Progress 03/01/19 to 02/29/24
Outputs
Target Audience:Molecular microbiologist, students with interest in research in planta depolymerization of lignin, microbial fermentation scientists, plant biotechnologists, and scientists, leaders in biotechnology companies interested in lignin recalcitrance Changes/Problems:During this period, the budget that was allocated to hire a Postdoctoral Fellow to perform these activities will be used to support the graduate student tuition and stipend and provide funding for equipment such as a PCR machine, an RT-PCR machine, a new Conviron plant growth facility, laboratory supplies and consumables, travel, and equipment repairs. This change is because, although a hiring committee selected by the Human Ecology Department met various times to make a selection for a suitable Postdoctoral Fellow to undertake these research activities and recommend for hiring, the employment offer did not get to these Postdoctoral Fellows promptly for them to accept employment. Such a significant delay in offering employment for selected candidates led to declining employment from all these candidates. Furthermore, this grant will hit the 5-year statutory limit on February 28, 2024, with no possibility for a no-cost extension, and the remainder of the grant activities will be performed by the PI, Dr. Bertrand Hankoua, graduate student Dr. Nancy Nichols (NCAUR-ARS), Mrs. Frazer Sarah (NCAUR-ARS). Although we could not hire the Postdoc to carry out critical tasks and milestones for the project, NIFA approved using the remaining unused funds to acquire essential equipment to strengthen our research capabilities. What opportunities for training and professional development has the project provided?The PI, Dr. Bertrand Hankoua, has the opportunity for training and professional development in biomass saccharification and ethanol fermentability at Dr. Nancy N. Nichols's Laboratory at the National Center for Agricultural Utilization Research, Bioenergy Research Unit, Peoria, Illinois. All these training and professional development activities were performed under the supervision of Mrs. Sarah Frazer, Biological Science Technician in Dr. Nancy N. Nichols's Laboratory from July 9 until August, 4th 2023. The PI also made two tripsto Dr. Crocker's laboratory at the University of Kentucky to learn GC-MS-micro pyrolysis to quantify lignin-degrading products from self-induced bioengineered biomass. How have the results been disseminated to communities of interest?The PI presented at NIFA's PI meetings in Virginia Beach. Undergraduate and graduate students supported by the projects made scientific presentations on topics related to the project at ARD conferences and Research symposiums. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Research activities during this period relate to performing ethanol fermentation trials using cellulase hydrolysates generated from various transgenic brachypodum expressing single or a combination of extremophile laccases and cellulose and hemicellulose degrading enzymes. Here are the transgenic bracypodium lines developed from Delaware State University, which were initially taken to Dr. Nancy Nichols at the NCAUR in Peoria to perform the ethanol fermentation trials in summer 2023: Brachypodium (non-bioengineered, wild type), dried biomass from engineered lines (6-HPT-H-MTLCC, 5-PZP-NPT-4Gene-MTLCC, 4-HPT-E1-MTLCC, 2-3 Gene Cellulase,1-3 Gene Cellulase, 3-HPT-E1-MTLCC, 11-PZP-NPT-3 Gene Cellulase,13-PZP-NPT-4 Gene,10-HPT-E1-MTLCC, 9-HPT-E1-MTLCC, 8-HPT-E1-MTLCC, 7-PZP-4 Gene-MTLCC13-HPT-E1-TTLCC, 13-PZP-NPT-4 Gene, 10-HPT-E1-MTLCC.9-HPT-E1-MTLCC, 7-PZP-4 Gene-MTLCC, 13-HPT-E1-TTLCC), and fresh biomass from engineered lines (8-HPT-E1-MTLCC,3-HPT-E1-MTLCC, 4-HPT-E1-MTLCC, 1-3-Gene Cellulase,6-E1-MTLCC-L1-3,5-PZP-4 Gene-MTLCC-L1-3, 2-PZP-3 Gene Cellulase-L1-4, 7-PZP-4 Gene-MTLCC, 11-PZP-4 Gene-MTLCC, 13-PZP-NPT-4 Gene-MTLCC-L-13, 9-HPT-E1-MTLCC, 