Using the OPEX value calculated and the mass of knotweed rhizomes required each year for 100 MT production of resveratrol, the cost of knotweed rhizome is determined to be $0.19 per kg. To give a better assessment of the CAPEX required for the upstream production, farm equipment cost was also calculated alongside the annual operating cost. Table 2.5 list the equipment deemed necessary for harvesting Japanese knotweed rhizomes. Values for the equipment cost were again retrieved from the UC Davis Agriculture and Resource Economics Center. Since the report published by the UC Davis Agriculture and Resource Economics Center was released in 2015, an inflation/cost adjustment of 1.125 was used to estimate the cost of the same farming equipment in 2021. Here, the total farming equipment cost was multiplied by a factor of 0.6 since some equipment purchased may be used rather than new equipment. Additionally, it is assumed that the equipment requirements scaled with cultivation acreage, thus estimating the number of each equipment required was multiplied by 7.4 to account for the larger cultivation acreage than use in the report. Using all this information, the total cost of equipment was to be $3.6 million.The utilization of natural plants as a source for chemical compounds is an ever-growing field. To date, pe grow bag there have been a variety of compounds which have been extracted from plants, including but not limited to, tetrahydrocannabinol from hemp 1 , phenolic acids from purple corn 2 , and flavonoids from chamomile flowers 2 .
Although natural plants offer a reduction of complexity in upstream bioprocessing in comparison to using genetically engineered microbes, the downstream and purification methods remain just as rigorous. A standardized process for the extraction of resveratrol from Japanese knotweed remains unestablished as novel technologies and methods are continuously being developed and researched. The most common unit operations seen utilized in current patent and scientific journal articles are shown in Figure 3.1 as a block flow diagram.The procedure needed for purification of resveratrol is understood, however, there is no consensus on what the best methods to use are since there remain numerous options for each step, each consisting with their own advantages and disadvantages. Notably, a crucial step during the downstream process is the deglycosylation and hydrolysis of polydatin performed after crushing and shredding the rhizome. Two methods can be employed to hydrolyze the polydatin compound found beside resveratrol, either using strong acids or enzymes. Performing either method has demonstrated conversion yields of polydatin to resveratrol above 90% 4,5. However, the use of acid hydrolysis may produce additional adducts in the reaction, thus decreasing the content of resveratrol present and prompting concerns for additional purification procedures. Furthermore, acid hydrolysis has been reported to require harsh processing conditions, often causing pollution as a result. Nevertheless, the large quantity and high cost of industrial cellulase enzymes serves as the biggest deterrent to the latter approach. Currently, there exists numerous published techno-economic analysis studies of plant based production focusing on bio-fuels, recombinant therapeutic proteins, industrial enzymes, and antimicrobial proteins for food safety.
Here, this chapter will describe the techno-economic analysis performed on the downstream processing required for the plant-based production of Japanese knotweed for the extraction of the biopolymer precursor, resveratrol, which has not been demonstrated before. This study will aim to establish a framework for more informed decisions on the development of a domestic production route for such polymer precursors.The simulation model and economic analysis was performed using a computer-aided process modeling and design software, SuperPro Designer® Version 12, Build 3 Special Build 1600. SuperPro was used to determine equipment sizing, specify equipment process parameters, and determine operations scheduling and raw material requirements. The economic analysis was used to determine the total capital expenditure , the total annual operating expenditure , and the cost of goods sold . A further detailed analysis provides a breakdown of all costs, e.g., raw materials, consumables, utilities, labor, and waste disposal- with the goal being to identify the materials and process steps which contribute most significantly to the total cost of production. Pricing for raw materials, consumables, and equipment within the model were calculated using publicly available commercial prices, unpublished personal communications with manufacturers of large-scale bio-processing equipment, previous SuperPro design files, and in some cases, SuperPro default values.The base case model was developed to process 100 metric tons of resveratrol per year, with the facility operating 330 days a year. A single batch duration is estimated to be 45.6 hours, totaling 1,295 batches per year with a cycle time of about 6 hours. Due to the short cycle time relative to the annual operating time, the process may be assumed to be operating under pseudo-continuous conditions. To attain the quantity of 100 MT of resveratrol, roughly 7.3 million kg of knotweed rhizomes a year are required for downstream processing.
