NON-TECHNICAL SUMMARY: This proposal advances research to address a critical challenge in the dairy industry - environmentally and economically sustainable management of dairy manure. Over 9 million dairy cows generate >226 billion kg/yr of wet manure in the U.S. Not only is the valuable source of carbon insufficiently recovered, nitrogen and phosphorus discharges are a challenge. The dairy industry is in need of engineered systems that provide opportunities to 'pivot' from a legacy waste management approach to solutions that concurrently achieve economic and environmental resilience. This proposal focuses on an engineered suite of processes to produce commercially viable products (bio-plastics, bio-power, bio-oil, and organic fertilizer) from dairy manure. Our technology ferments dairy manure to produce a volatile fatty acid-rich effluent, which is recovered to produce PHBV (a biodegradable bioplastic). Residual solids are digested to CH4-rich biogas for electricity production or vehicle fuel. Nutrients are upcycled to algal biomass, which is treated via hydrothermal liquefaction (HTL) to yield a bio-oil and a nutrient-rich aqueous fraction; alternately, algae is recovered as a fertilizer or as animal feed. HTL nutrients will be captured via struvite production or recycled to enhance algae production.
OBJECTIVES: Objective 1 - Establish Requirements to Generate Commercial Quantities of PHBV.Task 1.1 - Establish Engineering Operational Criteria to Maximize PHBV Synthesis. Research will focus on optimizing engineered operational parameters - OLR (15-30 CmmolVFAs/L-d) and SRT (2-4 days) - in the Enrichment reactor to maximize conversion of VFAs to PHBV in the Production reactor. ER investigations will be conducted through a factorial approach, designed based on Response Surface Methodology and constructed as a rotatable 2nd order central composite design (CCD). As necessary, results will be refined using a second factorial. Carbon will be measured as soluble COD  and as individual organic acids by GC/FID . PHBV will be quantified as methyl ester derivatives by gas chromatography/mass spectrometry (GC/MS) [83, 90]. N, P, and biomass concentrations will be analyzed per Standard Methods . For each ER scenario, biomass will be evaluated to determine the ability to synthesize higher PHBV concentrations. Production reactors will be operated consistent with our preliminary data [7, 32]. In addition, VFA-rich fermenter liquor will be augmented with VFA-rich sugar beet processing wastewater; our preliminary investigations indicate that this alternate byproduct stream, which is VFA-rich and nutrient poor, is an ideal co-substrate for enhanced PHBV production.Task 1.2 - Interrogate PHBV "Feast" Metabolome. The "feast" PHBV metabolism will be investigated and characterized at a metabolic level to develop a functional description of the process that has, to-date, been elusive [34, 60]. The metabolome will be interrogated through GC/MS and LC/MS methods consistent with Pan et al. . VFA and PHBV concentrations will be measured at the same frequency as the metabolome characterization.Objective 2 - Enhance PHBV for Targeted Applications.Task 2.1 - Establish Optimal PHBV Recovery and Purification Method. Concentrated PHBV-rich biomass will be dried and sequentially extracted with petroleum ether (to remove lipids) and chloroform (to extract PHBV). The solvent criteria are extraction efficiency, cost, and ease of recovery. A large Soxhlet extractor (0.5-kg biomass capacity) has been constructed for this work. Solvents will be recovered and reused.Task 2.2 - Enhance PHBV through Cross-Linking by Reactive Extrusion. The process-ability and structural properties of PHBV will be modified by free radical cross-linking using DCP via reactive extrusion using "green chemistry" methodology [100, 101]. Small-scale trials will be performed using a mixing extruder/molder; DCP will be mixed with PHBV and then injected into a mold or extruded into a strand for characterization . Once suitable conditions are chosen, these will be applied in sheet extrusion trials using a Leistritz twin-screw extruder with a sheet die and 3 roll-calendaring unit . Process conditions will be adjusted to obtain quality samples. Raw materials will be pre-blended and metered via weight loss feeders into the extruder. Specimens will be cut from the PHBV sheet for characterization.Task 2.3 - PHBV Material Characterization. Under this task we will establish the link between PHBV structure, cross-linked