12-HPT-E1-MTLCC (*3 Gene), PZP-NPT-4 Gene-MTLCCL13-2). Dried and fresh biomasses from these transgenic brachypodium lines were used for lignin self -digestibility, saccharification, and ethanol fermentability at Dr. Nancy N. Nichols Laboratory at the National Center for Agricultural Utilization Research, Bioenergy Research Unit, Peoria, Illinois. All research activities were performed under Mrs. Sarah Frazer, a Biological Science Technician, in Dr. Nancy N. Nichols's Laboratory. The overall research was supervised by both Dr. Nancy N. Nichols and Mrs. Sarah Frazer. Once the moisture content of all biomasses was recorded, approximately 1 gram of each was first pre-induced in the laccase reaction buffer containing copper sulfate and hydrogen peroxide for 72 hours under a shaking incubator set at 280 rpm and 75oC. Afterward, all samples' biomass pellets were saccharified with cellulase enzyme at 50oC for 72 hours. Biomass hydrolysates were used first to estimate the content of C5 and C6 sugars using HPLC and compare these data with control samples to assess the potential of each line to release sugars. HPLC data from saccharified samples are as follows. Data generated from these experiments were reported during the last reporting period. During this reporting period, 5g of dried from engineered lines (6-HPT-H-MTLCC, 5-PZP-NPT-4Gene-MTLCC, 4-HPT-E1-MTLCC, 2-3 Gene Cellulase,1-3 Gene Cellulase, 3-HPT-E1-MTLCC, 11-PZP-NPT-3 Gene Cellulase,13-PZP-NPT-4 Gene,10-HPT-E1-MTLCC, 9-HPT-E1-MTLCC, 8-HPT-E1-MTLCC, 7-PZP-4 Gene-MTLCC13-HPT-E1-TTLCC, 13-PZP-NPT-4 Gene, 10-HPT-E1-MTLCC.9-HPT-E1-MTLCC, 7-PZP-4 Gene-MTLCC, 13-HPT-E1-TTLCC), and 5 g of fresh biomass from engineered lines (8-HPT-E1-MTLCC,3-HPT-E1-MTLCC, 4-HPT-E1-MTLCC, 1-3-Gene Cellulase,6-E1-MTLCC-L1-3,5-PZP-4 Gene-MTLCC-L1-3, 2-PZP-3 Gene Cellulase-L1-4, 7-PZP-4 Gene-MTLCC, 11-PZP-4 Gene-MTLCC, 13-PZP-NPT-4 Gene-MTLCC-L-13, 9-HPT-E1-MTLCC, 12-HPT-E1-MTLCC (*3 Gene), PZP-NPT-4 Gene-MTLCCL13-2) were self -digested for lignin self-depolymerization at Delaware State University using protocols the PI learned during his stay in Dr. Nichols laboratory in summer 2023. These experiments were performed with the assistance of the graduate student supported by the project. The resulting self-induced biomasses were saccharified at our laboratory at Delaware State University using biomass saccharification protocols the PI learned in Dr. Nichols's laboratory in the summer of 2023. Biomass hydrolysates from all these samples were sent back to Dr. Nichols's laboratory for fermentability trials and to estimate the content of C5 and C6 sugars using HPLC and compare these data with control samples to assess the potential of each line to release sugars. HPLC data from saccharified dry samples and corresponding ethanol fermentation are as follows: Dry Fermentation Results Starting Glucose Consumed Glucose Starting Xylose Consumed Xylose Ethanol Yield (Ethanol/Glu + Xly) (g/100ml) (g/100ml) (g/100ml) (g/100ml) (g/100ml) (g) 2=3 Gene Cellulase-D-S 0.6621 ± 0.0000 0.6621 0.1679 ± 0.0000 0.1679 0.268 ± 0.0018 0.323 13 PZP-NPT-4 Gene MTLCC-D 0.7624 ± 0.0000 0.7624 0.2061 ± 0.0000 0.2061 0.3222 ± 0.0004 0.333 10 HPT-E1-MTLCC-D 0.7245 ± 0.0000 0.7245 0.182 ± 0.0000 0.1820 0.3219 ± 0.0029 0.355 11:PZP-NPT-3-Gene-Cellulase-D 0.7488 ± 0.0000 0.7488 0.1935 ± 0.0000 0.1935 0.3315 ± 0.0049 0.352 8-HPT-E1-MTLCC-D 0.7001 ± 0.0000 0.7001 0.1897 ± 0.0000 0.1897 0.2879 ± 0.0091 0.324 9-HPT-E1-MTLCC-D 0.6757 ± 0.0000 0.6757 0.1712 ± 0.0000 0.1712 0.289 ± 0.0131 0.341 13-HPT-E1-TTLCC-D 0.756 ± 0.0000 0.7560 0.1964 ± 0.0000 0.1964 0.3338 ± 0.0147 0.350 1=3 Gene Cellulase-D 0.6682 ± 0.0000 0.6682 0.1769 ± 0.0000 0.1769 0.294 ± 0.0001 0.348 6 HPT-H-MTLCC-D 0.7069 ± 0.0000 0.7069 0.1863 ± 0.0000 0.1863 0.2659 ± 0.0039 0.298 5 PZP-NPT-4-Gene-MTLCC-D 0.6684 ± 0.0000 0.6684 0.1748 ± 0.0000 0.1748 0.2972 ± 0.0021 0.352 4 HPT-E1-MTLCC-D 0.7145 ± 0.0000 0.7145 