Progress 09/01/23 to 08/31/24
Outputs
Target Audience:Students and postdocs are the target audience in this project since it provides a training and research platform for them. The project provided training opportunities for two graduate students and one postdoctoral scholar. On a larger scale, the emerging biorefinery industries and the whole biofuels community are our target audience. We are generating new technology and knowledge that lay the foundation for bio-conversion science and engineering. Changes/Problems:The postdoc researcher joined the lab about 10 months later than expected due to Visa issues, which caused the delay in the project's progress. We have been working on constructing the new strain in year 2, and the strain was recently constructed and is available for fermentation studies. The fermentation studies planned in year 2 will be delayed and will be conducted in year 3 instead. What opportunities for training and professional development has the project provided?The project trained two graduate students and one postdoctoral researcher during this reporting period. How have the results been disseminated to communities of interest?We have published two peer reviewed journal articles to disseminated results to the scientific community. What do you plan to do during the next reporting period to accomplish the goals?Task 1 Engineer Myceliophthora thermophila for cellobionate production from cellulose We plan to finish the strain engineering work. Task 2. Develop a pretreatment process using cellobionic acid as the pretreatment agent. Study the cellobionic acid degradation kinetic during pretreatment and test cellobionic acid as a pretreatment agent for wheat straw samples. Task 3. Develop a solid-state fermentation process for cellobionate production We will study the cellobionic acid production in solid-state fermentation in flasks. Task 4. Recover cellobionic acid from the fermentation broth using RW-EDI technology We will screen the ion-exchange resin wafers first and then study the CBA recovery using the mock solution first and then using the fermentation broth.
Impacts
What was accomplished under these goals?
Task 1 Engineer Myceliophthora thermophila for cellobionate production from cellulose We have engineered Myceliophthora thermophila 001 for cellobionate production. Starting from Myceliophthora thermophila 001, which is a super cellulase producer, we have successfully deleted eight ?-glucosidase genes and one cellobionate phosphorylase gene via three rounds of gene deletion using the CRISPR-Cas9/RNP technology, yield the strain M. thermophila 001 ?2bgl?cbap, ?5bgl?cbap, and ?8bgl?cbap. We have characterized the produced strain for cellobionate production. Strain ?8bgl?cbap) produced cellobionate from NaOH pretreated wheat straw at more than 50 mM in 5 days. We have obtained a functional recombinant strain for cellobionate production. Task 2. Develop a pretreatment process using CBA as the pretreatment agent We have optimized the process for potassium cellobionate production using cellobiose as the substrate. The potassium cellobionate was converted to CBA via anstrong acid cation resin, Amberlite™ IRC120 H resin. The purity of the produced cellobionic acid was verified using HPLC.We have studied the CBA degradation kinetic at the concentration of 50g/L using the produced CBA. Experiments were carried out at 160, 170, 180, and 190 C in tubular reactors inside an oil bath. The 50g/L CBA solution was heated to 160, 170, 180, and 190 C for different periods. The remaining CBA concentration was measured. The reaction rate constants at different temperatures and the activation energy were calculated based on the experimental data. The activation energy was about 123 kJ/mol. The reaction rate constants at 160, 170, 180, and 190 C were 0.039, 0.082, 0.154 and 0.270 (1/min) respectively. We also tested the pretreatment of the wheat straw at a concentration of 50 g/L. In the presence of the wheat straw, the CBA degradation was slower than without the wheat straw. Cellobionic acid was effective in improving the digestibility of the wheat straw. We are optimizing the cellobionic acid pretreatment process. Task 4. Recover cellobionic acid from the fermentation broth using RW-EDI technology We analyzed ionic conductivities of resin wafers in the lab using jar testing of the beads by varying several different cat ion-an ion compositions of resin wafers using Amerberlite ion exchange resins compositions ranging from 90:10 to 10:90 (cat ion: an ion ratios). We prepared synthetic CBA fermentation broth by mixing commercially available salts of lactobionic acid with essential inorganic salts and a surrogate gluconic acid feed solution by combining gluconic acid with sugars to simulate hemicellulose hydrolysate. We identified optimal compositions with the 20:80 (cation: anion) composition consistently showing the highest ionic conductivity and optimal ion adsorption rates for lactobionic and gluconic acid solutions. This 20:80 mixture outperformed other compositions, exhibiting superior ionic conductivity and efficient ion transport. Using the optimal compositions, we fabricated resin wafers (polyethylene:sucrose: resin beads ratio of 1:1.5:4.6) and tested them under lab conditions. We validated the resin wafer performance under various synthetic CBA and GA concentrations and actual CBA fermentation broth, confirming the robustness of the selected compositions. In a synthetic fermentation broth with the following feed composition: Calcium Lactobionate: 10 g/L, Sodium Chloride: 0.25 g/L, Potassium Phosphate: 0.75 g/L, Calcium Chloride: 0.05 g/L, Iron Sulphate: 0.025 g/L. The EDI system operated at an initial current of 21.3 mA, a voltage of 28 V, and a feed flow rate of 11.2 mL/min in the dilute stream and 10.6 mL/min in the concentrate stream, achieving a current efficiency of 57.87%. Approximately 59% of the lactobionate ions were successfully transported into the concentrate stream, where they were recovered in pure acid form due to the water-splitting capability of the bipolar membranes. However, increasing the feed concentration reduced current efficiency and resulted in lower recovery rates. To address this, we are exploring various membrane types with optimized permeability to enhance the efficient transport of lactobionate ions and improve overall recovery. EDI performance, however, showed that separation was consistent even at higher concentrations.