controlled hydrolysis combined with the fermentation to produce high yields of bioplastic poly(3-hydroxybutyrate) (PHB). In Objective 2, we will employ metabolic engineering methods to biosynthesize a novel bioplastic co-polymer by incorporating underutilized Whey Permeate Phospholipid Concentrate (WPPC), which supplies the necessary fatty acids into the process. In Objective 3, we will evaluate the commercial scale economic feasibility and environmental impact of the process through techno-economic assessment (TEA) and life cycle analysis (LCA) approaches. Besides increasing the feedstock availability for bioplastic production, this innovative approach of utilizing AW and WF as co-substrates for bioplastic production addresses several challenges associated with bioconversion of these individual streams, maximizes carbon utilization efficiency, and improves economic and environmental sustainability of the process. Altogether, the results of these efforts will give a lab scale demonstration of this sustainable bioprocess for generating two types of bioplastics from negative-cost wastes and have prepared the system to be ready for future testing at pilot scale. This approach also addresses concerns of environmental and human health, waste disposal, and the need to diversify products and markets for the forest product and dairy industries.
Project Methods
The methods in this project span bioprocess engineering, microbiology metabolic engineering and economic assessment of the process. Efforts and their respective Evaluations are herein described chronologically.Objective 1: Optimization of fermentation for maximizing PHB production from acid whey and waste fines mixThe first part of this objective involves optimizing the media composition during fermentation by investigating several important experimental factors, including the total carbon, carbon/nitrogen (C/N) ratio, and sugar/lactate ratio. The effect of parameters will be evaluated on the PHB yield, and the parameters will optimized. Initial experiments will be performed using pure sugars (glucose and lactose) and lactic acid. The nitrogen content will be controlled by adding bovine serum albumin (BSA). The fermentation will be performed for 48 h using recombinant E. coli. The microbial biomass (containing PHB) will be harvested at the end of fermentation and the cell yield will be determined using gravimetric methods. The PHB content of the cell biomass will be measured using GC-FID (gas chromatograph equipped with a flame ionization detector) using standard protocols. The second part of this objective focuses on producing desired media through controlled hydrolysis of acid whey (AW) and waste fines (FW) mix. The ratio of sugars to lactate in the solution will be controlled by adjusting the mixture of acid whey and waste fines at the start of hydrolysis. Various enzyme dosages will also be investigated to evaluate their impact on the carbohydrate conversion during hydrolysis. The next part involves PHB production using this hydrolysate. The media composition will be adjusted to achieve similar PHB yields as that with the fermentation of pure sugars and lactic acid. In the last part, the scale-up hydrolysis and fermentation experiments (6-L working volume) will be performed under the optimum conditions, and the results will be used to perform techno-economic analysis and further used for the life cycle analysis (Objective 3).Objective 2: Metabolic engineering of PHA co-polymer production with the addition of WPPCTo produce a co-polymer with more elastic properties than PHB from our mixed waste stream, we will add WPPC as a source of long-chain fatty acids in test flasks with our targeted bioplastic-producing microbes. We will perform growth and co-polymer production batch trials by inoculating the five targeted microbes into flasks containing at least three different WPPC to AW-PH ratios (0:100, 50:50, 100:0) held at 10 g/L sugar content, which promoted higher PHB yields in our preliminary data. After the cultures reach the stationary phase, the cells will be harvested, and biopolymers extracted and analyzed as described in Objective 1. To determine if a true co-polymer with desired properties has been biosynthesized compared to simultaneous homopolymer strands, we will perform chromatographic separation and collect fractions before identification by GCMS and NMR and other polymer analytical methods such as Elongation at Break. For our metabolic engineering strategy of our top PHA co-polymer-producing microbe, we will first determine which reaction in the biosynthesis pathway is the most limiting with our established PHA biosynthesis qPCR assay. Upregulation of gene expression during PHA co-polymer biosynthesis compared to non-polymer producing conditions will indicate which genes are involved in generating this co-polymer. We will generate a Flux balance Analysis Model (FBA) in KBase to predict which reactions are limiting. We will manually add any PHA biosynthesis genes or modifications. In the FBA model, we will run simulations varying substrates and nutrient loading, specifically carbon and nitrogen. Biosynthesis bottleneck reactions are identified by a lack of final PHA product and accumulation of initial substrate or intermediates. We then examine the responsible transporter or enzyme for that reaction. Based on the enzyme parameters listed in the BRENDA database, we can predict what is causing the inhibition or decreased turnover of limiting enzyme in specific AW-WF-WPPC process condition. We will then verify this limitation prediction by growing the microbe in that condition and perform our established enzyme activity or uptake assays. The magnitude of the polymer yield and change in enzyme activity should match the FBA model prediction. This will pinpoint which reaction and which protein contributes the most to the limitation of PHA co-polymer biosynthesis.Objective 3: Techno-economic analysis and life cycle assessment of the proposed processComprehensive process models will be developed for the complete process including hydrolysis, fermentation, PHB recovery, and other downstream operations, using SuperPro Designer (Intelligen, Inc., NJ, USA). The experimental results from objectives 1 and 2 will be used as inputs for the model simulations. The outcomes from the model simulations will include PHB and co-polymer yields, total capital investment, total operational cost and its breakdown, total energy consumption, and bioplastic production cost. The profitability will be estimated assuming a selling price of bioplastics, and results will be compared with alternate technologies. A sensitivity analysis will be performed by varying the purchase price of feedstocks to evaluate the potential gain to the agro-processing industries. A cradle-to-gate life cycle assessment model will be developed to quantify the environmental impact of the process. This assessment will follow the framework defined by the International Standardization Organization that includes goal and scope definition, inventory analysis, impact assessment, and results interpretation. The mass and energy balance data generated from process simulations and the existing life cycle inventory database (e.g., USLCI, Ecoinvent) will be used to conduct the analysis. The LCA results will be interpreted in relation to the double environmental benefits of biobased PHB: 1) utilization of a negative wastes stream that would otherwise create an environmental problem, and 2) substitution of fossil-based materials.