0.1959 ± 0.0000 0.1959 0.2945 ± 0.0095 0.323 3 HPT-E1-MTLCC-D 0.7189 ± 0.0000 0.7189 0.1881 ± 0.0000 0.1881 0.3364 ± 0.0157 0.371 WT Brachypodium-D 1.0867 ± 0.0000 1.0867 0.2126 ± 0.0000 0.2126 0.4646 ± 0.0096 0.358 Similarly, HPLC data from saccharified fresh samples and corresponding ethanol fermentation are as follows: Wet Fermentation Results Starting Glucose Consumed Glucose Starting Xylose Consumed Xylose Ethanol Yield (Ethanol/Glu + Xly) (g/100ml) (g/100ml) (g/100ml) (g/100ml) (g/100ml) (g) 2 PZP-3 Genes Cellulase L1-4-W 0.6259 ± 0.0000 0.6259 0.1269 ± 0.0000 0.1269 0.2301 ± 0.0024 0.306 4 HPT-E1-MTLCC-W 0.7266 ± 0.0000 0.7266 0.1408 ± 0.0000 0.1408 0.2768 ± 0.0048 0.319 6 E1-MTLCC-L1-3-W 0.6997 ± 0.0000 0.6997 0.1108 ± 0.0000 0.1108 0.2714 ± 0.0001 0.335 3 HPT-E1-MTLCC1-W 0.8128 ± 0.0000 0.8128 0.1245 ± 0.0000 0.1245 0.3101 ± 0.0059 0.331 1-3-Gene Cellulase-W 0.798 ± 0.0000 0.7980 0.1128 ± 0.0000 0.1128 0.2987 ± 0.0086 0.339 11-PZP-4 Gene-MTLCC-W 0.6774 ± 0.0000 0.6774 0.1101 ± 0.0000 0.1101 0.251 ± 0.0045 0.319 7-PZP-4 Gene-MTLCC-W 0.7098 ± 0.0000 0.7098 0.1113 ± 0.0000 0.1113 0.2683 ± 0.0021 0.327 WT Brachypodium-W 0.9247 ± 0.0000 0.9247 0.1591 ± 0.0000 0.1591 0.3348 ± 0.0113 0.309 5 PZP-4 Genes-MTLCC-L1-3-W 0.731 ± 0.0000 0.7310 0.142 ± 0.0000 0.1420 0.2573 ± 0.0049 0.295 8 HPT-E1-MTLCC-W 0.7471 ± 0.0000 0.7471 0.144 ± 0.0000 0.1440 0.2919 ± 0.0024 0.328 One of the primary goals of this study was to show whether the saccharified bioengineered biomasses were fermentable by a bioengineered strain of E. coli FBR5: In conclusion, based on these data, fermentations of the wet biomasses (the average of all samples) yielded 0.321 g ethanol/g glucose plus xylose, which is 62.9% of the theoretical 0.51 g/g. Likewise, the dry samples' fermentations averaged 0.341 g ethanol/g glucose plus xylose, which is 66.9% of the theoretical value. Therefore, we can conclude that all bioengineered materials tested were successfully fermented. However, they yielded lower than theoretical or lower than what you would typically see in fermentations of pure sugar.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Ahlawat, Y.K., Biswal, A.K., Harun, S. et al. Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus. Biotechnol Biofuels 17, 5 (2024). https://doi.org/10.1186/s13068-023-02452-7
|
Progress 03/01/22 to 02/28/23
Outputs
Target Audience:Molecular microbiologist, students with interest in research in planta depolymerization oflignin, microbial fermentation scientists, plant biotechnologists, scientists interested in lignin recalcitrance Changes/Problems:During this period, the budget which was allocated to hire a Postdoctoral Fellow to perform these activities will be used to support the graduate student tuition, and stipend and provide funding for equipment such as aPCR machine, a RT-PCR machine, a new Conviron plant growth facility, laboratory supplies and comsumables, travel, and equipment repairs. This change is due to the fact that, although a hiring committee selected by the Human Ecology Department met varioustimes to make a selection for a suitable Postdoctoral Fellow to undertake these research activitiesand recommend for hiring, the employment offer did not get to thesePostdoctoral Fellows in the timely manner for them to accept employment. Such significant delay to offer employment for selected candidate led to decline employment fromall these selected candidates. Furthermore, this grant will hit the 5-years statutory limit on Febrary 28, 2024with no possibilty for a no cost extension and the remainder of the grant activities will be performed by the PI, Dr. Bertrand Hankoua, graduate student, Dr. Nancy Nichols (NCAUR-ARS), Mrs. Frazer sarah (NCAUR-ARS). What opportunities for training and professional development has the project provided?The PI, Dr. Bertrand Hankoua, has the opportunity for training and professoionla development in biomass saccharification, and ethanol fermentatbility at Dr.NancyN. Nichols Laboratoryat the National Center for Agricultural Utilization Research, Bioenergy Research Unit, Peoria, Illinois. All these training and professional develoment activities were performed under the supervision of Mrs.Sarah Frazer, Biological Science Technician inDr.NancyN. Nichols Laboratory from July 9 until August, 4th 2023. 