Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Biological & Agricultural Engineering
Non Technical Summary
It has been 25 years since the first heterologous protein was expressed in tobacco followed by an antibody expression in microalgae (Fisher et al., 2003; Nikolov and Woodard 2004; Woodard et al., 2009; Mayfield et al., 2003, Rasala et al. 2010). Compared to microbial, animal, and mammalian cell cultures, plants and microalgae are attractive systems for production of native and recombinant proteins because they do not propagate human pathogens and are inexpensive to cultivate (Buyel, 2015). However, lower than expected protein expression levels have been a major obstacle for the development of economical bioprocesses. In addition, the low protein accumulation levels have hindered the design of novel protein extraction and recovery methods from plant or algal biomass. The recent technology advancements such as direct gene transcription in cell organelles suited for protein accumulation, targeting proteins to subcellular locations for optimal and stable accumulation as well as transient expression have opened a new chapter in the exploration of plants and algal systems for protein production. These latter developments have solved the accumulation challenge and reignited industry interest in alternative, cost-competitive plant - and algae-derived protein products. The goal of this project is to develop bioprocessing solutions that would make for protein production from plants and microalgae economically viable.A similar bioprocess interest surge is been observed (i.e., development of effective bioprocessing methods) with native (non-recombinant) microalgal protein products due to the need to fulfill the anticipated dietary protein demand in the coming decades. The current world population of 7.3 billion has a protein consumption demand of 202 million tons (MT), which is projected to increase to 360-1250 MT by 2050 (Henchion et al., 2017; Ritala et al., 2017). These projections underscore the need to fill the protein gap for the growing population's needs with an additional protein source. High-protein content (50%), nutritional properties, and low allergenicity (Becker, 2007a; Ritala et al., 2017) compare microalgae favorably to common protein sources such as soy (37%), milk (26%), meat (43%), and yeast (39%) (Barka & Blecker, 2016; Becker, 2004; Wells et al., 2017). In addition to nutritional quality attributes, current lifestyle trends, such as the increased consumption of health-promoting, vegan and sustainable food products, and diets that support sporting activities for health preservation (Bomgardner, 2015) have brought microalgae at the forefront of non-animal protein sources. Although the demand for non-animal sourced protein products has increased in the past 5 years, development of scalable protein extraction and recovery methods from algal biomass has not materialized. Consequently, suboptimal microalgal bioprocessing typically results in suboptimal product yield and high product cost. For example, current microalgal bioprocessing costs account for almost 60% of total product costs, while the industry benchmark for other protein feedstock (soybeans, peas, milk) is less than 30% (Lam et al., 2018). To match this industry benchmark, multiple, expensive, and time-consuming unit operations, which are currently used to produce different protein products from microalgae, have to be replaced, modified and/or optimized (Lam et al., 2018).Regardless of intended application (i.e., food or therapeutic protein products), the common principle to reduce protein manufacturing cost is to design a high-yielding and simple downstream process (Nikolov and Woodard, 2004; Buyel, 2014). The current downstream processing of recombinant proteins from mammalian and microbial systems relies on selective and rather costly bioseparations methods that typically contribute to 90% of the manufacturing cost (Evangelista et al., 1998). Bioprocessing cost of food protein isolates and hydrolysates are much lower, about 30% of the total manufacturing cost, but their economic value is also more than two orders of magnitude lower than that of therapeutic proteins. For the latter protein products process yields are important and so is the opportunity to capture by-product(s) value of algal and plant biomasses such as omega fatty acids, antioxidants, plant oils, and polysaccharides. Therefore, to reap the benefits of low-cost plant and algal biomass production one needs to develop bioprocess technologies that are less expensive than those currently used for microbial and mammalian cell culture platforms. Several reviews of protein production platforms (Nikolov et al., 2009; Rasala et al., 2010; Wilken and Nikolov, 2012, Soto-Sierra et al., 2018) indicate that no single plant or algal platform is capable of delivering desired products at a cost comparative to current protein production systems (crops, cell cultures, and bacteria). Thus, process development goals for a specific product would vary consistent with product application and market size. For plant-derived therapeutic products, such as vaccines and antibodies, novel and less expensive methods for protein purification are desirable. In the case of industrial and food proteins from microalgae, efficient fractionation of plant tissue and extracts to capture high-value co-products (antioxidants, polysaccharides, pigments and other bioactive molecules) is critical.In summary, the current world population of 7.3 billion has a protein consumption demand of 202 million tons (MT), which is projected to increase to 360-1250 MT by 2050. These projections underscore the need to fill the protein gap for the growing population's needs with an additional protein source.To reap the benefits of low-cost plant and algal biomass production we haveto havebioprocess technologies at our disposal that are less expensive than those are currently used for microbial and mammalian cell culture platforms. The goal of this project is to develop bioprocessing solutions that would make for protein production from plants and microalgae economically viable.