The model was scaled using publicly available patent literature, scientific journal articles and working process knowledge. Bioprocessing operations and conditions for certain unit operations within the simulation were adopted from scientific literature focusing on resveratrol production from various plant sources . Table 3.1 and Table 3.2 list the various processing techniques utilized for resveratrol purification from Japanese knotweed in patents and scientific journals, respectively.In the design of the downstream production process model, certain key bioprocessing parameters were extrapolated from publicly available information on resveratrol production. In particular, the quantity of enzymes per batch required to hydrolyze polydatin to resveratrol was tuned to match production methods found in patents. Patent literature detailing the extraction of resveratrol have suggested amounts of 2 – 4 weight % of enzymes per knotweed per batch should hydrolyze polydatin to resveratrol effectively for processing. However, scientific literature focused on optimizing the enzymatic hydrolysis process for resveratrol in knotweed have reported using enzyme concentrations closer to 10% of the total processed knotweed13. An average of these values resulted in 6.5 weight % of enzymes per the total knotweed rhizomes per batch; this percentage was used in the process simulation. Another parameter which was adjusted in the model to match resveratrol production methods described in patents was the percent recovery of resveratrol after undergoing the extraction process. Multiple sources have denoted the use of the ultrasonic technology and Ultrasonic Assisted Extractions to remove resveratrol from knotweed rhizomes but fall short by failing to provide key parameters such as percent recovery. UAEs are becoming a common operation for extracting polyphenols from plant biomass. Its application has already been utilized to extract resveratrol from other plant sources such as grape stems and grape leaves18. These two studies demonstrate the capability of UAE technology in extraction of resveratrol while noting a percent recovery of 78.8% and 80%, respectively. A conservative approach was taken, and the former of these values was used to define the resveratrol recovery in the UAE operation used in the process model. In an effort to accurately model the UAE operation in SuperPro, the other parameters associated with UAE such as power, temperature, and duration of agitation were aligned to Japanese knotweed roots processing conditions. Traditional methods of purification involve using silica gel resins in chromatography columns. However, silica gel resins are predominantly used for smaller scale production and there is often complication scaling up for large scale manufacturing. More recently, growing bags macroporous resins have been utilized to serve as a replacement as they hold several advantages over their silica gel counterparts. First, silica gel resins utilize mixtures of acids, such as chloroform and methyl alcohol, to serve as their eluents, whereas macroporous resins only require mixtures of ethanol and water. Additionally, the cost of using silica gel resins is higher compared to using macroporous resins while the recovery of using silica gel remains lower. In attempt to accurately model an industrial chromatography unit in SuperPro, an unpressurized vessel filled with macroporous resin was initialized to operate as an adsorption mixing tank. The resin is exchanged with fresh resin every 100 batches. All process flow specifications and assumptions used in the development of the downstream facility model is shown in Table 3.3 along with its source.
A detailed downstream process flow sheet depicting the purification of resveratrol from Japanese knotweed is shown in Figure 3.2. Each batch begins with the harvested knotweed rhizomes being transported from a designated storage warehouse to a silo bin using a conveyor belt . Approximately 5,635 kg of knotweed rhizomes are retrieved from the silo bin and transferred to a washer where they are washed with water at a 1:1 w/w ratio. Next, the knotweed rhizomes are mixed with water at a 1:1 w/w ratio andare grounded into a slurry solution using an industrial grinder operating at a throughput of 11,327 kg/hr. The slurry is pumped down the process line where 100 kg of citric acid is mixed with the solution to adjust the pH value down to 5.0. Following the pH adjustment step, a stream of enzymes consisting of cellulase and ß-glucosidase at an 80:20 ratio enters the process line where it is mixed with the solution before being pumped into a batch vessel reactor . Within the reactor , the solution is agitated for fifteen minutes and heated to 55 C to allow the enzymatic deglycosylation and hydrolysis of polydatin to occur, converting 90% of existing polydatin to resveratrol. To deactivate the enzymes and halt the reaction, the reactor is heated to 85 C using steam. Once the deactivation step is complete, the slurry is transferred to the ten ultrasonic assisted extractor units running in parallel . The ultrasonic assisted extraction units are first charged with ethanol at a 1:1 mass ratio with the plant biomass where it is then agitated for sixty minutes, allowing for efficient polyphenol extraction from the plant biomass . The slurry solution is then sent to a belt press filtration unit where the plant biomass can be separated from the liquid solution and disposed of properly . An additional separation unit in the form of a plate and frame filter is used to capture and separate any residual plant biomass within the solution before being sent for further processing. The filtrate is then pumped into a batch adsorption vessel containing NKA-II, a macroporous adsorption resin. After the binding step occurs, the resin is first rinsed with water to remove any impurities which may have been captured. Following the washing step, the adsorption vessel is set to charge in a stream of ethanol to elute the resveratrol from the macroporous resin . After the elution step, the eluate is then pumped into a crystallization column for further processing. The crystallizer operates under reduced pressure, an evaporation temperature of 98 C, and a crystallization temperature of 60 C, removing most of the residual ethanol and water present from before and yielding solids crystals . These crystals undergo a mixing step where they are resuspended in ethanol . This liquid-solid mixture is then sent to another crystallizer unit operating under similar conditions as before, but this time yielding 77.3 kg of resveratrol at 99% purity.A summary of the economic analysis results from the base case model set to produce 100 MT resveratrol is presented in Table 3.4. The CAPEX is the sum of the direct fixed capital , working capital , and startup and validation cost . The DFC for the facility was calculated by using a distributed set of purchase cost factors shown in Table 3.5 to estimate the facility direct costs , indirect costs , and other costs . Here, the purchase cost is the sum of all major equipment purchase costs shown in the SP flowsheet and the unlisted equipment purchase cost is assumed to be 20% of the PC. A list of all the major equipment, their composition, and purchase prices can be found in Table 3.6. The price of unlisted equipment was also included in Table 6 for reference. The working capital was estimated to two percent of the total DFC. The startup and validation cost combined were estimated to be about 1% of the DFC. Most of the equipment purchase costs were estimated using SuperPro default values. The composition of a majority the equipment are carbon steel except for the UAE, which is composed of Stainless steel 316L and the silo bin which is composed of concrete. The justification for using equipment fabricated using carbon steel instead of more costly stainless steel is recognizing that the process is being designed for polymer applications and not for a more regulated pharmaceutical application.