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?During the next reporting period, we will repeat the laccase self-induction, saccharification, yeast bioscreen, and fermetability using 6 grams instead of 1 gram of biomass of these lines as reported in this period: Brachypodium (non-bioengineered, wild type), dried biomass from engineered lines (6-HPT-H-MTLCC,5-PZP-NPT-4Gene-MTLCC,4-HPT-E1-MTLCC,2-3 Gene Cellulase,1-3 Gene Cellulase, 3-HPT-E1-MTLCC, 11-PZP-NPT-3 Gene Cellulase,13-PZP-NPT-4 Gene,10-HPT-E1-MTLCC, 9-HPT-E1-MTLCC, 8-HPT-E1-MTLCC, 7-PZP-4 Gene-MTLCC13-HPT-E1-TTLCC,13-PZP-NPT-4 Gene,10-HPT-E1-MTLCC.9-HPT-E1-MTLCC,7-PZP-4 Gene-MTLCC,13-HPT-E1-TTLCC), and wetbiomass from engineered lines (8-HPT-E1-MTLCC,3-HPT-E1-MTLCC,4-HPT-E1-MTLCC,1-3-Gene Cellulase,6-E1-MTLCC-L1-3,5-PZP-4 Gene-MTLCC-L1-3,2-PZP-3 Gene Cellulase-L1-4,7-PZP-4 Gene-MTLCC,11-PZP-4 Gene-MTLCC,13-PZP-NPT-4 Gene-MTLCC-L-13,9-HPT-E1-MTLCC,12-HPT-E1-MTLCC (*3 Gene),PZP-NPT-4 Gene-MTLCC-L13-2). This will allow to produce a good level of sugars in the hydrolysates so that meaning metabolic yield data will be generated from the fermentation trial. Data will be published and a graduate student will participate in this next experimental steps.
Impacts
What was accomplished under these goals?
Research activities undertaken during this period relate essential on performing ethanol fermentation trials using cellulase hydrolysates generated from various transgenic brachypodum expressing single or a combination of extremophiles laccases and cellulose and hemicellulose degrading emzymes. Here are the transgenic bracypodium lines developed from Delaware State University which were taken toDr. Nancy Nichols at the NCAUR in Peoria to perform the ethanol fermentation trials: Brachypodium (non-bioengineered, wild type), dried biomass from engineered lines (6-HPT-H-MTLCC,5-PZP-NPT-4Gene-MTLCC,4-HPT-E1-MTLCC,2-3 Gene Cellulase,1-3 Gene Cellulase, 3-HPT-E1-MTLCC, 11-PZP-NPT-3 Gene Cellulase,13-PZP-NPT-4 Gene,10-HPT-E1-MTLCC, 9-HPT-E1-MTLCC, 8-HPT-E1-MTLCC, 7-PZP-4 Gene-MTLCC13-HPT-E1-TTLCC,13-PZP-NPT-4 Gene,10-HPT-E1-MTLCC.9-HPT-E1-MTLCC,7-PZP-4 Gene-MTLCC,13-HPT-E1-TTLCC), and wetbiomass from engineered lines (8-HPT-E1-MTLCC,3-HPT-E1-MTLCC,4-HPT-E1-MTLCC,1-3-Gene Cellulase,6-E1-MTLCC-L1-3,5-PZP-4 Gene-MTLCC-L1-3,2-PZP-3 Gene Cellulase-L1-4,7-PZP-4 Gene-MTLCC,11-PZP-4 Gene-MTLCC,13-PZP-NPT-4 Gene-MTLCC-L-13,9-HPT-E1-MTLCC,12-HPT-E1-MTLCC (*3 Gene),PZP-NPT-4 Gene-MTLCC-L13-2). Dried and wet biomasses from these transgenic brachypodium lines were used for lignin self -digestibility, saccharification, and ethanol fermentatbility at Dr.NancyN. Nichols Laboratoryat the National Center for Agricultural Utilization Research, Bioenergy Research Unit, Peoria, Illinois. All research activities were performed under the supervision of Mrs.Sarah Frazer, Biological Science Technician inDr.NancyN. Nichols Laboratory. The overall research was supervised by bothDr.NancyN. Nichols andMrs.Sarah Frazer. Once the moisture content of all biomasses were recorded, approximately 1 gram of each biomasses were first pre-induced in the laccase reaction buffer containing cupper sulfate and hydrogen peroxide for 72 hours under a shaking incubator set at 280 rpm and at 75oC. After, biomass pellets from all samples were saccharified with cellulase enzyme at 50oC for 72 hours. Biomass hydrolysates were used first to estimate the content of C5 and C6 sugars using HPLC and compare these data with control samples with the goal to estimate the potential of each lines to release