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Goals / Objectives
The overall objective of this project is to design economical processes for protein recovery from plants and microalgae. The specific project objectives are to:Investigate and correlate properties of plant and algal source materials relevant to product extractability and stability.Identify process conditions that are essential for achieving optimal protein purification.Use high-throughput process development methods and process modeling to synthesize optimal, scalable, and economical bioprocess.
Project Methods
Objective 1. Investigate and correlate properties of plant and algal source materials relevant to product extractability and stability.To understand how to improve bioprocess efficiency, we will analyze first the effect of plant and microalgal extracts on the quality and stability of the target protein product. Because extract complexity depends on biomass (cell) structure and process conditions, high-throughput screening and optimization of extraction parameters will be performed. The selected plant and algal biomass will be processed and extracted under different conditions (pH, temperature, mixing time, ionic strength, biomass-to-water ratio, etc.). Extracted native proteins, carbohydrates, phenolics, and proteases will be identified and quantified. To assess target protein stability and interactions with the native extract components that could alter its activity/quality, extracts from non-transgenic (native) tissue will be spiked with a purified protein of interest from a different source or with a protein of similar properties (size, pI, activity, etc.). Spiking experiments conducted with a well-characterized protein molecule will eliminate complications such as incomplete in vivo protein processing and presence of degradation products of unknown origin often found in plant or algal extracts.Objective 2. Identify processing conditions that are essential for achieving an optimal protein purification.The purpose of this objective is to determine: 1) plant- or algae-derived impurities in the clarified extracts that may interfere with protein purification; and 2) process conditions (i.e., pH, salt, ionic strength, temperature, etc.) that can be manipulated to reduce the negative impact of impurities on downstream processing. The working hypothesis of this objective is that understanding the complexity of clarified plant or algae extracts and identifying physico-chemical properties of the mixture will enable the development of strategies for systematic removal of impurities and design of efficient (high-yielding) bioseparation methods.To assess the effect of impurities and their potential interference during downstream processing, native plant or microalga extracts, characterized in Objective 1, will be processed and fractionated by membrane filtration and adsorption chromatography - the two most common unit operations that are currently employed in the biotech industry. Extract molecules and processing conditions that result in fouling of ultrafiltration membranes and/or chromatographic resins will be identified first. Then, biomass tissue pre-treatment and extraction methods will be investigated and optimized to reduce the membrane and/or resin fouling. When prudent, other unit operations such as salt and isoelectric precipitation and two-phase aqueous extraction would be included as pretreatment or primary purification methods. High-throughput process development methods will be used to determine adsorption kinetics and dynamic binding capacity of chromatography resins (Nfor et al. 2010; Bergander et al. 2008). For industrial and food protein products, less expensive methods such as membrane filtration and protein precipitation will be screened as ultimate protein purification tools. The experimental design to achieve this objective will be flexible and open-ended.Objective 3. Use high-throughput process development methods and process modeling to synthesize optimal, scalable, and economical bioprocess.The data compiled from Objective 2 will be used to assemble bioprocess train alternatives (Hanke and Ottens, 2014). Feasibility and potential process bottlenecks of assembled downstream trains will be examined using an expert simulation software (SuperPro Designer, Intelligen, 2000). The most plausible and economical process designs will be experimentally tested at the lab and then scaled-up. The process simulation analysis will allow us to decide what unit operations, extract properties, and process conditions may need additional re-evaluation. Another task within this objective is to develop heuristic design rules-of-thumb ("bioprocessing philosophy") for translating collected data into product-tailored bioprocessing strategies (Hanke and Ottens, 2014). The data for the development of heuristics will be collected in a database with inputs that will include regulatory constraints, physico-chemical properties of critical extract components, market requirements, and process economics. Relevant and reliable published literature data will also be included in the database. The ultimate goal is to develop a prototype expert modeling system for bioseparations development of high-value bioproducts (Hanke and Ottens, 2014; Steffens et al., 2000).