PHBV characteristics, and material properties. PHBV tensile and three-point flexural properties will be determined on injection molded, cast film, or extruded sheet specimens [103, 104]. Polymer crystalline morphology will be analyzed on CHCl3 cast films . Thermal properties (Tm and Tg) will be determined on samples by differential scanning calorimetry (DSC) [12, 105-107]. Dynamic mechanical analysis (DMA) will be performed on sheets to obtain thermal (Tm and Tg) and viscoelastic properties [103, 108]. PHBV thermal stability will be determined by thermogravimetric analysis (TGA) . Steady shear and dynamic rheological measurements will be made to determine melt strength/viscosity, of the random, co-block, and cross-linked PHBV .Objective 3 - Integrate and Optimize an Algal Reactor System for Production of High-Value Commodities from PHBV Reactor Effluent Nutrients while Minimizing or Removing Exogenous Process Water Requirements.Task 3.1 - Develop continuous operation conditions for the algal production system. Experiments will be performed using the algal cultivation systems currently housed in the BSU greenhouses. The algae cultivation system will be inoculated with a naturally occurring polyculture of green algae isolated from the Boise River. Each algal cultivation system will be modified to support both suspended and attached growth on a nylon mesh installed in the algal cultivation systems. Algae biomass will be regularly harvested. pH, periodicity of pH control, effluent concentrations and amount of HTL aqueous phase nutrients, and retention times will be modulated in a sequential factorial design to measure effects on biomass production, growth rates, and nutrient capture. Process rates associated with nutrient capture will be measured daily, and then weekly as parameters reach steady state by measuring dissolved concentrations in the raceways. Biomass growth rates will be determined. Standard ash free dry weight measures will be employed to quantify algal production (see ). We will characterize how the N/P removal changes with fluctuating PHBV effluent quality (i.e., optical characteristics, N/P content, bacterial loads, etc.). Suspended algal biomass nutrient content will be assessed by capturing the biomass via filtration (2µm filter). Filtered and attached biomass will be lyophilized and N, P, K, and other trace element content determined by ICP-MS. Dissolved nutrients in the PHBV effluent and primary stage algal reactor will be monitored by filtering liquid samples (0.45µm filter), followed by ion chromatography analysis for NH4+, NO3-, PO42-. Total N will be determined using Hach Method 8075.We will utilize NH4+ from the HTL aqueous stream to introduce periodic low?intensity NH3 treatment within the primary stage as a means to reduce grazing pressure and limit competition by non-target algae and heterotrophic prokaryotes. The precise duration of pH elevation and/or cycle times will be varied to maximize effects on grazer densities and minimize effects on algal production and nutrient capture. Grazer responses will be determined by direct microscopic counts using a Sedgwick-Rafter chamber.Task 3.2 - HTL for bio-crude production and nutrient recovery process. Hydrothermal liquefaction will be assessed as a potential technology to produce a bio-oil from the algal biomass, while concurrently yielding a nutrient stream of high fertilizer value. The concentrated algae will be processed by HTL in a Parr pressure reactor under nominal conditions of 300ºC (heating rate of 20ºC?min-1) for a 20-minute holding period . The product liquid phase will be collected and separated, where the lighter oil will be removed from the heavier aqueous; both will be measured gravimetrically. The oil phase will be analyzed directly and also converted to fatty acid methyl esters (biodiesel) for analysis by GC-MS and calorific value. The aqueous phase will be analyzed for chemical oxygen demand , TOC, pH, and ammonia.Objective 4 - Expand the Integrated DAIRIEES Engineering Process Model for Pre-Commercialization Evaluations and Technology Operations. We have constructed a Microsoft Excel-based techno-economic system model  for the integrated technology, referred to as Decision-support for Digester-Algae IntegRation for Improved Environmental and Economic Sustainability (DAIRIEES). Work under this Objective will integrate data from Objectives 1-3 and build on the functionality of the DAIRIEES to include assessment capabilities for pre-commercialization analysis.