sugars. HPC data from saccharified samples are as follow. g/g dw Sucrose Glucose Xylose Galactose Arabinose Ethanol Glycerol Arabitol Xylitol Brachypodium 0 0.0760 0.0395 0.0029 0.0044 0 0 0 0 6-HPT-H-MTLCC 0 0.0939 0.0432 0.0030 0.0060 0 0 0 0 5-PZP-NPT-4Gene-MTLCC 0 0.1056 0.0446 0.0030 0.0059 0 0 0 0 4-HPT-E1-MTLCC 0 0.1024 0.0475 0.0030 0.0059 0 0 0 0 2-3 Gene Cellulase 0 0.1033 0.0472 0.0030 0.0059 0 0 0 0 1-3 Gene Cellulase 0 0.1036 0.0474 0.0030 0.0059 0 0 0 0 3-HPT-E1-MTLCC 0 0.0924 0.0411 0.0029 0.0059 0 0 0 0 11-PZP-NPT-3 Gene Cellulase 0 0.1027 0.0455 0.0029 0.0059 0 0 0 0 13-PZP-NPT-4 Gene 0 0.1020 0.0503 0.0030 0.0059 0 0 0 0 10-HPT-E1-MTLCC 0 0.1021 0.0444 0.0030 0.0059 0 0 0 0 9-HPT-E1-MTLCC 0 0.1037 0.0459 0.0030 0.0059 0 0 0 0 8-HPT-E1-MTLCC 0 0.0934 0.0438 0.0029 0.0058 0 0 0 0 7-PZP-4 Gene-MTLCC 0 0.0906 0.0468 0.0029 0.0058 0 0 0 0 13-HPT-E1-TTLCC 0 0.1106 0.0472 0.0029 0.0059 0 0 0 0 8-HPT-E1-MTLCC 0 0.1134 0.0625 0.0078 0.0117 0 0 0 0 3-HPT-E1-MTLCC 0 0.0948 0.0531 0.0076 0.0076 0 0 0 0 4-HPT-E1-MTLCC 0 0.1051 0.0683 0.0105 0.0105 0 0 0 0 1-3-Gene Cellulase 0 0.1217 0.0608 0.0087 0.0087 0 0 0 0 6-E1-MTLCC-L1-3 0 0.0891 0.0576 0.0105 0.0105 0 0 0 0 5-PZP-4 Gene-MTLCC-L1-3 0 0.1130 0.0633 0.0090 0.0090 0 0 0 0 2-PZP-3 Gene Cellulase-L1-4 0 0.1050 0.0573 0.0095 0.0095 0 0 0 0 7-PZP-4 Gene-MTLCC 0 0.0802 0.0519 0.0094 0.0094 0 0 0 0 11-PZP-4 Gene-MTLCC 0 0.0718 0.0538 0.0120 0.0120 0 0 0 0 13-PZP-NPT-4 Gene-MTLCC-L-13 0 0.1247 0.0794 0.0113 0.0113 0 0 0 0 9-HPT-E1-MTLCC 0 0.1116 0.0691 0.0106 0.0106 0 0 0 0 12-HPT-E1-MTLCC (*3 Gene) 0 0.1151 0.0704 0.0128 0.0128 0 0 0 0 PZP-NPT-4 Gene-MTLCC-L13-2 0 0.1708 0.1252 0.0228 0.0228 0 0 0 0 Data demonstratedthat the majority of transgenic Brachypodium release more glucose, xylose, arabinose, and glactose than the non transgenicBrachypodium lines possibly to reduce lignin recalcitrance of these transgenic due to lignin self-depolymerization duing laccase self-induction experiment. These encouraging data prompted the run of the Bioscreen Experiment using YRH2066 a haploid yeast engineered for xylose utilization. Data from this experiment demontrated thatthis yeast strain YRH2066 effectively utilized glucose and zylose from all the saccharified brachy podiumsamples inenzyme digested supernatants. Dr.Ron Hector, a Research Scientist at the REE-ARS and collaborator of Dr. Nancy Nichols provided some guidances to perform and interprete data from theBioscreen Experiment. Encouraging data from the bioscreen experiment led to perform ethanol fermentation experiment with all saccharified trasngenic biomass samples including samples from non transgenic brachypodium control. Data from the fermentation trial is a fellow. Metabolic Yield* Production Yield Ethanol Produced EtOH/(Consumed Glu+Xyl) EtOH/(Starting Glu + Xyl) EtOH/gdw g/100ml g/g g/g g/g 1-D 0.059 0.347058824 0.279620853 0.063415304 11-D 0.091 0.348659004 0.296416938 0.09913901 14-D 0.084 0.302158273 0.257668712 0.091076886 15-W 0.028 0.259259259 0.204379562 0.080471929 18-W 0.031 0.322916667 0.254098361 0.099037267 24-W 0.024 0.289156627 0.218181818 0.100052628 25-W 0.019 0.25 0.188118812 0.074212398 26-W 0.011 0.174603175 0.129411765 0.051736803 27-W 0.012 0.210526316 0.155844156 0.100458152 *theoretical yield is 0.51 Data shows that only two transegnic lines 11-D and 14-D produced more ethanol in 100 ml reaction volume compared to the non-transgenic lines. The metabolic yield differences data are all fairly low and might be due to thelow starting glucose present in the all the hydrolysates.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Ali Parsaeimehr, Ifti Iftekhar Ahmed, Myriam Loretta Kouemo Deumaga, Bertrand Hankoua, Gulnihal Ozbay,
Enhancement in phycobiliprotein accumulation in Aphanothece sp. using different carbon sources and flashing frequency,
Algal Research,
Volume 66,
2022,
102805,
ISSN 2211-9264,
https://doi.org/10.1016/j.algal.2022.102805.
