Progress 06/15/17 to 02/14/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The rate-limiting limonene production pathway enzymes necessitate either significant enzyme engineering efforts or the use of high-cell density fermentation. Engineering efforts to improve the rate at which an enzyme is capable of "turning over" a substrate has historically also resulted in a reduction in the specificity of the enzyme and, concomitantly, an increase in the formation of undesired byproducts. The preferred approach for the production limonene will utilize a high cell density fermentation. Efforts to demonstrate maintained specific productivity with increasing biomass concentration are in progress. A key challenge in downstream recovery of limonene is its high volatility, which makes handling samples of limonene challenging. Sample storage equipment which is commonly used for aqueous cell culture proved unsuitable for samples containing limonene, because such storage methods are not completely air tight and limonene escaped as vapor. Testing of 96-well sample plates, for example, showed significant evaporation, with most or all limonene evaporating in a matter of hours in many cases. This made storage and measurement of limonene a challenge. Sample-handling was modified to use only highly inert and airtight vials, resulting in improved ability to quantify samples at the expense of increased sampling handling time and complexity (and reduction in analysis throughput). Additionally, the fact that limonene is insoluble in water makes its sampling from fermentation culture challenging. Initial testing showed that under fermentation conditions limonene evaporated as quickly as it can be produced. Subsequently, sampling procedures were adopted which either sampled limonene from a dodecane layer added to the culture (for small-scale shake flask experiments) or sampled limonene from the fermentation off-gas (for bioreactor experiments). Limonene yield in both cases was sufficient to compare the productivity of different strains and process conditions. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Task 1. Strain Optimization. Work in this task focused on optimizing the specific rate (g/gCDW-hr) and yield of D-limonene production through the intermediate mevalonic acid. The strain development effort for this project utilized existing chassis strains that had been developed previously for mevalonate production. Efforts focused on engineering 2 key enzymes in the D-limonene production pathway. Chassis strains previously optimized for mevalonate production were further engineered to produce D-limonene. Strain variants were evaluated for limonene production in 2-stage phosphate depleted shake flask cultures with a 10% v/v dodecane overlay. Target strain performance metrics were achieved in these small scale studies. Task 2. Fermentation Development. Work in this task focused on developing a fermentation process suitable for the physical properties of D-limonene. Specifically, D-limonene is a volatile, non-polar hydrocarbon that is insoluble in water. Two strategies were explored to manage these challenges: 1) Use of a 2-phase fermentation by inclusion of an organic layer, and; 2) Collection of D-limonene from the fermentation off-gas. Methods were successfully developed to capture limonene at laboratory scale. Unlike most products of fermentation processes, limonene is insoluble in water, is volatile, and evaporates readily from both small-scale fermentations (e.g. shake flasks) and large-scale fermentation (e.g. bioreactors). This was demonstrated in "spike-recovery" experiments, where limonene was added to both shake flasks and bioreactors operating under typical process conditions (stirring, air flow, temperature, etc.) and over 95% of the limonene was demonstrated to evaporate. The approaches used to recover the limonene were specific to the scale of production. For small-scale shake flasks, recovery of the gas leaving the vessels was impractical due to their large number and small size. For these cultures, a "solvent overlay" approach was used. Dodecane, an inert oil, was added at a concentration of 10% by volume to the culture once growth was complete and limonene production was initiated. Although limonene is insoluble in water, it dissolves readily in dodecane, and this reduces its rate of evaporation dramatically. Although evaporation is not suppressed completely by this technique, it allowed relative comparisons of different strain and process variables. Recovery from bioreactors must be performed economically at commercial scale for bio-based limonene production. A solvent-overlay approach does not completely suppress limonene evaporation and solvents are challenging to recover at high yields from fermentation culture at large scale. Therefore, the focus was on recovery of limonene from the vapor phase. Task 3. Purification and Downstream Recovery. This task was closely linked to Task 2. Work in Phase 1 focused on evaluating the feasibility of economical strategies