Recipient Organization
SUSTAINABLE BIOPRODUCTS LLC
3132 HILLCREST DR
BOZEMAN,MT 597150689
Performing Department
(N/A)
Non Technical Summary
Acid-whey, a pollutant generated during the production of Greek yogurt, has become a significant environmental problem due to rapid increase in demand for yogurt, with U.S. alone producing more than 100 million tons annually. Current methods of disposing growing quantities of acid-whey are either inadequate or cost prohibitive, consequently, alternative methods are actively being sought. As human population increases and demand for higher-quality foods grows, the need to supply greater amounts of nutritional foods is rising. Current food-production, cannot sustain the increasing demand, thus, alternative high-protein food-sources are being investigated. Sustainable Bioproducts has developed a highly-efficient and cost-effective method for production of high-protein-biomass with an exceptional nutritional profile directly from acid-whey with little need of pre-processing. The novel methodology is based on surface fermentation under low pH conditions in combination with an extremophilic fungus (MK7). Preliminary Studies demonstrated that MK7 thrives on untreated, undiluted acid-whey. Additionally, MK7 consistently formed robust biomats when grown under these conditions. Our initial results showed that biomat growth on acid-whey can remove nearly all carbon from the liquid and rapidly neutralize pH.The proposal's objectives fall within Program-Area-Priority e. Agriculture Systems and Technology, 2. Bioprocessing and Bioengineering.We propose to:• Optimize acid-whey conversion into high protein biomass using our production-platform and use this biomass to determine optimal conditions for high-quality-food production.• Improve our products' value by testing methods for separating biomass into various components and analyze their properties for food/industrial applications.• Genetically engineer strains of MK7 for improved nutritional value of biomass grown on acid-whey
Animal Health Component
45%
Research Effort Categories
Basic
10%
Applied
45%
Developmental
45%
Goals / Objectives
The goal of this proposed research is to develop our process for the production of high-protein biomat and products suitable for human food, food supplements, animal feed, and as ingredients for subsequent food and feed preparation.Project Supporting ObjectivesObjective 1: Optimize the conversion of acid whey into high protein biomats using SB's production platform and determine nutritional composition of the produced biomats.Objective 2: Evaluate and improve methods for separating biomass into valuable nutritional components (protein, oils, flavorings, beta-glucan, etc.).Objective 3: Improve nutritional value of the produced biomass by developing genetically engineered strains of strain MK7 targeting high-value compounds desirable for food, food ingredients and food supplements.Objective 4: Techno-economic analysis of producing high-protein biomass and food components from acid whey using the SB platform.HypothesesHypothesis 1: The SB platform can be used to convert nearly all of the carbon in acid whey into valuable food and food ingredients.Hypothesis 2: Strain MK7 biomats produced from acid whey can be fractionated into valuable components, thus, adding value to the process.Hypothesis 3: The production of valuable food ingredients can be controlled by adjusting culture conditions and acid whey media composition.Hypothesis 4: Genetic engineering of strain MK7 can improve its nutritional properties, and thus, add value to the process.
Project Methods
Task 1: Process Optimization.Experiments to optimize acid whey conversion to biomats will initially focus on testing different sources of acid whey using SB bioreactor systems. Additional experiments will optimize pretreatment method, pH, macro and micronutrient nutrient composition, temperature, air flow rates and humidity.Subtask 1.1: Analysis of acid whey feedstocks, Months 0-2. Acid whey batches will be obtained from Dannon Greek yogurt manufacturing facilities located throughout the U.S. and the composition of macro and micro nutrients as well as carbohydrate, protein and lipid profiles will be thoroughly analyzed by Eurofins and by SB.Subtask 1.2: Biomass production as a function of acid whey source, pretreatment and pH, Months 0-8. Growth experiments using the suite of acid whey feedstocks obtained in Subtask 1.1 will be conducted with SB's production platform. Standard operating procedures developed for producing biomats from crude glycerol will be applied. Briefly, inoculum will be grown under submerged fermentation conditions in 10 L aerated reactors using a salts medium designed specifically for strain MK7. Cultures in the late exponential phase will be used as inoculum for acid whey experiments in tray reactors. Optimization parameters in this experimental matrix will include: i) volumes of inoculum, ii) acid whey will be used directly, or pretreated by autoclaving and iii) pH. Cultureswill be added to trays and allowed to incubate for 4-7 days. Conversion efficiencies and rates of biomat growth will be determined by measuring dry weights over time. Detailed compositional analysis of the produced biomats as well as the liquid remaining in the trays after harvesting the biomass will be performed at the SB laboratoryand by Center for Biorenewable Chemicals, Eurofins and IMPROVe.Subtask 1.3: Nutrients, Months 6-12. Nutrient optimization experiments will be conductedinclude for N, P, K, Ca, Mg, S and Fe.Subtask 1.4: pH, temperature, air flow and humidity, Months 12-18. Other process parameters (temperature, air flow, humidity) will be evaluated under optimized nutrient