(https://www.sciencedirect.com/science/article/pii/S221192642200176X)
|
Progress 03/01/21 to 02/28/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Dr. Ali Parsaeimehr, a Postdoctoral fellow initialliy hired to work on the projectleft the laboratory in November 2021. The co-PD, Professor Tom Richard of Penn State University also could not accept a sub-award due to its retirement. Dr. Nancy Nichols from ARS agree to take over all research activities ofProfessor Tom Richard as co-PD in this project. This process of co-PD change as well as re budgetting is currently being process at NIFA. The University has conducted a search to hire a new Postdoctoral Fellow to replaceDr. Ali Parsaeimehr. This process is concluded and a suitable Postdoc has been identified and will be joining the laboratory to continue deliverying on project milestones. 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?1) To compare the cellulose and lignin composition of biomass of parental and transformed lines; 2) To evaluate the saccharification, auto-hydrolysis, micropyrolysis of the engineered biomass as well as evaluate the fermentability of the saccharified samples. 3) Develop an efficient Agrobacterium transformation system for Miscanthus x Giganteus; 4) To train a new generation of minority students in advanced biofuel research, molecular biotechnology; 5) To increase capacities for biotechnology and bio-energy research at Delaware State University. Depending on the outcome of this feasibility phase, future work will include increasing the activity of these enzymes in Brachypodium through enzyme evolution, characterizing the best transgenic lines for biofuel production and evaluate their field performance. This approach will be transferred toMiscanthus x Giganteus, a high biomass-yielding energy grass as a strategy to develop novel grass with enhanced bioprocessing characteristics once validated in model Brachypodium.
Impacts
What was accomplished under these goals?
During this period, the PI and co-PIs were seeking tio hire a postdoctoral fellow to engage in the research activities as outlined in the project. The reporting period coincided with the Covid-restrictions and the teh PI was not able to recruit students to get trained in the project. The University process to hire a postdoctoral fellowship scientist was initiated in late 2019 as soon as the award was made and this process was completed on July 6, 2020 when Dr. Ali Parsaeimehr joined the plant science laboratory from the University of Florida to start investigating in the project.Dr. Ali Parsaeimehr left the laboratory in November 2021. The co-PD, Professor Tom Richard of Penn State University also could not accept a sub-award due to its retirement. Dr. Nancy Nichols from ARS agree to take over all research activities ofProfessor Tom Richard as co-PD in this project. This process of co-PD change is currently being process at NIFA. The University has conducted a search to hire a Postdoctoral Fellow to replaceDr. Ali Parsaeimehr. This process is concluded and a suitable Postdoc has been identified and will ne joining the laboratory to continue deliverying on project milestones.