for the purification of D-limonene from the aqueous fermentation broth. D-limonene rich streams from each of the approaches described in Task 2 were further purified using one or more unit operations, including decantation, centrifugation, and or distillation. Methods were developed to successfully capture limonene from bioreactors at laboratory scale. Unlike most fermentation products, limonene can be recovered from the gas stream (primarily air and water vapor) exiting the fermenter. The ability of this vapor-liquid separation to purify the product is very attractive from a cost and product-purity perspective, and limonene was demonstrated to evaporate at very high rates from bioreactors. For these reasons, downstream recovery focused on recovery of limonene from the vapor exiting the fermenter during the bioprocess ("off-gas"). The two primary approaches tested were condensation (cooling of the gas stream to condense the limonene) and adsorption (binding of the limonene to solid particles which have a high affinity for hydrocarbons such as limonene). In both cases, the primary engineering challenges are the presence of large quantities of water vapor and the dilute nature of the limonene in the off-gas stream (due to a high rate of gas sparging in the bioreatctor). Successful proof-of-concept was demonstrated for both the condensation and adsorption approaches, allowing them to be applied to the production of limonene at laboratory scale. For the condensation approach, custom heat exchangers were constructed which allowed cooling to cryogenic temperatures and were able to accommodate the build-up of ice which occurs due to rapid cooling of water vapor. These were tested with "spike-recovery" experiments to have a recovery >75% compared to control conditions. Proof-of-concept was also demonstrated successfully for the adsorption approach. Activated carbon, an inexpensive industrial-grade adsorbent, was tested for its ability to bind limonene. Although previous research indicates that limonene can be adsorbed at high yield, adsorption in the bio-based production of limonene is impacted by the presence of water (a key component of fermentation off-gas). However, even under representative fermentation conditions, a capacity of 15 grams limonene per kg of carbon was demonstrated, indicating that limonene adsorption in the presence of water vapor is feasible. This approach is practical at laboratory scale and represents a useful option at commercial scale as well. Apparatus of both types was used successfully on 6-liter bioreactors as part of the limonene scale-up effort. Additionally, 3rd-party engineering firms have been engaged to evaluate the potential for scaling up these processes. For example, firms which produce cryogenic-condensation systems capable of capturing limonene were engaged to obtain quotes for the cost of limonene condensation at pilot scale. This combination of laboratory results and 3rd-party discussions provides valuable data necessary for the design of limonene recovery at commercial scale. Task 4. Technoeconomic Analysis (TEA) Process modeling and techno- economic analyses were deployed throughout the program to continually assess progress against program metrics and to prioritize future development efforts. Techno-economic analyses were completed to inform commercialization efforts. The goal of this effort was to evaluate the impact of key process metrics (upstream and downstream) at commercial scale. The basis of the model was a plant capable of producing 5,000 metric tonnes per year of bio-based limonene. For recovery and purification, condensation with a mechanical refrigeration cycle and shell-and-tube heat exchangers was modeled, followed by gravity settling and centrifugation to separate limonene from water and back-extract water-soluble impurities. Of the total estimated capital cost, approximately one third is attributed to product recovery and purification, with most of the remainder being the fermentation equipment. The total capital investment required (direct and indirect costs) for a "brown-field" plant design (i.e. exclusive of utilities infrastructure) is within the realm of economic feasibility for industrial biotechnology processes. Operating costs were estimated using engineering calculations based on the operations involved in the complete bio-based production process. Operating and capital costs were then integrated into a discounted cash-flow analysis to estimate the minimum selling price that would provide investors in the plant a return on investment (IRR) of 30%. Various technical parameters were evaluated quanitfy their impact on this price. Product titer (limonene produced per liter of fermentation capacity) has the greatest impact on this selling price, followed by the cost of glucose (the most important raw material). However, bio-based limonene remains highly cost-competitive, even after attractive investment returns are factored in, for the wide range of metrics shown in the sensitivity analysis above.