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Quach, V., Mahaffey M., Chavezb, N., Kasuga, T., Fan, Z. Dilute gluconic acid pretreatment and fermentation of wheat straw to ethanol. 2024 Bioprocess and Biosyetms Engineering 47:5 623-632
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Wang, J., Kasuga, T., Fan, Z. 2024 Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10. Bioprocess and Biosystems Engineering 47: 10 1683-1690
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Students and postdocs are the target audience in this project since it provides a training and research platform for them. The project provided training opportunities for two graduate students and one postdoctoral scholar. On a larger scale, the emerging biorefinery industries and the whole biofuels community are our target audience. We are generating new technology and knowledge that lay the foundation for bio-conversion science and engineering. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project trained two graduate students and one postdoctoral researcher during this reporting period. One graduate student attended a professional conference and presented her work. How have the results been disseminated to communities of interest?A graduate student presented the work at the 45th Symposium on Biotechnology for Biomaterials, Fuels and Chemicals, Portland, May 2023. What do you plan to do during the next reporting period to accomplish the goals?Task 1 Engineer Myceliophthorathermophila for cellobionate production from cellulose We plan to finish the strain engineering work. Task 2. Develop a pretreatment process using cellobionic acid as the pretreatment agent. Study the cellobionic acid degradation kinetic during pretreatment and test cellobionic acid as a pretreatment agent for wheat straw samples. Task 3. Develop a solid-state fermentation process for cellobionate production We will study the cellobionic acid production in solid-state fermentation in flasks. Task 4. Recover cellobionic acid from the fermentation broth using RW-EDI technology We will screen the ion-exchange resin wafers first and then study the CBA recovery using the mock solution first and then using the fermentation broth.
Impacts
What was accomplished under these goals?
Task 2. Develop a pretreatment process using CBA as the pretreatment agent. We have studied the gluconic acid's potential as a wheat straw pretreatment agent at different concentrations (0.125-1 M) and temperatures (160-190°C) for 30 minutes, followed by enzymatic hydrolysis. 0.125 M gluconic acid, 170°C, yielded the highest xylose output, while 0.5 M gluconic acid at 190°C yielded the best glucose yield. A fraction of gluconic acid decomposed during pretreatment. Detoxified hemicellulose hydrolysate from 0.125 M gluconate at 170°C for 60 minutes showed promise for ethanol production. The gluconate contained in the de-toxified hemicellulose hydrolysate can be fermented to ethanol along with other hemicellulose sugars present by Escherichia coli SL100. The ethanol yield from gluconate and sugars was about 90.4±1.8%. The pretreated solids can be effectively converted to ethanol by Saccharomyces cerevisiae D5A using via simultaneous saccharification and fermentation with the cellulase and -glucosidase addition. The ethanol yield achieved was 92.8±2.0% of the theoretical maximum. The cellulose conversion was about 70.8±0.8%. Task 3. Develop a solid-state fermentation process for CBA production We have studied the production of cellobionate from the lignocellulosic substrate by the strain Neurospora crassa HL10 in submerged culture first. When NaOH-pretreated wheat straw was used as the substrate, it was found that adding an exogenous redox mediator was unnecessary because the lignin in the pretreated wheat straw could serve as the redox mediator. However, the amount of laccase produced by strain HL10 on pretreated wheat straw was relatively low, which led to slow cellobionate production. Cycloheximide successfully induced high-level laccase production in N. crassa HL 10. With the addition of 3 M of cycloheximide, the strain N. crassa HL10 produced about 57 mM cellobionate from pretreated wheat straw containing the equivalent of 20g/L cellulose without the addition of any enzyme or redox mediator, and the conversion time was shortened from 8 days to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Cellobionate Production from Sodium Hydroxide Pretreated Wheat Straw by Engineered Neurospora crassa HL10. 45th Symposium on Biotechnology for Biomaterials, Fuels and Chemicals, Portland, OR, May 2023.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Quach, V., Mahaffey M., Chavezb, N., Kasuga, T., Fan, Z. Dilute gluconic acid pretreatment and fermentation of wheat straw to ethanol. Bioporcess and Biosyetms Engineering
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Wang, J., Kasuga, T., Fan, Z. Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10. Bioporcess and Biosyetms Engineering
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