Publications
|
Progress 03/01/20 to 02/28/21
Outputs
Target Audience:Worldwide scientists and chemists from various institutions of higher learning working in the field of lignin recalcitrance; industries including biotechnology and bio-based companies involved in developing biotechnological and biochemical tools to reduce or eliminated lignin recalcitrance in dedicated feedstocks; collaborative institutions; local and state stakeholders interested in solving a national problem impeding the commercialization of cellulosic biomass; Students and Postdoctoral scientists interested to be involved in state-of-the-art and innovative science aiming to develop novel biomass with reducing lignin recalcitrance Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In December 2021, and in Collaboration with Dr. Eduardo Santillan-Jimenez of the Center for Applied Energy Research (CAER) of the University of Kentucky, the PI, Dr. Bertrand Hankoua, spend three weeks as a visiting scientist in (CAER) to perform GC-MS-Pyrolysis of biomasses originating from 13 bio-engineered Brachypodium lines which were fully characterized morphologically and molecularly by the Postdoctoral Fellow Dr. Ali Parsaeimehr. These bio-engineered Brachypodium lines are: 1(a) HPT-E1-MTLCC- L15; PZP-NPT- 3 gene cellulases- L2; HPT-E1-MTLCC-L2; PZP-NPT- 4 gene MTLCC-L2; HPT-E1-MTLCC-3 gene-L2; PZP-NPT-4 gene-MTLCC-L13;HPT-E1-TTLCC-L1; PZP-NPT-3gene-cellulase-L1; HPT-E1-MTLCC-L1; HPT1-MTLCC-L1; PZP-NPT-4 gene-MTLCC-L2; HPT-H-MTLCC-L1; PZP-NPT-3gene-cellulase-L1. It is important to note that the University of Kentucky is a co-PI institution for this project. How have the results been disseminated to communities of interest?During my visit to the Center for Applied Energy Research (CAER) of the University of Kentucky in December, I had a lot of interactions with graduate students, scientists, and Postdoctoral Fellow working inCAER and it was an opportunity to discuss the importance and state of the art science behind developing designer biomass using biotechnology tools to reduce biomass recalcitrance. What do you plan to do during the next reporting period to accomplish the goals?During the next period, the following objectives will be investigated: a)- Complete the self-saccharification for all the 13bio-engineered Brachypodium lines using HPLC Dionex; b) Perform compositional analysis and fermentability of self-induced biomass; c) Determine lignin removal capacity of this lignin self-depolymerization; d) Determine transgenic cellulase and laccase activities isolated from apoplast of these13bio-engineered Brachypodium lines; e)Complete the GC-MS-Micropyrolysis of theremainingbio-engineered Brachypodium lines in collaboration with CAER; f) Start the Agrobacterium compatibility screening using embryonic callus of Giant Miscanthus derived from young in vitro leaves; g) Trained undergraduates and graduate students in these molecular and biochemical tools and their application in bioenergy research. h) Prepare manuscripts and provisional patent applications.
Impacts
What was accomplished under these goals?
Dr. Ayalew Osena, a Postdoctoral scientist hired in Phase I of this project generated 77bio-engineered Brachypodiumlines with various combinations of expression vectors containing cellulases and laccases in single or multiple combinations. Once Dr. Ali was hired in July 2020, he performed germination and soil establishment trials of these transgenic Brachypodium lines. But out of the 77bio-engineered Brachypodiumlines, only 13 bio-engineered Brachypodiumlines successfully germinated and were established in the greenhouse. These 13 lines are 1(a) HPT-E1-MTLCC- L15; PZP-NPT- 3 gene cellulases- L2; HPT-E1-MTLCC-L2; PZP-NPT- 4 gene MTLCC-L2; HPT-E1-MTLCC-3 gene-L2; PZP-NPT-4 gene-MTLCC-L13;HPT-E1-TTLCC-L1; PZP-NPT-3gene-cellulase-L1; HPT-E1-MTLCC-L1; HPT1-MTLCC-L1; PZP-NPT-4 gene-MTLCC-L2; HPT-H-MTLCC-L1; PZP-NPT-3gene-cellulase-L1. The phenotypes of these 13 bio-engineered Brachypodiumlines were fully characterized morphologically under greenhouse conditions and biomasses were harvested from all the lines for molecular characterization through PCR and RT-PCR. The phenotypic characterization of B. distachyon transformed with different lignocellulose-degrading genes cloned in pPZP-NPTI vector. A maximum height was achieved from HPT1-MTLCC-L1, and the minimum height was achieved from PZP-NPT-4 gene-MTLCC-L2 treatments with an observation of early flowering. The number of spikes were recorded on 150 days of germination. HPT-E1-MTLCC: Acidothermus cellulolyticus endoglucanase E1 and Myceliophthora thermophila LCC1, PZP-NPT- 3 gene cellulose: Acidothermus cellulolyticus endoglucanase E1, Thermotoga maritima b-glucosidase and Caldicellulosiruptor bescii exoglucanase CBH1, PZP-NPT- 4 gene MTLCC: Acidothermus cellulolyticus endoglucanase E1,Thermotoga maritima, b-glucosidase and Caldicellulosiruptor bescii exoglucanase CBH1 and Myceliophthora thermophila Lcc1, HPT-E1-TTLCC-3 gene: Acidothermus cellulolyticus endoglucanase E1, Thermotoga maritima b-glucosidase and Caldicellulosiruptor bescii exoglucanase CBH1, HPT-E1-TTLCC: Acidothermus cellulolyticus endoglucanase E1, Thermotoga maritima b-glucosidase, HPT-MTLCC: Myceliophthora