Publications
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Progress 06/15/17 to 06/14/18
Outputs
Target Audience:The target audiences that were reach during this reporting period include: Customers seeking more a sustainable and cost-effective supply of limonene. Changes/Problems:As part of Task 3, Purification and Downstream Recovery, it was confirmed that the product was highly volatile in the aerated fermentation environment. Volatility was significantly greater than reported in the literature or was modeled using first principles. At the commercial scale, this will enable a highly efficient product recovery process - specifically, the condensation of the product from the fermenter off-gas. Technoeconomic modeling of the proposed recovery process supports that this is a very capital-efficient approach. At the laboratory scale, the volatility of D-limonene makes it difficult to accurately quantify Key Performance Metrics (KPM; rate, titer, yield). Our standard development procedure is to conduct development at the microfermentation scale - which is essentially a modified 96-well plate format. Accurate quantification of the product is impossible in this format. As such, strains will be evaluated using bench scale fermentation integrated with a custom-built off-gas condenser to collect and quantify D-limonene. Product formation will be determined for the fermentation batch as a whole, as opposed to measuring product concentration as a function of time during the run. As such, the KPMs will reflect the average performance over the duration of the fermentation. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall objective of this program is to demonstrate a lab-scale bioprocess for the production of D-limonene at performance metrics that support a modeled minimum selling price that is 15-25% less than current market price. Towards this end, the following accomplishments have been demonstrated during the reporting period: Creation of engineered strains that produce D-limonene. The significant volatility of the product has identified the need for improvements in sample handling to accurately quantify rate, titer, and yield (in progress). Design and technoeconomic modeling of an economically viable downstream recovery and purification process. Construction of a lab-scale process suitable for accurate product recovery. Completion of technoeconomic analyses for the production of D-limonene in two commercial strategies: 1) Use of a Contract Manufacturing Organization (CMO), and 2) Build-Own-Operate commercial model. Key Performance Metrics necessary to support a minimum selling price 15-25% less than current market have been identified for each commercial strategy.
Publications
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Progress 06/15/17 to 02/14/18
Outputs
Target Audience:The target audiences that were reach during this reporting period include: Customers seeking more a sustainable and cost-effective supply of limonene. Changes/Problems:As part of Task 3, Purification and Downstream Recovery, it was confirmed that the product was highly volatile in the aerated fermentation environment. Volatility was significantly greater than reported in the literature or was modeled using first principles. At the commercial scale, this will enable a highly efficient product recovery process - specifically, the condensation of the product from the fermenter off-gas. Technoeconomic modeling of the proposed recovery process supports that this is a very capital-efficient approach. At the laboratory scale, the volatility of D-limonene makes it difficult to accurately quantify Key Performance Metrics (KPM; rate, titer, yield). Our standard development procedure is to conduct development at the microfermentation scale - which is essentially a modified 96-well plate format. Accurate quantification of the product is impossible in this format. As such, strains will be evaluated using bench scale fermentation integrated with a custom-built off-gas condenser to collect and quantify D-limonene. Product formation will be determined for the fermentation batch as a whole, as opposed to measuring product concentration as a function of time during the run. As such, the KPMs will reflect the average performance over the duration of the fermentation. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall objective of this program is to demonstrate a lab-scale bioprocess for the production of D-limonene at performance metrics that support a modeled minimum selling price that is 15-25% less than current market price. Towards this end, the following accomplishments have been demonstrated during the reporting period: Creation of engineered strains that produce D-limonene. The significant volatility of the product has identified the need for improvements in sample handling to accurately quantify rate, titer, and yield (in progress). Design and technoeconomic modeling of an economically viable downstream recovery and purification process. Construction of a lab-scale process suitable for accurate product recovery. Completion of technoeconomic analyses for the production of D-limonene in two commercial strategies: 1) Use of a Contract Manufacturing Organization (CMO), and 2) Build-Own-Operate commercial model. Key Performance Metrics necessary to support a minimum selling price 15-25% less than current market have been identified for each commercial strategy.
Publications
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