Progress 08/01/19 to 04/30/20
Outputs
Target Audience: enzyme manufacturers and developers bioethanol and other biofuel producers manufacturers using enzymes as catalysts Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?We have been directly contacting representatives of bioethanol manufacturers on both research and development and production sides regarding our results to gauge their interest and better understand how our advances would impact their production processes. We have been seeking feedback to further improve our material, to understand what testing we would need to perform to meet their requirements, and to better simulate production conditions. We have discussed the market opportunity with potential distributors. Additionally, we have been discussing the potential for other similar enzyme-based manufacturing opportunities based on the results from our testing. We may also seek to present results of our testing at a scientific conference in the future to share our results with interested researchers. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project focused on reducing the costs associated with the use of enzymes in the production of biofuels such as bioethanol. In order to provide a cost advantage to the bioethanol industryenzyme materials need to be reusable; which means they must maintain enzymatic activity over weeks of continuous reaction in a fermentation or liquefaction environment, and they must be easily separated following completion so they can be used in subsequent reactions. Two of the primary enzymes used in corn-to-ethanol fermentation, alpha-amylase - which breaks down starches during liquefaction and glucoamylase - which converts small carbohydrates and polysaccharides into sugars usable by yeast, were enhanced using a patented immobilization method (ImmobiZyme™) developed by PI, Dr. Smiechowski. The immobilization method is a process where enzymes are chemically bound to inactive, low-cost, biological support materials. This immobilization enhances the stability of the enzymes in challenging environments and enables the enzymes to easily be collected at the end of a fermentation. The goals achieved during this project can best be summarized in 3 broad objectives: 1) Demonstrate that alpha-amylase and glucoamylase maintain their functionality when immobilized, 2) Evaluate the stability of the immobilized enzymes under simulated production conditions, and 3) Validate the performance of immobilized enzymes with lab-scale fermentations. Objective 1: Several formulations were studied to generate enzyme pellets to meet Phase I activity and stability requirements. Testing focused on the composition of the support matrix, balancing the pellet's strength, available surface area, and projected raw material cost. A significant factor noted in the success of a support matrix was the total solids content. If the solids content was too low the matrix cannot form pellets, and if it was too high the material was too viscous to pump. Between these limits, pellets could be formed. However, as the solid content approached the upper working limit pellets would transition to a continuous noodle-like shape. This transition was noted for future work, as it suggests as the matrix could be extruded and cut with a die providing access to common and scalable manufacturing processes along with the opportunity for greater control of the final pellet dimensions. Pellet durability was measured bybreaking strength. Pellets were studied dry and after 24 hours of immersion in water. Under dry conditions the strongest pellets would break at 16-20 N, and between 5.5-7.5 N when wet. The final goal for formulation development was that the enzymes must retain more than 50% of their starting activity after immobilizationto the pellets. Candidates for further testing were selected based the combined consideration of enzymatic activity and pellet strength. A selected candidate needed to demonstrate a retained enzyme activity of greater than 50% and be in the top 1/3 of all pellet strength results. 3 immobilized glucoamylase (IG) candidates met these criteria along with 2 immobilized alpha-amylase (IA) candidates. Objective 2: The second objective evaluated IA and IG performance under simulated use conditions to determinetheir retained activity and recoverability. For IA testing, the immobilized enzyme was run in 30 wt. % corn flour solutions at 80 C for 6-8 hours. Following the reaction IA was recovered from the mixture using a screen filter and washing with DI water. One of the candidates retained more than half of its original activity following the testing,maintaining all its starting activity. However, only half of the starting material from this candidate was recovered. While this meets the activity goal for this effort, not enough material was retained for a practical application.The largematerial loss was attributed to the particle size being too close to the size of larger corn flour particles.Using IA pellets with larger diameters is expected to improverecovery. IG performance was evaluated by simulating fermentation conditions. IG pellets or stock glucoamylase was addedto 10 mL buffer solutions at pH 5.5, containing 5-15% ethanol by volume. The test solutions were incubated at 35 C and agitated at 60 RPM for periods of 24, 48, and 72 hours. Following exposure, the samples were tested for enzymatic activity and compared to unexposed enzymes as controls. As expected, stock glucoamylase lost activity when exposed to ethanol, losing all enzyme activity after 48 hours of exposure for all tested concentrations. The activity of IG also decreased with ethanol exposure time and increasing ethanol concentration, but all candidates still retained on average 10% of their original activity after 72 hours of exposure. Examination of the IG pellets following each ethanol exposure showed thatall pellets retained their integrity throughout the test period. Following this testing it was determined ethanol interfereswith the performance of the enzyme assay for both IG and stock enzyme and is a notable factor in why IG's retained activity was lower than the expected 50%. Objective 3: The third objective evaluated IG candidates in lab-scale (1.5 L) ethanol fermentations for retained activity, material recovery, and for reusability. The fermenter was operated at 35 C, pH 5.25, and 300 RPM with no aspiration, and each fermentation was run for at least 60 hours to emulate bioethanol plant conditions. Following a completed fermentation, IG pellets were recovered using wire mesh screens and washed with DI water. Of the three candidates IG candidates 2 and 3 both exceeded expectations in terms of material recovery and retained enzymatic activity. The best performing candidate, IG 3, was then evaluated by running it through multiple consecutive fermentations. Material losses per fermentation continued to be small and well within acceptable ranges. Candidate 3's also maintained a high enzymatic activity after reacting for several hundred hours in the fermentation vessel, exceeding our original performance expectations. The third technical objective thus validated that an IG material was capable of being used in a fermentation and being easily recovered using a mesh filter and exceeded the remaining goals of the Phase I project. Conclusions: The Phase I project met its primary goals of validating that enzyme reuse in bioethanol production through our immobilization process was viable. While IA materials were able to react with corn flour, the process was not optimal. The separation was hampered because the pellets were too small which lead to a significant amount of enzyme material loss. The reaction process itself also was difficult to control, which had a negative effect on test results. Thereis value in revisiting IA materials in the future using pellets with larger diameters to simplify the separation and scaling up the test apparatus for better reaction control. IG's ability operate undercorn-to-ethanol fermentation conditions was verified. Pellet stability, ease of collection between operations, and reusability were validated. Testing showed the best performing material successfully operating through 6 consecutive fermentations, while still maintaining enough enzyme activity for even further use. The IG enzyme as tested has the potential to reduce bioethanol plant enzyme expenses by a factor of 5. These technical results strongly support the feasibility of success in moving forward to Phase II.
Publications
|