thermophila LCC1, PZP-NPT-3gene-cellulose: Acidothermus cellulolyticus endoglucanase E1, Thermotoga maritima b-glucosidase and Caldicellulosiruptor bescii exoglucanase CBH1. Total protein was obtained from all 13 lines to perform laccase and cellulase activities. The laccase activities were measured by screening the oxidation of model lignin compounds (ABTS, DMP, Veratryl alcohol), and the lignin (Kraft). (a) ABTS (2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid) laccase activity test was performed at OD420 using 0.5 mM ABTS, 0.05 mM H2O2 in 50 mM sodium acetate (pH=5.0). (b) 2,6-DMP (2,6 Dimethoxyphenol) laccase activity test was done at OD468 using 2mM 2,6-DMP and 0.125 mM H2O2 in 50 mM sodium acetate buffer (pH= 5.0). (c) Veratryl alcohol laccase assay was performed using 0.25 mM Veratryl alcohol, 0.2mM H2O2 in 0.01 M citrate buffer (pH=4.8). (d) Lignin (Kraft) laccase activity was performed at OD465 by 0.5mg/ml of lignin, 4mM H2O2 in 50 mM succinic buffer (pH=5.5). Each reaction was supplemented with 100 mM concentrated crude protein extracts, which had been heated at 80°C for 25 min to eliminate mesophilic enzymes which may interfere with the thermophilic enzyme activity assay. Transgenic cellulases activities within this cocktail of enzymes obtained from all these lines are ongoing. Lignin activities with small molecules, lignin monomers, lignin model, and lignin from wood biomasses using total protein isolated from apoplast of biomasses isolated from all the transgenic lines were also performed. Colorimetric assay of biomass saccharification from wild type and transgenic B. distachyon using a different buffers at different concentrations of CuSO4 was performed. Buffers tested from biomass saccharification were: (a) 50 mM succinic buffer (b) 50 mM Sodium acetate buffer, (c) 10 mM citrate buffer, (d) 10mM phosphate buffer. The self-induction reaction started by adding H2O2 40 (mM), and the samples were shaken at 250 rpm at 75°C for 70 hours. pH of all the buffers was adjusted to 5.6. An increase of 222% in reducing sugar was achieved in line PZP-NPT- 4 gene MTLCC simultaneously expressing Acidothermus cellulolyticus endoglucanase E1, Thermotoga maritima, b-glucosidase, and Caldicellulosiruptor bescii exoglucanase CBH1 and Myceliophthora thermophila Lcc1compared to the wild type non-bioengineered B. distachyon and this dramatic increase in reducing sugars was achieved from the succinic buffer with 2mM CuSO4. The lowest reducing sugar was observed in phosphate buffer. A novel system of in vitro regeneration of Miscanthus X Giganteus was developed and this system is based on using young in vitro leaves from in vitro tillersof Miscanthus X Giganteusto induce and proliferate non-morphogenic callus and regenerate shoot from such proliferating embryogenic callus. This system will be key to successfully establishing Agrobacterium and biolistic mediated transformation of Miscanthus X Giganteus.
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Progress 03/01/19 to 02/29/20
Outputs
Target Audience:Plant biotechnologists, industries with interest to use biotechnologyb tools to address lignin recalcitrance, students and postdoctoral scientists interested to gain skill in bioconversion and development of novel biomass with self-digestable capabilities 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?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next period, the PIs and co-Pis in collaboration with the Postdoc and students planned to investigate the following objectives: 1) To compare the cellulose and lignin composition of biomass of parental and transformed lines; 2) To evaluate the saccharification, auto-hydrolysis, micropyrolysis of the engineered biomass as well as evaluate the fermentability of the saccharified samples. 3) Develop an efficient Agrobacterium transformation system for Miscanthus x Giganteus; 4) To train a new generation of minority students in advanced biofuel research, molecular biotechnology; 5) To increase capacities for biotechnology and bio-energy research at Delaware State University. Depending on the outcome of this feasibility phase, future work will include increasing the activity of these enzymes in Brachypodium through enzyme evolution, characterizing the best transgenic lines for biofuel production and evaluate their field performance. This approach will be transferred to Miscanthus x Giganteus, a high biomass-yielding energy grass as a strategy to develop novel grass with enhanced bioprocessing characteristics once validated in model Brachypodium.
Impacts
What was accomplished under these goals?
During this period, the PI and co-PIs were seeking tio hire a postdoctoral fellow to engage in the research activities as outlined in the project. The reporting period coincided with the Covid-restrictions and the teh PI was not able to recruit students to get trained in the project. The University process to hire a postdoctoral fellowship scientist was initiated in late 2019 as soon as the award was made and this process was completed on July 6, 2020 when Dr. Ali Parsaeimehr joined the plant science laboratory from the University of Florida to start investigating in the project.
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