concentrations. Process parameters will be tested using SB's controlled environmental chamber specifically built to evaluate the effects of temperature, air flow, evaporation rate and humidity on biomat growth.Task 2: Fractionation of Biomass.Methods will be developed to (A) enhance separation into two general fractions concentrated in (1) nucleotides and carbohydrates and (2) proteins and beta-glucan, (B) increase concentration and separation of proteins, beta-glucans and nucleotides by additional fractionation steps.Subtask 2.1: Cell disruption. Months 0-3. Several techniques for cell wall lysis will be evaluated. This will include freezing biomass at different rates and temperatures (e.g. slow freeze at -20 °C versus -80 °C) and different thaw temperatures (e.g. 30-75 °C) and incubation times.Subtask 2.2: Separation of biomass into various fractions. Months 0-30. (Fig. 6; Steps 3-7). A variety of separation/fractionation methods will also be evaluated. Filtering or blending the biomass after initial freeze/thaw cycle will be evaluated. Initially, a simple liquid extract/cell wall pellet fraction will be obtained after either pressing biomass after heat step or after blending and settling, centrifuging or filtering using a course filter (0.25-0.5 mm). Various pressing techniques will be evaluated. Liquid extract will be dried at 50 °C or lyophilized for analysis.The solid pellet fractionwill be dried at 50 °C or lyophilized and subjected to a 2:1 chloroform/hot methanol extraction following the Folch method. The polar fraction, insoluble transition phase and non-polar fraction will be analyzed for nutritionally relevant components along with mass balance determination. The transition phase and original pellet will be subject to a number of other separation methods to determine how nutritional components fractionate including:other solvents such as hexane, acetone, ethanol, enzyme treatments, supercritical CO2.Amino acids,glycerol, organic acids, alcohols and sugars,anions, cations,nutritionally important minerals: Ca, Fe, Mg, K, P, lipids, fatty acids, total protein, nucleic acids and ashTask 2.4 Extensively analyze the properties of protein and beta-glucans for food and industrial applications. IMPROVE will be utilized for understanding protein qualities desired for food products (e.g. texture, rheology, solubility). Detailed tests include: ileal digestibility of protein (Boisen method), ileal digestibility of dry mater (Boisen method), SDS Pages profile (fluorescent), emulsifying properties, foaming capacity and foam stability, solubility curves at 6 different pH units, viscosity (flowing properties), gelling properties, water holding capacity, oil holding capacity, proteins mass assessment using MALLS and particle size distribution. Furthermore, confocal and SEM microscopy will be utilized to understand the physical and spatial properties of our protein concentrates.Task 3: Genetic modification of strain MK7.Subtask 3.1: Genetic Modification Methods and Metabolic Targets, Months 0-30. CRISPR/Cas9 system will be employed using single gRNA (guide RNA) expressing cassettes and the multiplex gRNA expression method patented by Dr. Yang. CRISPR/Cas9 technology in brief, uses the Cas9 endonuclease coupled with gRNA targets the DNA sequence of 5′-N20-NGG-3′ (N indicates any base), where N20 is identical to the gRNA 5′ sequence and NGG is the protospacer-adjacent motif (PAM). This gRNA along with the high occurrence of PAM in genomes allows gRNA Cas9 to edit nearly any genetic element. Single and multiple gRNA's cassettes on a single plasmid will be introduced into cells to promote, knock-out and or introduce (move) regulatory genes into target sites of the strain MK7 genome. A draft genome on 8,000 contigs currently exists for strain MK7 and SB is preparing to send genomic DNA to the National Center for Genome Resources in Santa Fe, NM for higher quality sequencing, assembly and annotation. Genetic modification efforts will focus on improving the nutritional value of the biomass. Initial targets include enhanced production of beta-carotene (vitamin A) and ergosterol (vitamin D2 precursor) already produced by strain MK7.Subtask 3.2: Enhancement of vitamin D2 levels by photochemical conversion of ergosterol, Months 9-12. Strain MK7 contains high levels of naturally occurring ergosterol (a precursor to vitamin D2). Current levels of ergosterol are between 1.25-2.50 mg/g dry weight.SB will utilize UV techniques to convert ergosterol from MK7 into either previtamin D2 or vitamin D2. MK7 will be subjected to varying UV light wavelengths for various time intervals. Both fresh mat tissue as well as process dried powder will be used and levels of ergosterol, vitamin D2, and pre-vitamin D2 will be quantified using established methods (both SB and Eurofins).Task 4. Techno-economic analysis, Months 18-36.The analysis will include utilization of strains genetically edited to enhance production of specific products. Study level techno-economic analyses will be performed using data obtained in Tasks 1-3, and will include feedstock costs (transportation, storage), costs of additional nutrients (N, P), fixed and variable costs associated with the biomass production facility and equipment (inoculum production, trays, racks, maintenance of environmental growth conditions, utilities, labor, operation and maintenance of harvesting equipment, separation equipment, etc.), cost of packaging and storage of the product, and value of the product. This level of economic estimate is anticipated to assess profit margins for the process at the ±100% range.The analyses will be conducted by SB's resident engineer in collaboration with Dr. Raj Raman, at Iowa State University, CBiRC.